Case Studies of the Hot Dog-Fold and Acyl- Adenylate-Forming Superfamilies: Characterizing the Importance of Functional Divergence in Cellular Metabolism Lucas Zimney

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9-1-2015 Case Studies of the Hot Dog-Fold and Acyl- Adenylate-Forming Superfamilies: Characterizing the Importance of Functional Divergence in Cellular Metabolism Lucas Zimney

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This Dissertation is brought to you for free and open access by the Electronic Theses and Dissertations at UNM Digital Repository. It has been accepted for inclusion in Chemistry ETDs by an authorized administrator of UNM Digital Repository. For more information, please contact [email protected]. ! ! ! ! ! ! ! !!!!!Lucas!R.!Zimney! !!!!!!!Candidate!! !!!!!! !!!!!Chemistry!and!Chemical!Biology! !!!!!Department! !!!!!! ! !!!!!This dissertation is approved, and it is acceptable in quality and form for publication: ! !!!!!Approved!by!the!Dissertation!Committee:! ! !!!!!!!! ! ! ! ! ! ! ! !!!!!Debra!Dunaway;Mariano,!Chairperson! !! ! !!!!!Patrick!S.!Mariano! ! ! !!!!!Charles!E.!Melançon!III! ! ! !!!!!Karen!N.!Allen! ! ! !!!!!! ! ! !!!!!! ! ! !!!!!!! ! ! !!!!!!! ! ! !!!!!!! ! ! ! !

! i! ! ! ! ! ! ! ! ! CASE!STUDIES!OF!THE!HOT!DOG.FOLD!AND!ACYL.! ADENYLATE.FORMING!SUPERFAMILIES:! CHARACTERIZING!THE!IMPORTANCE!OF!FUNCTIONAL! DIVERGENCE!IN!CELLULAR!METABOLISM! ! ! ! ! by! ! ! ! ! ! LUCAS!R.!ZIMNEY! ! B.S.,!Chemistry,!South!Dakota!State!University,!2009! ! ! ! ! ! ! ! ! ! ! ! DISSERTATION! ! Submitted!in!Partial!Fulfillment!of!the! Requirements!for!the!Degree!of! ! Doctor!of!Philosophy! Chemistry! ! The!University!of!New!Mexico! Albuquerque,!New!Mexico! ! ! July,!2015! !

! ii! DEDICATION!

To!my!parents,!Barry!and!Maureen,!who!always!took!interest!in!what!I!was! doing,!even!if!they!thought!I!was!just!making!stuff!up.!Thank!you!for!the!constant! love,!support!and!the!steady!flow!of!baked!goods,!without!any!of!which!this!would! not!have!been!possible.!!

! iii! ACKNOWLEDGEMENTS!

If! the! past! six! years! have! taught! me! nothing! else,! it! has! shown! me! that! success! is! seldom! achieved! alone.! Throughout! this! journey! I! have! received! many! helping!hands!from!advisors,!mentors,!colleagues!and!friends.!Those!of!you!know! who!you!are!and!deserve!more!than!just!a!small!mention!on!this!page.!Nevertheless,! here!goes.!

First! and! foremost,! I! must! thank! my! advisor,! Dr.! Debra! Dunaway;Mariano,! for! her! significant! contributions! to! my! education! and! research.! Her! expertise! and! guidance! in! my! research! and! post;grad! life! has! proven! to! be! invaluable.! Also,! I! would!like!to!recognize!Dr.!Patrick!Mariano!and!my!remaining!committee!members!

(Dr.!Chad!Melançon!and!Dr.!Karen!Allen),!and!thank!them!for!all!of!their!advice!and! helpful!suggestions!along!the!way.!

During!my!time!in!DDM’s!lab,!I!was!fortunate!enough!to!work!with!a!truly! fantastic!group!of!people!that!I!am!proud!to!call!my!colleagues!and!friends.!I!want!to! thank! my! mentor,! Hong,! for! showing! me! the! ropes! and! teaching! me! all! of! the! molecular! biology! and! enzymology! basics.! Also,! many! thanks! to! Chunliang,! who! seemingly! had! an! unlimited! amount! of! patience! in! helping! me! with! organic! synthesis.!!To!my!two!best!friends!in!the!lab,!Tyrel!and!John!;;!thanks!for!all!of!the! intellectual!conversations,!research!discussions,!the!stupid!pranks!and!of!course,!the! dirty! jokes.! You! guys! made! life! in! and! out! of! the! lab! a! ton! of! fun.! To! my! two! undergraduate!minions,!Kaila!and!Rachel!;;!you!guys!put!in!a!lot!of!truly!spectacular!

! iv! work!on!the!ligase!project.!Without!your!hard!work!and!dedication,!we!would!not! have!accomplished!nearly!as!much!as!we!did.!!

Lastly,! to! Kaila! ;;! your! love! and! support! outside! of! the! lab! was! the! only! reason! I! kept! my! sanity! during! these! last! few! months.! Thank! you! for! keeping! me! motivated!and!focused!on!the!finish!line.!It!“litrally”!means!the!world!to!me!and!I! will!be!forever!grateful.!

! v! ! ! ! ! ! ! ! ! ! CASE!STUDIES!OF!THE!HOT!DOG.FOLD!AND!ACYL.! ADENYLATE.FORMING!SUPERFAMILIES:! CHARACTERIZING!THE!IMPORTANCE!OF!FUNCTIONAL! DIVERGENCE!IN!CELLULAR!METABOLISM! ! ! ! ! by! ! ! ! ! ! LUCAS!R.!ZIMNEY! ! ! ! ! ! ! ! ! ! ! ! ABSTRACT!OF!DISSERTATION! ! ! ! Submitted!in!Partial!Fulfillment!of!the! Requirements!for!the!Degree!of! ! Doctor!of!Philosophy! Chemistry! ! The!University!of!New!Mexico! Albuquerque,!New!Mexico! ! ! July,!2015!

! vi! Case!Studies!of!the!Hot!Dog.Fold!and!Acyl.Adenylate.Forming!Superfamilies:!

Characterizing!the!Importance!of!Functional!Divergence!in!Cellular!

Metabolism!

Lucas!R.!Zimney!

B.S.,!Chemistry,!South!Dakota!State!University,!2009!

ABSTRACT!

! !

! Some! of! the! biggest! contributors! to! cellular! respiration! (and! cellular! metabolism!in!general)!are!acyl;CoA!derivatives,!a!subclass!of!biological!thioesters.!

Known!to!function!in!a!variety!of!pathways,!the!regulation!of!their!formation!and! breakdown! are! critical,! carried! about! by! acyl;CoA! synthetases! and! thioesterases,! respectively.! The! work! reported! within! this! dissertation! will! focus! on! functional! divergence!within!two!enzyme!superfamilies!;;!the!hot!dog;fold!and!acyl;adenylate; forming!superfamilies!–!and!can!be!broken!down!into!two!main!parts.!

! Part!one!will!look!at!tracking!the!functional!divergence!within!the!hot!dog; fold!superfamily!thioesterases.!A!highly!evolved!thioesterase,!flK,!has!been!found!to! function!in!the!critical,!and!highly!specific!role!of!fluoroacetate!detoxification!within! the! fluorometabolite;producing! bacteria,! S.! cattleya.! Using! an! extensive! bioinformatics! analysis,! flK! orthologs! were! identified! and! tracked! throughout! all!

! vii! three! domains! of! life,! primarily! in! bacteria! that! make! up! the! gut! microbiome.!

Additionally,! sequence! and! structural! analyses! revealed! distinct! flK! scaffolds,! a! further! indication! of! divergent! functionality.! Various! flK! orthologs! were! then! isolated,!cloned!and!subjected!to!substrate!screening!by!measuring!their!individual! steady;state! kinetic! parameters! kcat,! Km! and! kcat/Km.! Combined! with! gene! context! analyses,! divergent! in! vivo! functionality! was! assigned! to! members! of! the! flK! subfamily,!as!they!were!proposed!to!be!involved!in!supplying!formate!for!the!one; carbon!pool.!

! Part! two! will! focus! on! the! functional! characterization! of! the! acyl;CoA! synthetases!(ligases)!in!Pseudomonas!aeruginosa,!the!dominant!pathogen!present!in! all! patients! with! cystic! fibrosis! and! the! leading! cause! of! morbidity! and! mortality! within!this!afflicted!population.!Nine!freestanding!ligases!were!cloned,!isolated!and! subjected!to!an!extensive!substrate!screening!for!acyl;CoA!synthetase!activity!using! a!novel!high;throughput!assay.!Individual!activities!were!verified!by!measuring!the! steady;state!kinetic!parameters.!Combing!these!results!with!extensive!gene!context! analyses,!in!vivo!functions!were!proposed!for!the!tested!ligases,!implicating!them!in! a!variety!of!nutrient!scavenging!pathways!as!well!as!in!virulence!factor!production.!

! !

! viii! TABLE!OF!CONTENTS!

LIST!OF!ABBREVIATIONS………………………………………………………………………………xii!

LIST!OF!TABLES..………………………………………………………………………………………...xviii!

LIST!OF!FIGURES…………………………………………………………………………………………xxvi!

CHAPTER!1:!The!Biological!Thioester!–!An!Overview!of!its!Function!in!Cellular!

Metabolism!and!the!Enzymes!that!Regulate!it………………………………..…………….....1!

1.1 Overview!of!Cellular!Metabolism………………………………………………………..…1!

1.1.1!Cellular!Respiration………………………………………………………………..1!

1.1.2!Biological!Significance!of!Thioesters………………………………………..5!

1.2!Acyl;CoA!Thioesterases………………………………………………………………………..6!

! 1.2.1!The!Hot!Dog;Fold!Superfamily!and!Hot!Dog!Thioesterases……….7!

! 1.2.2!Mechanism!of!Catalysis!of!HD!Thioesterases……………………………9!

! 1.2.3!Divergence!of!Function!Within!HD!Thioesterases…………………..11!

! 1.3!The!Acyl;Adenylate;Forming!Superfamily…………………………………………..14!

! ! 1.3.1!Functional!Roles!of!Acyl;CoA!Synthetases……………………………...18!

! ! 1.3.2!Structure;Function!Relationship!of!Acyl;CoA!Synthetases……....21!

! 1.4!Summary…………………………………………………………………………………………...22!

! References……………………………………………………………………………………………....23!

CHAPTER! 2:! Novel! Thioesterase! Activity! from! S.#cattleya#flK! Reveals! a! Basis!

For!Divergent!Biological!Function…………………………………………………………………30!

! 2.1!Introduction………………………………………………………………………………………30!

! ix! ! 2.2!Methods!and!Materials……………………………………………………………………….43!

! ! 2.2.1!Materials………………………………………………………………………………43!

! ! 2.2.2!Synthesis!of!Acyl;CoA!Substrates…………………………………………..44!

! ! 2.2.3!Cloning,!Expression!and!Purification!of!flK!and!Orthologs………44!

! ! 2.2.4!Determination!of!Steady;State!Kinetic!Parameters…………………46!

2.2.5! Crystallization! and! X;ray! Structure! Determination! of! MA0038!

and!BVU_1957………………………………………………………………………………46!

2.2.6!Bioinformatics!Analysis………………………………………………………...47!

! 2.3!Results!and!Discussion……………………………………………………………………….48!

! ! 2.3.1!Biological!Range!of!the!Fluorinase!Cluster……………………………..48!

! ! 2.3.2!Biological!Range!and!Sequence!Analysis!of!flK!Orthologs………..49!

! ! 2.3.3!Cloning!and!Isolation!of!flK!Orthologs……………………………………57!

! ! 2.3.4!Substrate!Specificity!of!flK!Orthologs……………………………………..57!

! ! 2.3.5!Structural!Analysis!of!flK!and!Isolated!Orthologs……………………61!

! ! 2.3.6!Gene!Context!Analysis…………………………………………………………..67!

! ! 2.3.7!Divergence!of!Function…………………………………………………………71!

! 2.4!Summary…………………………………………………………………………………………...73!

References………………………………………………………………………………………………76!

CHAPTER!3:!Characterizing!the!Contribution!of!Multiple!Acyl.CoA!Synthetases!

Towards! Enhanced! Virulence! in! Pseudomonas# aeruginosa…………………………………………………………………………………………………….84!

! 3.1!Introduction………………………………………………………………………………………84!

! 3.2!Methods!and!Materials……………………………………………………………………….94!

! x! ! ! 3.2.1!Materials………………………………………………………………………………94!

! ! 3.2.2!Bioinformatics!Analysis………………………………………………………...95!

! ! 3.2.3!Cloning,!Expression!and!Purification!of!P.!aeruginosa!Ligases…96!

! ! 3.2.4!High;throughput!Substrate!Screening………………………………….100!

! ! 3.2.5!Determination!of!Steady;State!Kinetic!Constants………………….101!

! 3.3!Results!and!Discussion……………………………………………………………………..102!

! ! 3.3.1!Selection!of!Standalone!Ligases…………………………………………...102!

! ! 3.3.2!Determination!of!Ligase!Substrate!Specificity!Profiles………….102!

! ! 3.3.3!Biological!Roles!of!P.!aeruginosa!Ligases……………………………...115!

! 3.4!Summary…………………………………………………………………………………………136!

! References…………………………………………………………………………………………….138!

Appendix…………………………………………………………………………………………………….153!

! A;1!Multiple!Sequence!Alignments…………………………………………………………153!!

! ! A;1;1!Alignment!of!flK!(F8JPF9)!ortholog!representatives!based!on!! ! CD;HIT!Clustering……………………………………………………………………….154!

! ! A;1;2!Alignment!of!flK!(F8JPF9)!with!Actinobacteria!representative! ! orthologs……………………………………………………………………………………170!

! ! A;1;3!Alignment!of!flK!(F8JPF9)!with!Bacteroidetes!representative! ! orthologs……………………………………………………………………………………173

A;1;4!Alignment!of!flK!(F8JPF9)!with!Proteobacteria!representative! ! orthologs……………………………………………………………………………………176!

! ! A;1;5!Alignment!of!flK!(F8JPF9)!with!Firmicutes!representative! ! orthologs……………………………………………………………………………………182!

A;1;6!Alignment!of!flK!(F8JPF9)!and!BVU_1957!(A6L1R5)!with! ! Deinococcus;Thermus!representative!orthologs…………………………..199

! xi! LIST!OF!ABBREVIATIONS!

°C! ! ! Degrees!Celsius!

4;CBA!! ! 4;chlorobenzoate!

4;CBA;CoA! ! 4;chlorobenzoyl;CoA!

4;FT! ! ! 4;fluorothreonine!

4;HBA!! ! 4;hydroxybenzoate!

4;HBA;CoA! ! 4;hydroxybenzoyl;CoA!

4;HBT!! ! 4;hydroxybeznoyl;CoA!thioesterase!

5;FDRP! ! 5;fluoro;5;deoxy;D;ribose;1;phosphate! ! !

5;FDRulP! ! 5;fluoro;5;deoxy;ribulose;1;phosphate!

5’;FDA!! ! 5’;fluoro;5’;deoxyadenosine!

AAc! ! ! Acetoacetate!

AAcCoA! ! Acetoacetyl;CoA!

Aacs! ! ! Acetoacetyl;CoA!synthetase!

Ac! ! ! Acetate!

AcCoA!! ! Acetyl;CoA!

ACP! ! ! Acyl!carrier!protein! acs! ! ! acetyl;CoA!synthetase!

Ala/A! ! ! Alanine!

AMP! ! ! Adeonsine!monophosphate!

Arg/R! ! ! Arginine! ! ! ! ! !

! xii! Asn/N!! ! Asparagine!

Asp/D!! ! Aspartate! ! ! !

ATP! ! ! Adeonsine!triphosphate!

Bis;Tris!Propane! 1,3;bis(tris(hydroxymethyl)methylamino)propane!

BLAST!! ! Basic!Local!Alignment!Search!Tool!

C! ! ! Carbon!

Ca! ! ! Calcium!

CF! ! ! Cystic!fibrosis!

CFTR! ! ! Cystic!fibrosis!transmembrane!conductance!regulator! cm! ! ! centimeter!

CO2! ! ! Carbon!dioxide!

CoA/CoASH! ! Coenzyme!A!

Cys/C! ! ! Cysteine!

DHNA!! ! 1,4;dihydroxy;2;naphthoate!

DHNA;CoA! ! 1,4;dihydroxy;2;naphthoyl;CoA!

DNA! ! ! Deoxyribonucleic!acid!

DPPC! ! ! Dipalmitidylphosphatidylcholine!

DTNB! ! ! 5,5’;dithio;bis;(2;nitrobenzoic!acid)!

EFI! ! ! Enzyme!Function!Initiative!

EMBL! ! ! European!Molecular!Biology!Laboratory!

F! ! ! Fluorine!

FAc! ! ! Fluoroacetate!

FAcCoA! ! Fluoroacetyl;CoA!

! xiii! FAD/FADH2! ! Flavin!adenine!dinucleotide!! ! !

FAh! ! ! Fluoroacetaldehyde!

FAS! ! ! Fatty!acid!synthase!

FPLC! ! ! Fast!Protein!Liquid!Chromatography! g! ! ! gram!

Gln/Q! ! ! Glutamine! !

Glu/E! ! ! Glutamate!

Gly/G! ! ! Glycine!

HAD! ! ! Haloalkanoate!dehalogenase!

HCoA! ! ! Formyl;CoA!

HD! ! ! Hot!Dog;Fold!

HEPES!! ! 2;[4;(2;hydroxyethyl)piperazin;1;yl]ethanesulfonic!acid!

HGT! ! ! Horizontal!(lateral)!gene!transfer!

His/H! ! ! Histidine!

HTn! ! ! 4;hydroxy;trans;aconitate!

HTS! ! ! High;throughput!screening!

Ile/I! ! ! Isoleucine!

IPTG! ! ! Isopropyl!β;D;1;thiogalactopyranoside

Iso! ! ! Isomerase! kcat! ! ! Turnover!number!

KCl! ! ! Potassium!chloride!

Km! ! ! Michaelis!constant!

L! ! ! Liter!

! xiv! LB! ! ! Luria;Bertani!

LCFA! ! ! Long;chain!fatty!acid!

Leu/L! ! ! Leucine!

Lys/K! ! ! Lysine!

MCL! ! ! Medium;chain!length!

MES! ! ! 2;(N;morpholino)ethanesulfonic!acid

Met/M!! ! Methionine! !

MFS! ! ! Major!Facilitator!Superfamily!

Mg! ! ! Magnesium! mL! ! ! milliliter! mm! ! ! millimeter! mM! ! ! millimolar! mRNA!! ! messenger!!

NaCl! ! ! Sodium!chloride!

NAD+/NADH! ! Nicotinamide!adenine!dinucleotide!

NCBI! ! ! National!Center!for!Biotechnology!Information!

NEB! ! ! New!England!Biolabs! nm! ! ! nanometer!

NMR! ! ! Nuclear!Magnetic!Resonance!

NRPS! ! ! Nonribosomal!polyketide!synthase!

PA! ! ! Phenylacetate!

PA;CoA! ! Phenylacetyl;CoA!

PAGE! ! ! Polyacrylamide!Gel!Electrophoresis!

! xv! PC! ! ! Phosphatidylcholine!

PCR! ! ! Polymerase!Chain!Reaction!

PFK;1! ! ! Phosphofructokinase!

PHA! ! ! Polyhydroxyalkanoate!

PHB! ! ! Polyhydroxybutyrate!

Phe/F! ! ! Phenylalanine!

PKS! ! ! Polyketide!synthase!

PLP! ! ! Pyridoxal!phosphate!

PPi! ! ! Pyrophosphate! ppm! ! ! Parts!per!Million!

PQS! ! ! Pseudomonas!quorum!signal!

Pro/P! ! ! Proline!

PSI! ! ! Pounds!per!Square!Inch!

RNA! ! ! Ribonucleic!acid!

RPM! ! ! Revolutions!per!Minute!

SAM! ! ! S;adenosylmethionine!

SCFA! ! ! Short;chain!fatty!acid!

SCL! ! ! Short;chain!length!

SDS! ! ! Sodium!dodecyl!sulfate!

Ser/S! ! ! Serine!

TB! ! ! Terrific!broth!

TCA!Cycle! ! Tricarboxylic!acid/Krebs!Cycle!

TE! ! ! Thioesterase!

! xvi! THF! ! ! Tetrahydrofolate!

Thr/T! ! ! Threonine!

Tran! ! ! Transaldolase!

Trp/W! ! Tryptophan!

Tyr/Y! ! ! Tyrosine!

V!! ! ! Initial!velocity!

Val/V! ! ! Valine!

Vmax! ! ! Maximum!velocity!

WT! ! ! Wild;type!

α! ! ! Alpha!

β! ! ! Beta!

Δ! ! ! Delta!

μg! ! ! microgram!

! xvii! LIST!OF!FIGURES!

Figure!1.1.!Overview!of!aerobic!respiration!showing!how!β;oxidation!can!feed!into! the!TCA!cycle!to!generate!NADH!and!FADH2………………………………………………………….2!

Figure!1.2.!Common!biological!thioesters!including!(top!to!bottom)!acyl;CoA,!acyl; holo;ACP,!acyl;cysteine!and!acyl;S;glutathione……………………………………………………...4!

Figure! 1.3.! Activation! (formation)! and! deactivation! (breakdown)! of! biological! thioesters………………………………………………………………………………………………………….…6!

Figure!1.4.!Comparison!of!the!tertiary!structures!of!the!α/β;hydrolase!fold!(A)!and! the! hot! dog;fold! (B).! (A)! Human! acyl;CoA! thioesterase! 4! (PDB:! 3K2I).! (B)! E.! coli!

FabA!(PDB:!1MKA).!Residues!are!colored!by!secondary!structure………………………….7!

Figure! 1.5.! Active! site! scaffolds! of! 4;HBT;I! from! Pseudomonas! sp.! CBS;3! (PDB:!

1LO8)! (left)! and! 4;HBT;II! from! Arthrobacter! sp.! Strain! SU! (PDB:! 3R3F)! (right).!

Active! sites! are! positioned! at! the! dimer! interface.! Monomer! units! are! colored! separately.!4;hydroxybenzoyl;CoA!substrate!analog!inhibitors!are!colored!in!orange! and!green…………………………………………………………………………………………………………….9!

Figure!1.6.!Mechanisms!of!catalysis!utilized!by!HD!thioesterases………………………10!

! xviii! Figure!1.7.!Pathway!for!4;chlorobenzoate!(4;CBA)!degradation.!The!enzymes!that! carry!out!each!step!are!listed!in!bold…………………………………………………………………12!

Figure! 1.8.! Abbreviated! pathway! for! PA! degradation! highlighting! the! functional! role!of!the!HD!thioesterase,!PaaI…………………………………………………………………………13!

Figure! 1.9.! Abbreviated! pathway! for! menaquinone! (Vitamin! K2)! synthesis! highlighting!the!role!of!a!hot!dog!thioesterase……………………………………………………..14!

Figure!1.10.!Reaction!scheme!of!two;step!adenylation!reaction.!(I)!Formation!of!the! acyl;AMP! intermediate.! (II)! Formation! of! the! acylated! product! with! CoASH! as! the! nucleophile………………………………………………………………………………………………………15!

Figure!1.11.!Crystal!structures!(from!left!to!right)!of!acetyl;CoA!synthetase!(acsA)! from! S.!enterica!(PDB:! 2P20),! 4;chlorobenzoate! ligase! (CBAL)! from! Alcaligenes!sp.!

AL3007! (PDB:! 3CW8)! and! long! chain! fatty! acyl;CoA! synthetase! (fadD)! from! T.! thermophilus! HB8! (PDB:! 1V26).! N;terminal! domains! are! colored! blue,! teal! and! magenta,! respectively.! C;terminal! domains! are! colored! gold! and! linker/hinge! residues!are!colored!red……………………………………………………………………………………..16!

Figure!1.12.!Depiction!of!C;terminal!domain!movement!in!acyl;CoA!ligases!using!4; chlorobenzoate!ligase!(CBAL)!crystal!structures!from!Alcaligenes!sp.!AL3007.!CBAL! with! 4;CBA;adenylate! bound! (PDB:! 3CW8)! (left).! CBAL! with! substrate! analog!

! xix! inhibitor!4;chlorophenacyl;CoA!bound!(PDB:!3CW9)!(right).!The!N;terminal!domain! is!colored!in!teal!and!the!mobile!C;terminal!domain!is!colored!in!yellow.!The!flexible! linker!or!“hinge”!is!colored!in!red………………………………………………………………………..17!

Figure! 1.13.! Relative!sizes!of!various!ligase!fatty!acid!binding!tunnels.!(A)!Short; chain! ligase! from! S.!enterica!(2P2F)! with! acetate! bound.! (B)! Medium;chain! ligase! from!M.!acetivorans!(3ETC)!with!acetate!modeled!in!the!active!site.!(C)!Long;chain! ligase!from!T.!thermophilus!(1V26)!with!palmitate!bound……………………………………20!

Figure! 2.1.! Three! of! the! top! ten! selling! drugs! in! 2011:! Lipitor! (Pfizer),! Crestor!

(AstraZeneca)!and!Fluticasone!(GlaxoSmithKline)……………………………………………….31!

Figure!2.2.!Common!biogenically;produced!organofluorine!compounds……………..32!

Figure!2.3.!Effect!of!fluoroacetate!(FAc)!poisoning!on!the!TCA!cycle.!Black!arrows! represent!normal!TCA!cycling!with!acetyl;CoA!(AcCoA)!while!red!arrows!represent!

TCA!cycling!with!FAc.!TCA!cycle!inhibition!is!represented!by!a!red!“X”.!Blue!arrows! represent!energy;generating!byproducts…………………………………………………………….33!

Figure! 2.4.! The! fluorinase! pathway! in! S.! cattleya.! (A)! Reaction! steps! for! the! biosynthesis! of! 4;fluorothreonine! and! fluoroacetate.! (B)! Organization! of! fl! gene! cluster!with!functional!predictions!based!on!homology.!The!genes!encoding!Iso,!Deh! and!Tran!are!not!found!in!close!proximity!to!the!fl!gene!cluster……………………………36!

! xx! Figure! 2.5.! Active! site! comparison! of! 4;HBT;II! thioesterase! (PDB:! 1Q4T),! MaoC! dehydratase! (PDB:! 1IQ6)! and! flK! thioesterase! (PDB:! 3KV8)! with! each! subfamily’s! corresponding!catalytic!mechanism!printed!below.!The!loop!between!β1!and!αHD! comprising! the! lid! structure! is! colored! pink,! yellow! or! green,! respectively.………………………………………………………………………………………………………40!

Figure! 2.6.! flK! topology! diagram! and! view! of! tertiary! structure.! Each! monomer! subunit!is!color!coded!from!N;terminus!(blue)!to!C;terminus!(red)……………………...42!

Figure! 2.7.! Representative! node! network! of! putative! bacterial! flK! orthologs.!

Representatives! are! color;coded! by! phylum:! Acidobacteria! (Dark! Green),!

Actinobacteria! (Orange),! Bacteroidetes! (Blue),! Chloroflexi! (Yellow),! Cyanobacteria!

(Lavender),! Deinococcus;Thermus! (Purple),! Firmicutes! (Green),! Fusobacteria!

(Pink),! Planctomycetes! (Salmon),! Proteobacteria! (Red),! Spirochaetes! (Teal),!

Verrucomicrobia! (Grey).! Cloned! orthologs! are! indicated! by! arrows:! (1)! flK,! (2)!

BVU_1957,!(3)!cgp_0542,!(4)!Galf_1995,!(5)!TTHA0967……………………………………….52!

Figure!2.8.!Representative!node!network!of!putative!bacterial!flK!orthologs!within!

Proteobacteria.!Representatives!are!colored!by!class:!Alpha!(red),!Beta!(blue),!Delta!

(green),!Epsilon!(yellow),!Gamma!(pink)…………………………………………………………….54!

Figure! 2.9.! Figure! 2.9.! Crystal! structure! overlays! of! flK! (PDB:! 3KV8)! (blue),!

MA0038! (pink),! BVU_1957! (green)! and! TTHA0967! (PDB:2CWZ)(orange).! Insert!

! xxi! shows! conservation! of! key! catalytic! residues.……………………………………………………………………………………………………………62!

Figure! 2.10.! Structural!comparison!of!flK!(teal)!(PDB:!3KV8),!MA0038!(magenta),!

BVU_1957! (green)! and! TTHA0967! (PDB:! 2CWZ)! (red).! The! helical! lid! motif! is! colored!in!yellow.!Active!site!volumes!are!colored!by!electrostatic!potential………...65!

Figure! 2.11.! Multiple! alignment! of! flK! and! cloned! flK;orthologs.! Completely! conserved! residues! are! highlighted! in! red! and! highly! conserved! residues! are! highlighted! in! yellow.! Secondary! structure! was! overlaid! using! the! flK! crystal! structure!(PDB:!3KV8_B)…………………………………………………………………………………….66!

Figure!2.12.!Gene!context!within!T.!potens!(Firmicutes)!showing!flK!ortholog!(TE)! co;localization! with! genes! involved! in! folate! biosynthesis! (red! box)! and! the! one! carbon!pool!by!folate!(blue!box)……………………………………………………………..…………..68!

Figure! 2.13.! Gene! context! within! A.! metalliredigens! (Firmicutes)! showing! flK! ortholog! (TE)! co;localization! with! a! gene! operon! for! de! novo! purine! biosynthesis! and!salvage………………………………………………………………………………………………………..70!

Figure! 2.14.! Reaction! scheme! depicting! the! potential! involvement! of! BVU;like! orthologs!in!the!one!carbon!pool!and!its!downfield!contributions!to!de!novo!purine! biosynthesis! and! salvage! (blue)! and! acetogenic! carbon! fixation! (Wood;Ljundahl!

! xxii! pathway)(red).! FTHFS:! formate;tetrahydrofolate! synthetase.! CODH/ACS:! carbon! monoxide/acetyl;CoA!synthase!complex……………………………………………………………72!

Figure! 3.1.! Comparison! of! wild;type! (I)! and! mutant! (II/III)! CFTR.! CF;causing! mutations!in!the!CFTR!gene!can!result!in!dysfunctional!protein!(II)!or!cause!CFTR! breakdown! before! implantation! into! the! plasma! membrane! (III).! Both! types! of! mutation! inhibit! ion! transport! across! the! membrane! and! lead! to! accumulation! of! thick,!dry!mucus…………………………………………………………………………………………………85!

Figure!3.2.!Quorum!signaling!regulatory!cascade!in!P.!aeruginosa!consisting!of!the! las! (I),! Rhl! (II)! and! pqs! (III)! systems.! Black! arrows! denote! autoinducer! synthesis!

(straight)! and! binding! (curved).! Transcriptional! activation! is! denoted! by! blue! arrows!(+)!and!repression!is!denoted!by!red!arrows!(;)………………………………………90!

Figure! 3.3.! Substrate!specificity!of!alkyl!fatty!acid;activating!ligases.!Bars!indicate! range! of! biologically! relevant! activity! of! each! ligase! in! terms! of! fatty! acid! carbon! length.! Colored! regions! are! defined! by! carbon! length! as! short! (green),! medium!

(blue)!and!long!(red)!chain!fatty!acids………………………………………………………………104!

!Figure! 3.4.!(A)!Gene!neighborhood!of!PA0887!(yellow)!showing!the!proximity!of! an! itaconate! degradation! operon! (blue)! as! well! as! arginine! uptake! (teal)! and! utilization! (red)! operons.! (B)! The! citric! acid! cycle! (black)! showing! the! glyoxylate!

! xxiii! bypass! (red).! The! key! enzyme! Isocitrate! lyase! (ICL)! is! shown! in! blue.! (C)! P.! aeruginosa!genes!involved!in!itaconate!degradation…………………………………………..117!

Figure! 3.5.! (A)!Gene!neighborhood!surrounding!PA1997!(red)!showing!proximity! to! genes! involved! leucine! (green),! tyrosine! (orange)! and! carnitine! (blue)! degradation.! Genes! involved! in! short;chain! fatty! acid! utilization! are! shown! in! purple.! (B)! Reaction! diagram! showing! late;stage! steps! of! leucine! and! tyrosine! degradation!as!well!as!carnitine!metabolism.!All!pathways!utilize!atoB!to!produce! the!critical!TCA!cycle!metabolite!acetyl;CoA!(yellow)………………………………………...121!

Figure! 3.6.! Potential! involvement! of! P.! aeruginosa! acyl;CoA! ligases! in! polyhydroxyalkanoate!(PHA)!cycling………………………………………………………………...125!

Figure! 3.7.! Potential! PA3860! involvement! in! phosphatidylcholine! (PC)! synthesis.!

Gene! context! shows! proximity! of! PA3860! to! pcs,! PA3859! (carboxylesterase),!

PA3853! (putative! acyltransferase),! and! downstream! genes! involved! in! PC! synthesis………………………………………………………………………………………………………….130!

Figure! 3.8.! PQS! biosynthetic! pathway! in! P.! aeruginosa.! (A)! Organization! of! the!

PA0996! gene! cluster! in! P.! aeruginosa! PAO1! to! show! co;localization! of! pqs!

(red/black)!and!phn!pathway!genes!(blue).!(B)!Reaction!steps!of!the!two!routes!for!

PQS!biosynthesis.!The!“tryptophan”!route!is!catalyzed!by!the!kynurenine!pathway!

! xxiv! (green)!while!the!“chorismate”!route!is!catalyzed!by!the!phn!pathway!(blue).!Both! pathways!converge!to!utilize!the!pqs!pathway!(red/black)………………………………..134!

! xxv! LIST!OF!TABLES!

Table! 2.1.! Taxonomic! information! of! flK! orthologs! selected! for! cloning! and! isolation…………………..………………..………………..………………..………………..…………………..58!

Table!2.2.!Steady!state!kinetic!constants!for!flK!and!ortholog;catalyzed!hydrolysis! of! various! acyl;CoA! substrates.! *kcat/Km! calculated! from! an! estimated! Km! of! 60! nM...... 60!

Table!3.1.!Ligase!domains!in!Pseudomonas!aeruginosa!PAO1.!PKS/NRPS!complexes! are! highlighted! in! red! and! previously! characterized! genes! are! highlighted! in! gray…………………………………………………………………………………………………………………103!

Table! 3.2.! High;throughput! screening! of! P.! aeruginosa! ligases! with! various! fatty! acid!and!amino!acid!substrates.!Activity!is!colored!on!a!gradient!scale!from!red!(high! activity)!to!white!(no!activity).!Untested!substrates!are!denoted!by!“n/a.”!…………108!

Table! 3.3.! Steady;state! kinetic! constants! for! P.! aeruginosa! ligase;catalyzed! activation!of!various!fatty,!aromatic!and!amino!acids.!Substrates!highlighted!in!grey! indicate!Km!values!too!high!for!biologically!relevant!activity.!Substrates!highlighted! in! red! represent! the! highest! overall! kinetic! efficiency.! ! (HB):! hydroxybutyrate…...... 110!

! xxvi! Chapter(1(

The(Biological(Thioester:(An(Overview(of(its(Function(and(Regulation(in(

Cellular(Metabolism(

(

1.1 Overview(of(Cellular(Metabolism(

1.1.1(Cellular(Respiration(

! Cellular! respiration! is! the! collection! of! metabolic! processes! that! convert! organic! carbon! sources! (nutrients)! into! ATP! (energy).! As! the! main! energy! generation! pathway! for! all! domains! of! life,! cellular! respiration! is! a! collection! of! redox! reactions! in! which! biological! fuels! (carbon! precursors)! are! oxidized! in! the! presence! of! an! inorganic! electron! acceptor.! Depending! on! the! type! of! electron! acceptor,! cellular! respiration! can! be! broken! down! into! three! different! categories:! aerobic! respiration,! fermentation! and! anaerobic! respiration.! Aerobic! respiration! utilizes!molecular!oxygen!(O2)!as!the!terminal!electron!acceptor!and!represents!the! pathway! utilized! by! all! eukaryotic! species! as! well! as! all! obligate! and! facultative! aerobes![1].!Aerobic!respiration!occurs!in!four!steps!(Figure!1J1).!In!short,!glucose!is! converted! to! two! molecules! of! pyruvate! in! a! process! known! as! glycolysis.! In! this! process,!two!molecules!of!NAD+!are!reduced!to!NADH!and!two!molecules!of!ATP!are! formed.! Pyruvate! is! then! oxidized! to! acetylJCoA! and! CO2! by! the! pyruvate! dehydrogenase! complex! (PDC)! in! a! step! known! as! oxidative! decarboxylation.!

Another!molecule!of!NAD+!is!reduced!to!NADH!in!the!process.!From!here,!acetylJCoA! enters!the!tricarboxylic!acid!(TCA)!cycle!where!it!is!oxidized!to!CO2!and!H20!in!an!

! 1! Glycolysis

ATP ADP ATP ADP

Glucose Glucose-6-P Fructose-6-P FBP Glyceraldehyde-3-P (x2)

NAD+

ADP ATP NADH

PEP 2-phosphoglycerate 3-phosphoglycerate 1,3-diphosphoglycerate

Oxidative1 Β(oxidation Decarboxylation Acyl-CoA (Δ2C) Pyruvate Acetyl-CoA

NAD+ NADH

NADH Oxaloacetate Citrate

+ Β-ketoacyl-CoA Lactate NAD Malate Isocitrate NAD+ NADH TCA1Cycle + NADH NAD Fumarate α-ketoglutarate Β-hydroxyacyl-CoA NAD+

FADH 2 Succinate Succinyl-CoA NADH FAD

GTP GDP trans-enoyl-CoA

Oxidative1 FADH 2 NADH & FADH 2 Fatty Acid Phosphorylation ADP FAD ATP Fatty acyl-CoA O NADH & FADH 2 2 AMP

NAD+ & FAD H2O

ATP (

Figure(1A1.(Overview!of!aerobic!respiration!showing!how!βJoxidation!can!feed!into! the!TCA!cycle!to!generate!NADH!and!FADH2.(

! 2! 8Jstep! process.! The! resulting! molecule! of! oxaloacetate! can! combine! with! another! molecule!of!acetylJCoA!to!restart!the!cycle.!During!the!TCA!cycle,!one!turn!produces!

3!NADH,!1!FADH2!and!1!GTP!(which!can!be!converted!to!ATP).!In!the!last!part!of!the! aerobic!respiration!pathway,!all!of!the!NADH!and!FADH2!molecules!produced!from! the! first! three! stages! are! oxidized! back! into! their! substituent! NAD+!and! FAD.! This! process,!known!as!oxidative!phosphorylation,!or!the!electron!transport!chain!(ETS),! utilizes!O2!as!the!electron!acceptor!and!creates!a!chemiosmotic!proton!gradient!that! is! used! to! drive! ATP! synthesis.! Overall,! one! molecule! of! glucose! is! converted! to! roughly!32!molecules!of!ATP!during!aerobic!respiration![2].!

Both! fermentation! and! anaerobic! respiration! are! utilized! when! oxygen! is! limiting!or!completely!absent.!In!the!case!of!fermentation,!pyruvate!(formed!from! glycolysis)!is!converted!into!small!molecule!waste!products,!such!as!lactate,!ethanol!

+ and!CO2.!During!this!process,!NADH!is!oxidized!back!to!NAD !so!it!is!available!to!be! reJused! in! glycolysis.! During! anaerobic! respiration,! neither! oxygen! nor! pyruvate! derivatives!are!available!as!the!final!electron!acceptor.!Instead,!nitrate!and!sulfate! are! the! most! common! acceptors.! While! both! processes! occur! in! the! absence! of! oxygen,! they! differ! in! their! mode! of! ATP! synthesis.! While! anaerobic! respiration! utilizes! an! electron! transport! chain! to! generate! a! proton! gradient! (as! in! aerobic! respiration),! fermentation! utilizes! substrateJlevel! phosphorylation! to! drive! ATP! synthesis.!Both!processes!are!common!in!gutJdwelling!bacteria!that!survive!under! oxygenJlimiting!conditions![3].(

! While! glucose! is! the! preferred! carbon! source! during! aerobic! respiration,! carbohydrates,!lipids!and!proteins!may!also!be!consumed!as!reactants!when!it!is!not(

! 3! NH2

N N O O O O H3C CH3 O P O P O S R N N N N O O O H H OH O

HO O

O P O

O O O H3C CH3 ACP Ser O O P O S R N N O H H OH O

O R

S S R O O O Cys H N O HO N OH H

NH2 O (

Figure(1A2.(Common!biological!thioesters!including!(top!to!bottom)!acylJCoA,!acylJ holoJACP,!acylJcysteine!and!acylJSJglutathione.!

! readily! available.! When! protein! levels! are! high,! amino! acids! released! from! the! breakdown!of!proteins!are!metabolized!by!a!variety!of!specialized!pathways!and!are! converted! into! useful! components! that! can! enter! respiratory! pathways! at( various! spots.! In! the! case! of! lipids,! stored! fat! molecules! (triacylglycerides)! can! be! broken! down!into!their!constituent!glycerol!and!longJchain!fatty!acid!(LCFA)!components!in! the! process! of! lipolysis! where! both! can! enter! the! respiration! pathway.! While! glycerol! can! be! converted! to! glucose,! the! fatty! acid! components! must! be! broken! down! into! acetylJCoA! precursors! in! the! process! of! βJoxidation! (Figure! 1J1).! Once! converted,!the!acetylJCoA!byproducts!enter!respiration!via!the!TCA!cycle.!During!the!

! 4! bacterial! fermentation! process,! the! breakdown! of! carbohydrates! produces! shortJ chain! fatty! acids! (SCFA)! like! acetate,! propionate! and! butyrate! along! with! CO2! and! molecular! hydrogen! (H2)! [3,! 4].! While! the! SCFA! ratio! is! dependent! on! diet,! their! absorption! has! been! shown! to! play! various! roles! in! colonic! health.! For! example,! acetate! can! be! converted! to! acetylJCoA! and! act! as! a! precursor! for! fatty! acid! biosynthesis!or!enter!the!TCA!cycle![4].!Butyrate!acts!as!the!main!energy!source!for! colonocytes! and! has! been! implicated! in! the! prevention! and! treatment! of! various! colonic! diseases! [3,! 4].! Overall,! fatty! acids! of! all! types! play! a! large! role! in! cellular! energy!production!across!all!domains!of!life.!However,!in!order!to!be!activated!for! usage!in!such!pathways,!fatty!acids!must!first!be!converted!to!thioesters.!

(

1.1.2(Biological(Significance(of(Thioesters(

Biological!thioesters!serve!the!cell!as!the!activated!form!of!free!organic!acids! and!are!made!up!of!two!main!components:!a!carboxylic!acid!and!a!free!thiol.!While! the! carboxylic! acid! (organic! acid)! can! vary! greatly! in! shape,! size! and! polarity,! the! free!thiol!component!is!typically!the!free!pantotheine!arm!of!coenzyme!A!(though! acyl! carrier! protein! (ACP),! a! free! cysteine! residue! or! glutathione! may! be! used! as! well)! (Figure! 1J2).! As! an! activated! form! of! fatty! acids,! thioesters! play! important! roles!in!cellular!metabolism!that!include!lipid!synthesis!and!degradation,!amino!acid! catabolism,! and! central! carbon! metabolism! [5].! Additionally,! thioesters! have! been! implicated! in! polyketide! biosynthesis! and! in! the! degradation! of! aromatic! compounds! and! acylated! proteins! [6J8].! Beyond! roles! in! regulating! cellular! metabolism,!thioesters!have!also!been!found!to!function!in!solubility!and!transport,!

! 5! O O Acyl%CoA(synthetase + CoA SH CoA R OH Thioesterase R S (

Figure( 1A3.( Activation! (formation)! and! deactivation! (breakdown)! of! biological! thioesters.!

! cell!cycling,!signal!transduction!pathways!and!even!in!gene!regulation![9].!Given!the! functional! importance! of! these! roles,! the! regulation! of! thioester! formation! and! breakdown! are! critical! functions! carried! out! in! the! cell.! AcylJCoA! synthetases!

(ligases)!are!responsible!for!the!formation!(activation)!of!thioester!linkages!while! thioesterases! are! responsible! for! their! degradation! (Figure! 1J3).! The! reactions! carried! about! by! both! acylJCoA! ligases! and! thioesterases! will! be! covered! in! more! detail!in!the!following!sections.!

1.2(AcylACoA(Thioesterases(

! Thioesterases!catalyze!the!hydrolysis!of!acylJCoA!into!its!free!fatty!acid!and! coenzyme! A! (CoA)! constituents.! Given! this! role,! thioesterases! are! considered! regulators!of!free!CoA,!fatty!acid!levels!within!the!cell.!Two!enzyme!superfamilies,! the! α/βJhydrolaseJfold! and! hotdogJfold,! have! evolved! this! function! over! time! [10,!

11].! Interestingly! enough,! while! the! overall! tertiary! structures! of! the! two! superfamilies!are!completely!different,!they!carry!out!the!same!function,!utilizing!

! 6! (

Figure(1A4.(Comparison!of!the!tertiary!structures!of!the!α/βJhydrolase!fold!(A)!and! the! hot! dogJfold! (B).! (A)! Human! acylJCoA! thioesterase! 4! (PDB:! 3K2I).! (B)! E.& coli&

FabA!(PDB:!1MKA).!Residues!are!colored!by!secondary!structure.!

! similar!modes!of!catalysis!(Figure!1J4).!While!both!α/βJhydrolase!thioesterases!and! hot! dogJfold! thioesterases! have! been! reported! to! play! critical! roles! in! cellular! processes,! this! report! will! focus! solely! on! the! hot! dogJfold! superfamily! of! thioesterases.!

(

1.2.1(The(Hot(DogAFold(Superfamily(and(Hot(Dog(Thioesterases(

The! first! member! of! the! hot! dogJfold! superfamily! was! reported! in! 1996! as!

FabA,! a! dehydrataseJisomerase! involved! in! the! E.& coli& type! II! fatty! acid! synthase!

! 7! (FAS)! [12].! The! superfamily! was! given! its! name! based! on! the! FabA! tertiary! structure,! which! was! described! as! a! long,! αJhelix! surrounded! by! a! highly! curved,! antiJparallel!βJsheet!resembling!a!hot!dog!in!a!bun!(Figure!1J4B)![12].!All!members! of! the! hot! dog! superfamily! share! this! conserved! core! topology,! with! the! highly! curved!βJsheet!wrapping!around!the!rightJhanded,!αJhelix!(αHD)!in!the!order!of!β1J

αHDJβ3Jβ4Jβ5Jβ2.! Additionally,! some! hot! dogJfold! family! members! contain! extra! sequence! motifs! inserted! between! β1! and! the! αHD,! as! seen! in! the! hydratase/dehydratase!subfamily![13].!The!minimal!functional!unit!of!the!hot!dogJ fold! family! is! the! dimer,! as! the! active! site! is! formed! at! the! dimer! interface! and! utilizes! residues! from! both! monomer! units! (Figure! 1J5).! The! hot! dogJfold! is! ubiquitous!in!nature,!found!in!all!domains!of!life.!To!date,!over!1300!members!of!the! hot! dogJfold! superfamily! have! been! identified,! with! over! 60! crystal! structures! deposited!in!the!protein!database!(PDB)![11,!14].!These!enzymes!are!part!of!over!15! distinct!subfamilies,!which!include!enzymes!functioning!as!hydratase/dehydratases,! isomerases! and! acetyltransferases! [15].! However,! the! majority! of! hot! dogJfold! superfamily!members!are!thioesterases.!

The!first!hot!dogJfold!thioesterase!crystal!structure!was!reported!in!1998!by! the!DunawayJMariano!and!Holden!groups![16].!Isolated!from!Pseudomonas&sp.&CBSJ

3! and! characterized! as! a! 4JhydroxybenozylJCoA! thioesterase! (4JHBT),! it! was! discovered! to! function! in! a! pathway! for! the! degradation! of! 4Jchlorobenzoate! (4J

CBA)!(Figure!1J7).!In!2003,!another!TE!crystal!structure!was!reported!by!the!same! groups! from! Arthrobacter& sp.& SU! [17].! While! it! was! found! to! be! another! 4JHBT! functioning!in!an!orthologous!4JCBA!degradation!pathway,!the!crystal!structure!

! 8! (

Figure( 1A5.( Active! site! scaffolds! of! 4JHBTJI! from! Pseudomonas& sp.& CBSJ3! (PDB:!

1LO8)! (left)! and! 4JHBTJII! from! Arthrobacter& sp.& Strain! SU! (PDB:! 3R3F)! (right).!

Active! sites! are! positioned! at! the! dimer! interface.! Monomer! units! are! colored! separately.!4JhydroxybenzoylJCoA!substrate!analog!inhibitors!are!colored!in!orange! and!green.!

! revealed! a! completely! different! active! site! architecture! from! the! Pseudomonas& 4J

HBT.! Soon! after,! the! 4JHBT! from! Pseudomonas&came!to!be!known!as!4JHBTJI! and! from! Arthrobacter& as! 4JHBTJII.! Since! then,! other! HD! TE! subfamilies! have! been! identified,! though! the! majority! of! HD! thioesterases! have! been! discovered! to! resemble!either!4JHBTJI!or!4JHBTJII,!depending!on!their!active!site!scaffolds!(Figure!

1J5).((

(

1.2.2(Mechanism(of(Catalysis(of(HD(Thioesterases(

! In! general,! the! majority! of! HD! thioesterases! (regardless! of! active! site! scaffold)!utilize!one!of!two!distinct!mechanisms!to!catalyze!the!hydrolysis!of!acylJ!

! 9! Nucleophilic0Catalysis

acyl%enzyme O O -CoA O O R SCoA R O E/D R OH O O O O E/D O E/D H

General0Base0Catalysis

O O R SCoA R OH

O O H H O E/D O

O E/D (

Figure(1A6.(Mechanisms!of!catalysis!utilized!by!HD!thioesterases.!

(

CoA! substrates! (Figure! 1J6).! Both! mechanisms,! nucleophilic! catalysis! and! general! base!catalysis,!rely!on!an!active!site!glutamate!or!aspartate.!In!nucleophilic!catalysis,! the! active! site! Glu/Asp! attacks! the! thioester! carbonyl,! forming! a! tetrahedral! intermediate.! Collapse! of! the! intermediate! to! reform! the! carbonyl! leads! the! expulsion!of!the!CoA!leaving!group!and!the!formation!of!an!acylJenzyme!anhydride! intermediate.!In!the!final!step,!an!activated!water!molecule!positioned!in!the!active! site! attacks! either! carbonyl! group! of! the! acylJenzyme! intermediate,! leading! to! formation! of! the! free! carboxylate! product.! In! general! base! catalysis,! instead! of!

! 10! attacking! the! substrate! carbonyl! directly,! the! active! site! Glu/Asp! deprotonates! a! water! molecule,! activating! it! for! attack.! Formation! and! subsequent! collapse! of! a! tetrahedral!intermediate!results!in!the!concerted!formation!of!both!free!carboxylate! and!CoA!products.!

(

1.2.3(Divergence(of(Function(Within(HD(Thioesterases(

! A! central! trait! to! HD! thioesterases! and! all! members! of! the! HD! family! in! general! is! degeneracy! of! sequence.! Due! to! the! robustness! of! the! HD! fold,! a! large! degree! of! sequence! degeneracy! is! allowed! while! maintaining! the! same! overall! tertiary!structure.!As!sequence!plasticity!is!a!necessary!trait!for!the!divergence!of! function,!the!HD!superfamily!(and!thioesterases!specifically)!has!evolved!to!perform! a!myriad!of!functional!roles!within!critical!metabolic!pathways.!

As! widely! distributed! substrates! in! nature,! many! bacterial! organisms! have! evolved! metabolic! pathways! for! the! utilization! of! aromatic! compounds! as! carbon! sources![18,!19].!Two!such!aromatic!utilization!pathways!involve!the!degradation!of!

4Jchlorobenzoate!(4JCBA)!and!phenylacetate!(PA).!

! The!dehalogenation!of!4JCBA!proceeds!through!two!acylJCoA!intermediates! before! its! final! conversion! to! 4Jhydroxybenzoate! (4JHBA)! (Figure! 1J7).! The! first! step! involves! the! activation! of! 4JCBA! to! 4JchlorobenzoylJCoA! (4JCBAJCoA)! by! 4J chlorobenzoate!ligase!(CBAL).!The!dehalogenation!step!follows,!as!a!dehalogenase! substitutes!the!chloro!group!for!a!hydroxyl!group,!forming!4JhydroxybenzoylJCoA!

(4JHBAJCoA).! The! final! step! is! carried! out! by! the! 4JhydroxybenzoylJCoA! thioesterase!(4JHBT)!to!form!4JHBA.!As!mentioned!earlier,!the!4JHBT!thioesterase!

! 11! O O O SCoA O SCoA O O ATP H2O H2O

Cl Cl OH OH AMP, PPi Cl CoA CBAL Dehalogenase 4-HBT (

Figure(1A7.(Pathway!for!4Jchlorobenzoate!(4JCBA)!degradation.!The!enzymes!that! carry!out!each!step!are!listed!in!bold.!

! from!the!Pseudomonas&sp.&CBSJ3!4Jchlrobenzoate!degradation!pathway!was!the!first!

HD!thioesterase!ever!characterized.!

! Another! widely! distributed! aromatic! compound! that! can! be! utilized! by! bacteria!as!a!viable!carbon!source!is!phenylacetic!acid!!(PA).!The!genes!that!mediate! the!utilization!of!PA!are!clustered!together!in!what!is!known!as!the!PAA!degradation! operon!(Figure!1J8).!The!operon!consists!of!14!separate!genes,!made!up!of!various! aromatic! oxygenases! (PaaABCDE),! genes! analogous! to! βJoxidation! enzymes!

(PaaFGHIJ)!and!a!phenylacetylJCoA!ligase!(PaaK).!PaaI!has!been!shown!to!hydrolyze!

PAJCoA!and!its!various!hydroxylated!derivatives.!A!hot!dogJfold!thioesterase!of!the!

4JHBT! clade,! PaaI! is! thought! to! function! in! rescuing! CoA! after! spontaneous! dehydration! leads! to! the! formation! of! the! deadJend! products! 2J! and! 3J hydroxyphenylacetylJCoA![19].&

! Menaquinone,!also!known!as!vitamin!K2,!is!an!essential!cofactor!in!both!!

! 12! O O O

SCoA SCoA SCoA OH OH OH OH O

OH dehydration PaaI

O O O

SCoA SCoA SCoA

OH OH OH !

Figure( 1A8.( Abbreviated! pathway! for! PA! degradation! highlighting! the! functional! role!of!the!HD!thioesterase,!PaaI.!

& eukaryotic!and!prokaryotic!organisms,!shown!to!play!crucial!roles!in!blood!clotting,! calcium! binding! and! cell! cycle! regulation! [20].! Mammals,! unable! to! synthesize! menaquinone,! rely! on! its! acquisition! from! bacterial! species! within! the! gut! microbiome!and!also!from!the!ingestion!of!leafy!vegetables.!Menaquinone!synthesis! in!bacterial!systems!has!been!extensively!studied!due!its!potential!as!a!drug!target.!

The! synthetic! pathway! is! a! 9Jstep! route! that! converts! isochorismate! to! menaquinone,!catalyzed!by!a!gene!cluster!known!as!the!Men!operon!(Figure!1J9).!

Through! the! actions! of! MenF,! D,! H,! C,! E! and! B,! choristmate! is! converted! to! 1,4J dihydroxyJ2JnaphthoylJCoA!(DNHAJCoA).!The!subsequent!step!is!the!hydrolysis!of! the!thioester!bond,!resulting!in!1,4JdihydroxyJ2Jnaphthoate!(DHNA).!In!2009,!after! much!confusion!about!which!thioesterase!was!responsible!for!was!this!reaction!

! 13! OH OH O OH O

O MenFDHCEB SCoA HD4TE O

O O OH OH O O Chorismate DHNA6CoA DHNA

O MenA H MenG n4=41613

O Menaquinone (

Figure( 1A9.( Abbreviated! pathway! for! menaquinone! (Vitamin! K2)! synthesis! highlighting!the!role!of!a!hot!dog!thioesterase.!

! step,! it! was! discovered! that! a! HD! thioesterase! of! the! 4JHBTJI! clade! catalyzed! the! hydrolysis! of! DHNAJCoA! [21].! More! recently,! the! E.&coli&HD! thioesterase! YdiI! was! also!shown!to!function!in!the!Men!pathway,!catalyzing!this!particular!reaction!step!

[22].!

1.3(The(AcylAAdenylateAForming(Superfamily(

! Adenylation! is! the! process! by! which! semiJreactive! carboxylate! compounds! are!activated!by!condensation!with!amines,!thiols!or!alcohols!to!give!highly!reactive! amide,! thioester! or! ester! constituents.! The! enzymes! that! carry! out! adenylation! reactions!within!biological!systems!are!ubiquitous!in!nature,!functioning!across!all! domains!of!life,!in!metabolic!pathways!(such!as!fatty!acid!degradation!and!aromatic!!

! 14! I NH2 NH2 N N N N

O O O O N N O O N N + O P O O P O P O P O O O R O R O O O O Carboxylate OH OH OH OH

ATP O O Acyl& O P O P O O O

PPi II

NH2 N N O O O N N O P O O + CoASH R SCoA R O Acyl&CoA OH OH NH2 Acyl& N N

O N N

O P O O O OH OH AMP !

Figure(1A10.(Reaction!scheme!of!twoJstep!adenylation!reaction.!(I)!Formation!of!the! acylJAMP! intermediate.! (II)! Formation! of! the! acylated! product! with! CoASH! as! the! nucleophile.!

! compound! degradation),! secondary! metabolite! in! pathways! (PKS/NRPS! systems),! survival! and! virulence! mechanisms! and! DNA! translation! [23J27].! Some! members! are! even! involved! in! the! production! of! light! [30].! Members! of! the! acylJadenylateJ! forming!superfamily!are!divided!into!three!classes:!(I)!adenlation!domains!within!!

! 15! !

Figure(1A11.(Crystal!structures!(from!left!to!right)!of!acetylJCoA!synthetase!(acsA)! from! S.&enterica&(PDB:! 2P20),! 4Jchlorobenzoate! ligase! (CBAL)! from! Alcaligenes&sp.&

AL3007! (PDB:! 3CW8)! and! long! chain! fatty! acylJCoA! synthetase! (fadD)! from! T.& thermophilus& HB8! (PDB:! 1V26).! NJterminal! domains! are! colored! blue,! teal! and! magenta,! respectively.! CJterminal! domains! are! colored! gold! and! linker/hinge! residues!are!colored!red.!

PKS/NRPS! modules,! (II)! acyl/arylJCoA! synthetases! and! (III)! luciferase! oxidoreductases![28].!!While!the!separate!adenylation!classes!are!found!to!function! primarily! in! separate! biochemical! pathways,! the! reaction! they! carry! out! is! essentially! the! same! (Figure! 1J10).! An! ATPJdependent! reaction,! adenylation! is! a! twoJstep! process! in! which! the! driving! force! is! the! production! of! pyrophosphate!

2+ (PPi).! Additionally,! these! reactions! have! been! found! to! be! Mg Jdependent,! as! the! divalent!cation!neutralizes!the!charge!on!ATP!and!PPi!as!well!as!stabilizes!the!!

! 16! 140ο

Figure(1A12.(Depiction!of!CJterminal!domain!movement!in!acylJCoA!ligases!using!4J chlorobenzoate!ligase!(CBAL)!crystal!structures!from!Alcaligenes&sp.&AL3007.!CBAL! with! 4JCBAJadenylate! bound! (PDB:! 3CW8)! (left).! CBAL! with! substrate! analog! inhibitor!4JchlorophenacylJCoA!bound!(PDB:!3CW9)!(right).!The!NJterminal!domain! is!colored!in!teal!and!the!mobile!CJterminal!domain!is!colored!in!yellow.!The!flexible! linker!or!“hinge”!is!colored!in!red.!

! transition! state! [29].! In! the! first! reaction,! the! enzyme! catalyzes! the! nucleophilic! attack! of! the! substrate! carboxylate! on! the! αJphosphate! group! of! ATP,! leading! to! formation! and! release! of! PPi.! The! second! reaction! step! involves! the! binding! of! anucleophile!(an!amine,!thiol,!or!alcohol),!which!attacks!the!carbonyl!carbon!of!the!

! 17! acylJAMP!intermediate,!releasing!AMP!and!forming!the!acylated!product.!In!addition! to!being!characterized!by!functional!class,!members!of!the!acylJadenylateJforming! superfamily! are! further! classified! by! their! adopted! tertiary! structure.! The! class! I! tertiary!structure!(as!seen!in!the!acyl/arylJCoA!synthetase!class)!is!composed!of!a! large!NJterminal!domain!and!small!CJterminal!domain!connected!by!a!flexible!linker!

(Figure! 1J11).! The! NJterminal! domain! has! been! shown! to! contain! 3! subdomains! with!an!α/β!topology.!The!CJterminal!domain,!also!with!an!!α/β!topology,!sits!on!top! of! the! NJterminal! domain! like! a! lid! [30].! During! the! course! of! the! reaction,! these! enzymes! under! go! a! drastic! domain! movement,! as! the! CJterminal! domain! rotates! nearly!140°!(Figure!1J11)![24,!31J33].!This!soJcalled!“domain!alteration”!occurs!in! the!middle!of!the!twoJstep!reaction,!after!the!acylJadenylate!intermediate!has!been! formed.!The!conformational!change!creates!a!new!active!site!scaffold!that!binds!the!

CoA!nucleophile.!Upon!binding,!the!second!reaction!step!can!then!commence.!In!the! acyl/arylJCoA!synthetase!class,!the!nucleophile!in!the!second!reaction!step!is!thiol,! coenzyme!A.!These!enzymes!play!critical!roles!in!various!metabolic!pathways!and! will!be!the!focus!for!the!remainder!of!this!section.!

(

1.3.1(Functional(Roles(of(AcylACoA(Synthetases(

! The!majority!of!acylJCoA!ligases!through!all!domains! of! life! can! be! defined! within!four!main!functional!roles!based!on!the!carboxylate!substrates!they!activate.!

These!roles!are!acetylJCoA!ligase,!mediumJchain!and!longJchain!fatty!acylJCoA!ligase! and!aromatic!acylJCoA!ligase.!

! 18! ! AcetylJCoA! synthetase! (acs)! is! responsible! for! the! conversion! of! acetate! to! acetylJCoA!and!plays!a!large!role!in!acetate!metabolism!and!utilization.!As!acetylJ

CoA!is!the!key!component!of!the!citric!acid!cycle,!acs!genes!are!found!throughout!all! domains! of! life.! Beyond! that! scope,! however,! acs! has! been! found! to! function! in! a! other!pathways.!For!example,!in!eukaryotic!organisms,!acs!has!been!shown!to!play!a! role! in! gene! regulation,! providing! acetylJCoA! for! histone! acetylation! by! histone! acetyltransferase![34].!

! MediumJchain! fatty! acylJCoA! ligases! (MCFACS)! are! responsible! for! the! activation!of!fatty!acids!between!the!carbon!lengths!of!C6JC12.!While!there!are!few! reports! in! the! literature! characterizing! their! function! directly,! the! utilization! of! mediumJchain! fatty! acids! (MCFAs)! in! βJoxidation! has! been! described! before,! indicating!their!functionality!in!this!regard![35].!Some!mediumJchain!ligases!have! been!shown!to!be!promiscuous!towards!aromatic!acylJCoA!substrates,!indicating!a! potential!crossover!function!for!these!ligases![36].!

! The! main! function! role! of! longJchain! fatty! acylJCoA! ligases! (LCFACS)! is! to! activate!longJchain!fatty!acids!(LCFAs)!for!degradation!via!βJoxidation!(Figure!1J1).!

As!a!critical!source!of!acetylJCoA!for!cellular!respiration,!longJchain!fatty!acylJCoA! ligases! (generally! active! with! C16! and/or! longer! chain! lengths)! are! ubiquitous! in! nature,!found!throughout!all!domains!of!life.!Additionally,!in!prokaryotic!organisms,!

LCFACS!has!been!also!been!found!to!function!in!the!transport!of!exogenous!LCFAs! across! the! cell! membrane! for! entry! into! βJoxidation! [37].! In! eukaryotes,! LCFACS! activity!has!been!shown!to!regulate!a!number!of!cellular!processes,!including!!

! 19! (A) (B) (C) (

Figure( 1A13.( Relative!sizes!of!various!ligase!fatty!acid!binding!tunnels.!(A)!ShortJ chain! ligase! from! S.&enterica&(2P2F)! with! acetate! bound.! (B)! MediumJchain! ligase! from!M.&acetivorans!(3ETC)!with!acetate!modeled!in!the!active!site.!(C)!LongJchain! ligase!from!T.&thermophilus&(1V26)!with!palmitate!bound.!

! protein! transport,! enzyme! activation,! cell! signaling! and! transcriptional! regulation!

[37].!

! As!discussed!previously,!the!wide!availability!of!aromatic!compounds!in!the! environment! has! driven! the! evolution! of! aromatic! utilization! pathways.! Two! such! examples!discussed!in!section!1.2.4!were!the!utilization!of!4Jchlorobenzoate!and!! phenylacetate.!In!both!pathways,!activation!of!both!4JHBA!and!PA!via!aromatic!acylJ

CoA!ligases!was!required!for!utilization!as!a!carbon!source.!Another!critical!function! of! aromatic! ligases! is! in! virulence! and! survival,! as! seen! in! the! anthranilateJCoA! ligase! (PA0996)! in! P.& aeruginosa.! An! infamous! pathogen! known! for! its! enhanced! virulence! factor! production,! P.& aeruginosa! is! found! to! utilize! PA0996! in! the!

! 20! biosynthesis!of!PQS,!one!of!three!main!quorum!signals!produced!for!virulence!factor! regulation![38,!39].!This!ligase!is!discussed!in!further!detail!in!Chapter!3.!

(

1.3.2(StructureAFunction(Relationship(of(AcylACoA(Synthetases(

! Given!the!variety!of!functional!roles!that!acylJCoA!ligases!carry!out,!it!is!no! doubt!that!ligase!active!sites!have!undergone!a!large!amount!of!structural!evolution! to!accommodate!such!diverse!range!of!carboxylate!substrates.!As!seen!in!hot!dogJ fold! thioesterases,! a! large! degree! of! sequence! degeneracy! defines! the! acylJCoA! ligases,!with!little!sequence!homology!outside!of!key!conserved!ATP!and!carboxylate! binding!motifs![ref].!Once!again,!sequence!plasticity!has!allowed!for!the!evolution!of! distinct! active! site! shapes! and! volumes,! as! seen! from! the! comparison! of! short,! medium! and! longJchain! ligase! active! sites.! The! crystal! structure! of! acetylJCoA! synthetase!(acs)!(Figure!1J13A)!reveals!a!short,!narrow!tunnel,!restricting!both!the! length! (parent! chain)! and! width! (substituent! groups)! of! the! fatty! acid! substrate.!

While!the!fatty!acid!binding!tunnels!in!medium!chainJligases!are!also!restricted!in! length,!they!have!an!increased!width!compared!to!shortJchain!ligases,!allowing!for! mediumJchain!fatty!acids!to!wind!around!(Figure!1J13B).!This!active!site!shape!also! resembles! that! of! an! aromatic! ligase,! wide! enough! to! accommodate! a! (potentially! substituted)! aromatic! ring! system.! LongJchain! ligases,! on! the! other! hand,! tend! to! have! long,! narrow! binding! tunnels! that! not! only! allow! for! the! accommodation! of! long!chain!fatty!acids!but!help!provide!substrate!specificity!as!well!(Figure!1J13C).!

! 21! 1.4(Summary!

! Both! the! hot! dogJfold! and! acylJadenylateJforming! superfamiles! are! ancient! collections! of! enzmes! evolved! to! carry! out! a! variety! of! biochemical! functions.! As! seen! in! both! superfamilies,! the! strict! conservation! of! a! robust! tertiary! structure! combined!with!the!enhanced!plasticity!of!sequence!degeneration!has!allowed!for!a! wide!divergence!of!function!while!still!keeping!maintaing!a!common!catalytic!theme.!

! The! work! discussed! within! this! disseration! will! focus! on! the! divergence! of! function!within!both!the!hot!dogJfold!and!acylJadenylateJforming!superfamilies!in! the! context! of! metabolic! pathways.! The! goal! of! this! work! is! to! study! members! of! both!superfamilies!to!discover!how!the!divergence!of!function!has!led!to!a!selective! metabolic!advantage.!Chapter!two!will!explore!the!flK!thioesterase!from!S.&cattleya! and!an!attempt!will!be!made!to!map!its!functional!divergence!as!it!has!evolved!to! perform!such!a!remarkable!role.!In!chapter!three,!I!investigate!the!standalone!acylJ

CoA!ligases!in!Pseudomonas&aeruginosa&in!an!attempt!to!discover!how!such!a!large! number! of! ligase! may! contribute! to! pathogenesis! and! enhanced! virulence! in! the! lungs!of!cystic!fibrosis!patients.!

! The! goal! of! this! work! is! to! further! understand! the! critical! role! that! evolutionary!divergence!of!function!plays!in!metabolic!pathways!and!advance!our! knowledge!of!novel!chemistries!being!carried!out!in!biological!systems.!

(

! 22! References(

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Biol.,&373(4):965E967.&

[34]!Takahashi,!H.,!McCaffery,!J.!M.,!Irizarry,!R.!A.,!Boeke,!J.!D.!(2006)!Nucleocytosolic!

acetylJcoenzyme! A! synthetase! is! required! for! histone! acetylation! and! global!

transcription.!Mol&Cell.,&23(2):207E217.&

! 28! [35]!Papamandjaris,!A.!A.,!MacDougall,!D.!E.,!Jones,!P.!J!H.!(1998)!Medium!chain!fatty!

acid! metabolism! and! energy! expenditure:! obesity! treatment! implications.! Life&

Sci.,&62(14):1203E1215.&

[36]!Kasuya,!F.,!Igarashi,!K.,!Fukui,!M.!(1996)!Inhibition!of!a!medium!chain!acylJCoA!

synthetase!involved!in!glycine!conjugation!by!carboxylic!acids.!

[37]&Weimar,!J.,!DiRusso,!C.!C.,!Delio,!R.,!Black,!P.!N.!(2002)!Functional!role!of!fatty!

acylJcoenzyme!A!synthetase!in!the!transmembrane!movement!and!activation!of!

exogenous!longJchain!fatty!acids.!J&Biol&Chem.,&277:29369E29376.&

[38]!Brendenbruch,!F.,!Nimtz,!M.,!Wray,!V.,!Morr,!M.,!Müller,!R.,!Häussler,!S.!(2005)!

Biosynthetic!pathway!of!Pseudomonas&aeruginosa&4JhydroxyJ2Jalkylquinolones.!

J&Bacteriol,&187(11):3630E3635.&

[39]!Farrow!III,!J.!M.,!Pesci,!E.!C.!(2007)!Two!distinct!pathways!supply!anthranilate! as!a!precursor!to!the!pseudomonas&quinolone!signal.!J.&Bacteriol.,&189(9):3425E3433.&

(

! 29! Chapter(2(

Novel(Thioesterase(Activity(from(the(flK(Thioesterase(Subfamily(Reveals(a(Basis(

For(Divergent(Biological(Function(

(

2.1(Introduction(

Fluorine! is! the! most! electronegative! element! in! nature! and! when! bonded! to! carbon,!forms!the!strongest!bond!in!organic!chemistry![1].!While!technically!covalent,! the! large! degree! of! polarization! of! the! CBF! bond! lends! it! a! significant! amount! of! electrostatic! character! and! provides! the! bond! (and! parent! molecule)! with! an! opportunity!at!a!number!of!interB!and!intraBmolecular!interactions!otherwise!reserved! for! ionic! linkages! [1].! Additionally,! the! atomic! radius! of! fluorine! is! similar! to! that! of! hydrogen,! allowing! the! presence! of! a! CBF! bond! to! provide! significant! electronic! alterations! to! a! given! molecular! structure! without! costly! steric! effects! [1].! Given! the! ability!of!fluorine!to!substantially!change!the!way!a!molecule!behaves,!its!introduction! to!the!pharmaceutical!industry!has!had!a!profound!effect!on!rational!drug!design!and! development.! Dramatically! increasing! the! efficacy! and! potency! of! many! disease! therapies,! fluorinated! pharmaceuticals! have! been! used! to! treat! a! variety! of! diseases! ranging!from!fungal!infections!and!anxiety!to!arthritis!and!cancer![2].!In!fact,!roughly!

30%!of!all!drugs!currently!on!the!market!contain!at!least!one!fluorine!atom,!including! some!of!the!top!sellers!(Figure!2B1)!in!recent!history![3,!4].!Thanks!to!the!development! of!newer!and!milder!fluorination!techniques,!more!and!more!synthetic!chemists!are!!

! 30! F OH OH O O O S N OH HO OH NH H

F H F O Atorvastatin*(Lipitor) F O Fluticasone*(Seretide) S N O N N

O OH F

HO OH

Rosuvastatin*(Crestor) !

Figure( 2D1.! Three! of! the! top! ten! selling! drugs! in! 2011:! Lipitor! (Pfizer),! Crestor!

(AstraZeneca)!and!Fluticasone!(GlaxoSmithKline).!

! trying! their! hand! at! fluorine! chemistry,! continually! adding! to! the! already! increasing! number!of!synthetic!organofluorine!compounds!in!existence.!!

In! contrast,! nature! is! a! much! more! modest! producer! of! organofluorines.! In! general,!naturally!occurring!organohalogen!compounds!are!relatively!common!BB!over!

3,700!are!known!to!be!in!existence![5].!However,!despite!the!fact!that!fluorine!is!the!

13th!most!abundant!element!in!the!earth’s!crust,!only!30!organofluorines!(less!that!1%! of!the!total!number!of!organohalogens)!are!known!to!occur!naturally![5].!This!shocking! revelation! is! explained! by! the! fact! that! the! majority! of! naturally! occurring! fluorine! is! contained! as! fluorite! (CaF2)! or! other! minerals! that! cannot! be! easily! converted! to! molecular!fluorine!(F2)![5].!Organofluorine!compounds!are!produced!from!both!abiotic!

! 31! OH O O NH2 F F OH H N N NH Fluoroacetaldehyde O 2 H2N S Fluorothreonine O N N O O O F F F OH OH OH HO OH

Fluoroacetate Nucleocidin HO O O O HO O Fluorocitrate

F OH

Fluoro.fatty1acids !

Figure(2D2.(Common!biogenicallyBproduced!organofluorine!compounds.!

!and! biogenic! sources,! the! latter! of! which! (i.e.! living! organisms)! producing! mainly! carboxylic!acid!derivatives!(Figure!202)![5].!Of!particular!interest!is!fluoroacetate!(FAc).!!

! The!most!common!naturally!produced!organofluorine!compound,!FAc!has!been! found!to!occur!in!a!variety!of!tropical!and!subtropical!plant!species!throughout!Africa,!

Australia! and! Brazil! [5].! Originally! discovered! in! the! plant! species! Dichapetalum, cymosum,! FAc! acts! as! a! selfBdefense! mechanism! to! deter! animals! like! rodents! and! livestock!from!feeding!on!the!plant!leaves![6,!7].!A!small!molecule!analog!of!acetate,!FAc! is!a!metabolic!poison,!highly!toxic!to!all!obligate!aerobic!organisms,!especially!mammals! and!insects![8].!A!potent!inhibitor!of!the!TCA!cycle,!FAc!is!first!activated!as!fluoroacetylB

CoA! (FAcCoA)! where! it! enters! the! cycle! in! place! of! acetylBCoA! (Figure! 2B3).! The! structurally! similar! FAcCoA! is! converted! to! 4Bfluorocitrate! by! citrate! synthase! and! subsequently!converted!to!4BhydroxyBtransBaconitate!(HTn)!by!aconitase![9].!HTn!!

! 32! FAc FAcCoA AcCoA O CoASH

SCoA AcCoA NADH Oxaloacetate Citrate/4+fluorocitrate O Aconitase F SCoA Isocitrate/ Malate FAcCoA 4+hydroxy+trans+aconitate

X NADHA+ACO2

Fumarate Ketoglutarate

Succinate Succinyl+CoA NADHA+ACO2 FADH2

GTPA+ACoASH !

Figure( 2D3.( Effect! of! fluoroacetate! (FAc)! poisoning! on! the! TCA! cycle.! Black! arrows! represent!normal!TCA!cycling!with!acetylBCoA!(AcCoA)!while!red!arrows!represent!TCA! cycling!with!FAc.!TCA!cycle!inhibition!is!represented!by!a!red!“X”.!Blue!arrows!denote! energyBgenerating!byproducts.!!

! irreversibly!binds!the!aconitase!active!site,!thereby!inhibiting!any!further!generation!of! isocitrate! and! bringing! the! cycle! to! a! complete! halt! [9].! The! consequence! is! a! lethal! decrease!in!energy!production!mainly!due!to!the!lack!of!NADH!and!FADH2,!which!drive!

ATP!synthesis!during!cellular!respiration.!Additionally,!accumulation!of!citrate!and!4B fluorocitrate!leads!to!the!inhibition!of!phosphofructokinaseB1!(PFKB1)![10].!Regarded!as!

! 33! the! most! important! step! in! glycolysis,! PFKB1! is! responsible! for! the! ATPBdependent! conversion! of! fructoseB6Bphosphate! to! fructoseB1,6Bbisphosphate.! As! a! result,! PFKB1! inhibition!leads!to!the!inability!to!utilize!glucose!as!an!energy!source.!Without!the!most! effective! means! of! ATP! production,! the! organism! cannot! sustain! and! eventually! succumbs!to!death.!In!fact,!the!lethal!effects!of!FAc!on!mammals!are!so!prominent!that! in!the!1940’s,!it!was!first!marketed!and!commercially!supplied!(under!the!brand!name!

“1080”)! as! a! rodenticide! and! predacide,! used! to! remove! unwanted! populations! of! rodents,!wild!dogs,!foxes,!wolves!and!feral!pigs![11].!!

Streptomyces, cattleya,! a! soilBdwelling! actinomycete,! has! long! been! a! known! producer! of! natural! products! and! has! been! found! to! naturally! synthesize! important! antibiotics! such! as! thienamycin,! penicillin! and! cephamycin! [12,! 13].! More! notably,! however,! has! been! the! discovery! of! a! biosynthetic! pathway! within! the! bacteria’s! genome!inferring!the!ability!to!produce!FAc!as!well!as!4Bfluorothreonine!(4BFT)![14].!

Extensive!research!has!been!conducted!to!discover!the!genes!directly!involved!in!the! pathway!and!a!mechanism!for!FAc!and!4BFT!synthesis!has!been!partially!elucidated.!The! first! step! is! regarded! as! the! hallmark! of! the! pathway! and! is! responsible! for! the! formation! of! the! CBF! bond! (Figure! 2B4A).! Catalyzed! by! 5’BfluoroB5’Bdeoxyadenosine! synthase! (flA),! the! SAMBdependent! reaction! proceeds! via! nucleophilic! attack! of! a! fluoride! ion! on! the! SAM! ribose! ring! at! the! C5! position! followed! by! expulsion! of! LB methionine! to! give! 5’BfluoroB5’Bdeoxyadenosine! (5’BFDA)! [15].! The! next! step! is! catalyzed! by! 5’BfluoroB5’Bdeoxyadenosine! phosphorylase! (flB)! and! involves! the! nucleophilic! attack! of! phosphate! on! the! 5’BFDA! ribose! ring! at! the! C1! position! and! subsequent! expulsion! of! the! adenosine! base! to! give! 5BfluoroB5BdeoxyBDBriboseB1B

! 34! phosphate! (5BFDRP)! [16].! Through! the! action! of! an! isomerase! (Iso),! 5BFDRP! is! then! converted!to!5BfluoroB5BdeoxyBribuloseB1Bphosphate!(5BFDRulP)![17].!An!aldolase!then! catalyzes! the! conversion! of! 5BFDRulP! to! fluoroacetaldehyde! (FAh).! At! this! point,! the! pathway!splits,!with!FAh!acting!as!the!last!common!precursor!for!both!FAc!and!4BFT!

[18].!At!one!branch!point,!an!NADH+Bdependent!dehydrogenase!(Deh)!converts!FAh!to!

FAc! while! at! the! other,! FAh! is! converted! to! 4BFT! by! a! PLPBdependent! transaldolase!

(Tran)![19,!20].!With!the!exception!of!the!aldolase,!all!of!the!genes!in!the!pathway!have! been! identified! and! their! function’s! verified.! While! four! putative! aldolases! have! been! identified!in!S.,cattleya,!the!one!responsible!for!the!conversion!of!5BFDRulP!to!FAh!still! remains!to!be!determined![21].!!

In!S.,cattleya,,the!enzymes!responsible!for!the!first!two!steps!of!the!pathway,!flA! and!flB,!are!found!in!the!middle!of!a!gene!cluster!consisting!of!12!open!reading!frames!

(Figure! 2B4B).! While! the! rest! of! the! cluster! does! not! appear! to! encode! any! of! the! remaining! enzymes! directly! involved! in! FAc! and! 4BFT! biosynthesis,! a! number! of! putative! auxiliary! roles! have! been! identified! within.! flI! has! been! shown! to! be! an! SB adenosylhomocysteine! hydrolase! and! is! thought! to! relieve! SBadenosylhomocysteine! inhibition!of!flA![16].!flH!is!designated!as!a!Na+/H+!antiporter!and!was!thought!to!be! responsible! for! the! uptake! of! cellular! fluoride! ions! into! the! cell! [21].! However,! flH! knockout!strains!showed!similar!levels!of!FAc!and!4BFT!production!compared!to!wild! type,! indicating! either! a! different! function! for! flH! or! the! presence! of! a! redundant! fluoride!transporter!elsewhere![21].!The!proteins!encoded!by!flE,!flF,!flG!and!flL!are!all! annotated!as!DNABbinding!regulatory!proteins!and!most!likely!play!some!sort!of!role!in! regulating!the!fluorinase!pathway.!However,!their!exact!function!as!not!been!confirmed.!!

! 35! A" NH2 NH2 N O N N flA N O O F flB P S F O N N O N N O O O O NH3 HO OH HO OH HO OH 5)FDRP SAM 5')FDA

O Iso

O F Deh O Aldolase O O F FAc P HO H O O F Tran HO OH OH O FAh 5)FDRulP O

F NH3

4)FT B" flL# flK# flJ# flI# flH# flG# flF# flA# flB# flC# flD# flE#

Gene Size'(aa) Annotated'Function flA 299 5'(fluoro(5'(deoxyadenosine4synthase flB 299 5'(fluoro(5'(deoxyadenosine4phosphorylase flC 397 MFS4permease flD 216 HAD4dehalogenase/phosphatase flE 222 DNA(binding4regulatory4protein flF 185 DNA(binding4regulatory4protein flG 234 DNA(binding4regulatory4protein flH 467 Na+/H+4antiporter flI 489 Adenosylhomocysteine4hydrolase flJ 131 Hypothetical4protein flK 139 Fluoroacetyl(CoA4Thioesterase flL 225 DNA(binding4regulatory4protein Iso 353 5(fluoro(5(deoxyribulose(1(phosphate4isomerase Deh 507 Aldehyde4dehydrogenase Tran 634 4(fluorothreonine4transaldolase !

Figure(2D4.!The!fluorinase!pathway!in!S.,cattleya.!(A)!Reaction!steps!for!the!synthesis!of!

4Bfluorothreonine! (4BFT)! and! fluoroacetate! (FAc).! (B)! Organization! of! fl! gene! cluster! with!homologyBbased!functional!annotations.!The!genes!encoding!Iso,!Deh!and!Tran!are! not!found!in!close!proximity!to!the!fl!gene!cluster.!

! 36! flC,! with! an! unconfirmed! function! as! well,! appears! to! be! a! member! of! the! major! facilitator!superfamily!(MFS)!and!is!proposed!to!be!involved!in!the!transport!of!small! metabolites! (possibly! FAc! and! 4BFT)! [16].! Interestingly! enough,! attempts! to! discover! the! fluorinase! biosynthetic! cluster! in! other! organisms! have! revealed! its! extremely! limited!biological!range.!To!date,!orthologous!clusters!have!only!been!discovered!in!four! other! Actinomycete! species:! Streptomyces, sp., MA37,! Actinoplanes, sp., N902B109,!

Nocardia,brasiliensis,HUJEGB1!and!Streptomyces,xinghaiensis,[36B38].!!

Given! the! lethal! toxicity! of! FAc,! the! need! for! a! resistance! mechanism! in! FAcB producing! organisms! is! paramount.! In! S., cattleya,! the! protein! encoded! by! flD! is! annotated! as! a! putative! HAD! dehalogenase/phosphatase.! Fluoroacetate! dehalogenase! has! been! described! before! as! a! mechanism! of! detoxification! in! FAcBproducing! plant! species!as!well!as!in!the!nonBfluorometaboliteBproducing!bacteria!Burkholderia,sp.,FA1!

[22,! 23].! The! deBfluorination! reaction! not! only! detoxifies! FAc! by! removing! the! fluoro! group,! but! creates! glycolate! in! the! process,! a! metabolically! useful! byproduct! [23].! In! comparison!to!known!FAc!dehalogenase!proteins,!however,!flD!does!not!share!a!large! amount!of!sequence!identity.!Additionally,!flD!does!not!appear!able!to!rescue!S.,cattleya! from!the!lethal!effects!of!FAc!poisoning,!indicating!its!lack!of!involvement!in!resistance!

[24].!Instead,!the!first!(and!only)!line!of!defense!in!S.,cattleya,resistance!appears!to!be!a! thioesterase.!

( Located! within! the! S., cattleya, fluorinase! biosynthetic! cluster! is! flK,! a! gene! encoding! a! hot! dogBfold! thioesterase.! The! flK! thioesterase! is! of! particular! interest! because! of! the! highly! specialized! role! it! plays! in! cellular! resistance! to! FAc.! While! HD! thioesterases! are! known! to! function! in! a! variety! of! metabolic! pathways! as! well! as! in!

! 37! other!cellular!processes!(discussed!in!chapter!1),!this!is!the!first!known!TE!to!function! in!a!resistance!capacity.!Multiple!reports,!including!experiments!within!the!body!of!this! work,! indicate! that! flK! is! highly! active! towards! FAcCoA! hydrolysis! [24,! 25].! The! hydrolysis!of!FAcCoA!into!its!constituent!FAc!and!CoASH!prevent!its!entry!into!the!TCA! cycle,!thereby!blocking!the!action!of!FAc!poisoning!and!conferring!cellular!resistance.!

What! makes! the! flKBcatalyzed! hydrolysis! of! FAcCoA! especially! impressive! is! the! requirement! to! distinguish! FAcCoA! from! AcCoA,! its! structurally! similar! (and! metabolically! priceless)! relative! (Figure! 2B3).! To! this! end,! flK! exhibits! remarkable! substrate! specificity,! preferring! FAcCoA! over! AcCoA! with! a! >105Bfold! difference! in! overall!kinetic!efficiency![24].!!

! The! high! degree! of! substrate! specificity! has! attracted! a! lot! of! interest! in! uncovering!flK’s!underlying!catalytic!mechanism!and!studies!have!indicated!that!both! catalysis!and!molecular!recognition!play!a!role!in!FAcCoA!discrimination.!Interestingly,! it! has! been! discovered! that! flK! utilizes! two! distinct! catalytic! mechanisms! for! FAcCoA! and! AcCoA! hydrolysis,! the! former! of! which! is! dependent! on! the! recognition! and! positioning!of!the!fluoro!group!(Figure!2B5)![26,!27].!SiteBdirected!mutagenesis!studies! within! the! flK! active! site! have! identified! a! catalytic! triad! (Glu50BHis76BThr42)! to! be! critical!for!turnover!in!both!mechanisms![24B27].!In!either!case,!hydrolysis!is!initiated! by! nucleophilic! attack! of! Glu50! on! the! substrate! carbonyl,! forming! an! acylBenzyme! intermediate.!At!this!point,!the!mechanisms!diverge,!depending!on!the!polarity!of!the! substrate! Cα! substituent.! In! the! case! of! acetylBCoA! (nonpolar! –! weakly! polar! Cα! substituents),!it!is!thought!that!His76!is!responsible!for!deprotonation!and!activation!of! a!water!molecule!to!attack!the!acylBenzyme!intermediate.!The!role!of!Thr42!appears!to!

! 38! be!in!coordinating!both!Glu50!and!His76,!as!it!is!positioned!within!hydrogenBbonding! distance!of!both.!Additionally,!Thr42!appears!to!be!within!hydrogenBbonding!distance! of!the!substrate!thiol!and!may!further!assist!in!expulsion!of!the!CoA!leaving!group.!For! hydrolysis! of! FAcCoA! (and! substrates! with! highly! polar! Cα! substituents),! His76! is! responsible!for!deprotonation!of!the!fluoroBcontaining!Cα,!resulting!in!the!formation!of!a! ketene!intermediate.!Attack!of!a!water!on!the!ketene!carbon!forms!an!enolate,!which!! subsequently!collapses!to!reform!the!carbonyl!and!deprotonate!the!histidinium!group.!

Once!again,!this!mechanistic!pathway!is!thought!to!utilize!Thr42!to!coordinate!and!align!

Glu50!and!His76.!However,!given!that!a!T42A!mutant!abolishes!enzyme!activity!for!both!

FAcCoA!and!AcCoA!hydrolysis,!it!could!be!acting!in!a!more!expanded,!yet!unknown!role!

[25].!!

While! a! keteneBforming! Cα! deprotonation! mechanism! has! not! been! described! before!in!a!hot!dogBfold!TE,!analysis!of!the!flK!crystal!structure!provides!further!support! and!a!basis!as!to!how!it!could!utilize!two!distinct!catalytic!mechanisms.!Interestingly! enough,! flK! appears! to! be! somewhat! of! a! chimera,! adopting! structural! traits! characteristic! of! both! the! 4BHBTBII! thioesterase! and! MaoC! dehydratase! subfamilies!

(Figure!2B5).!flK!adopts!the!prototypical!HD!fold:!a!highly!curved,!5Bstranded!βBsheet! wrapped!around!a!central!αBhelix!(Figure!2B6).!Glu50!is!found!positioned!on!the!αHD!of! the!opposing!monomer!subunit!(αHD’),!roughly!corresponding!to!the!same!position!of! the! catalytic! Glu/Asp! in! the! 4BHBTBII! clade! of! thioesterases! [28].! In! both! the! TE! and! dehydratase! subfamilies,! hydrogenBbonding! interactions! with! the! backbone! amide! of! the!terminal!αHD!residue!lead!to!polarization!of!the!substrate!carbonyl,!activating!it!for! attack![29,!30].!Additionally!the!oxyanion!hole!created!in!this!space!is!able!to!stabilize!a!!

! 39! 4"HBT"II' MaoC'

F25'

Q58' F32' H36'

G65' G54'

E73'

OH O OH OH O O acyl%enzyme -H O R SCoA 2 R SCoA R SCoA O O O -CoA O O O H R O R O R SCoA R O E/D E/D O N O N E/D H H

,lK'

F33' F36' E50' T42' H76'

R120'

HN O H H N H76 acyl)enzyme

O -CoASH O O R = H O O O R R H SCoA O H O E50 O E50 O O O E50 R = F H H

O O F O O F C O F F O E50 OH O H H

ketene HN N N N H H76 H H76 !

Figure( 2D5.( Active! site! comparison! of! 4BHBTBII! thioesterase! (PDB:! 1Q4T),! MaoC! dehydratase! (PDB:! 1IQ6)! and! flK! thioesterase! (PDB:! 3KV8)! with! each! corresponding!

! 40! catalytic!mechanism!below.!The!loop!between!β1!and!αHD!comprising!the!lid!structure! are!colored!pink,!yellow!or!green,!respectively.!

! negatively!charged!enolate!or!tetrahedral!intermediate.!flK!is!no!different,!utilizing!the! same!effect,!though!the!positioned!threonine!(T42)!is!different!from!the!conserved! glycine!in!the!4BHBTBII!and!MaoC!subfamilies.!This!is!not!abnormal!though,!given!the! same!effect!is!seen!in!the!4BHBTBI!clade,!whose!members!generally!have!a!conserved!

Tyr!positioned!at!the!αHD!terminus![29B31].!Perhaps!the!most!interesting!feature!of!the! flK!tertiary!structure!is!an!extended!loop!region!between!β1!and!the!αHD,!termed!the!

“helical! lid.”! While! not! a! conserved! structural! motif! in! either! 4BHBT! clade! or! HD! thioesterases! in! general,! an! extended! loop! region! is! characteristic! of! the! MaoC! dehydratase!subfamily![32].!This!region!generally!houses!a!catalytic!histidine!that!acts! in! a! Cα! deprotonation! pathway! to! initiate! loss! of! water! across! the! CαBCβ! bond! of! 3B hydroxyacylBCoA!substrates![29,!32].!Furthermore,!this!extended!loop!region!is!known! to!house!conserved!aromatic!residues!that!interact!with!the!catalytic!histidine!through!

πBstacking!interactions![29].!While!the!flK!catalytic!nucleophile!H76!is!not!positioned!in! the!lid!structure,!two!phenylalanine!residues!(F33!and!F36)!are!present,!potential!relics! of!a!MaoC!fold.!Additionally,!flK!contains!active!site!residues!not!conserved!throughout! either!4BHBT!or!MaoC!subfamily.!Arg120!is!positioned!in!the!bottom!of!the!active!site! within!hydrogenBbonding!distance!of!Glu50.!Given!the!polar!nature!of!the!CBF!bond,!it! has!been!postulated!that!Arg120!is!responsible!for!the!orientation!of!the!fluoro!group,!!

! 41! C’ α4' α2 N’

α3 (HD)

α1 β5' β4' β2' β3' β1'

β1 β3 β2 β4 β5 α3’ (HD) α1'

N α2' α4 C !

Figure(2D6.(flK!topology!diagram!and!view!of!tertiary!structure.!Each!monomer!subunit! is!color!coded!from!NBterminus!(blue)!to!CBterminus!(red).!

! potentially!aiding!in!substrate!specificity![25].!However,!attempts!to!probe!the!catalytic! role! of! Arg120! through! siteBdirected! mutagenesis! have! resulted! in! unstable! protein.!

While! its! catalytic! role! remains! inconclusive,! this! result! indicates! the! importance! of!

Arg120!in!a!structural!capacity![25].!!

Overall,! it! would! appear! that! flK! is! a! highly! evolved! member! of! the! HD! superfamily,! potentially! adopting! the! catalytic! scaffolds! of! at! least! two! separate! subfamilies!in!order!to!carry!out!a!highly!specialized!function.!Additionally,!given!the! limited!biological!range!of!the!fluorinase!cluster,!it!would!appear!as!if!this!functional! role! is! not! ubiquitous! in! nature,! but! born! of! necessity! through! functional! divergence.!

This!chapter!will!explore!the!evolutionary!path!of!the!flK!thioesterase!through!the!lens! of!the!structureBfunction!relationship,!and!using!a!combined!mechanistic,!structural!and! bioinformatics!approach,!will!attempt!to!track!the!divergence!of!function!within!the!flK! subfamily.!An!inBdepth!bioinformatics!analysis!is!reported!in!the!determination!of!the!

! 42! biological!range!of!the!flK!thioesterase!and!in!sequence!and!gene!context!analyses!of! putative! orthologous! genes.! Additionally,! we! report! an! expanded! substrate! screening! for! flK! as! well! as! the! isolation! and! screening! of! multiple! uncharacterized! orthologs.!

Lastly,!we!report!and!utilize!the!crystal!structures!of!two!previously!uncharacterized!flK! orthologs.! Combining! all! of! the! results! together,! we! propose! potential! divergent! functions!being!carried!out!by!the!flK!scaffold.!

(

2.2(Methods(and(Materials(

2.2.1(Materials((

All! restriction! enzymes,! T4! DNA! ligase! and! Deep! Vent! DNA! polymerase! were! purchased! from! NEB.! Pfu, Turbo! DNA! polymerase! was! purchased! from! Agilent! and! custom! oligonucleotide! primers! were! synthesized! by! Invitrogen.! Genomic! DNA! was! purchased! from! ATCC.! DNA! sequencing! was! performed! in! part! by! DNA! Sequencing!

Services! at! the! University! of! New! Mexico! as! well! as! by! GeneWiz.! All! protein! samples! were!purified!on!an!ÄKTA!FPLC!system!(GE!Healthcare)!by!monitoring!UV!absorbance! at! 280! nm.! Protein! concentrations! were! determined! using! the! Bradford! method.!

Various!acylBCoA!compounds!were!synthesized!or!purchased!from!Sigma.!Synthesized! acylBCoA! compounds! were! purified! on! a! Shimadzu! Prominence! UFLC! with! a! Restek!

Ultra! Aqueous! C18! reverse! phase! column! (250! x! 10! mm).! All! other! chemicals! were! purchased!from!Sigma!or!Fisher!unless!otherwise!specified.!Mass!spectrometry!analysis! was!performed!by!the!Mass!Spectrometry!Facility!at!the!University!of!New!Mexico.!NMR! analysis!was!carried!out!at!the!NMR!Facility!at!the!University!of!New!Mexico.!

! 43! 2.2.2(Synthesis(of(AcylDCoA(Substrates(

! FluoroacetylBCoA!and!formylBCoA!were!synthesized!as!previously!described![24,!

33].!Final!products!were!verified!by!1H!and!13C!NMR.(

(

2.2.3(Cloning,(Expression(and(Purification(of(flK(and(orthologs(

The! gene! encoding! flK! was! amplified! by! PCR! using! Streptomyces, cattleya, genomic!DNA!(ATCC!35852D)!as!the!template,!custom!oligonucleotides!as!primers!and!

Deep! Vent! DNA! polymerase.! The! gene! was! digested! using! NdeI! and! XhoI! restriction! endonucleases! and! ligated! into! pETB28a(+)! expression! vector! (NdeI/XhoI! digested)! using!T4!DNA!ligase.!Vector!containing!the!ligated!gene!was!used!to!transform!E.,coli,

BL21(DE3)! competent! cells! (Invitrogen).! The! transformed! cells! were! grown! in! kanamycinBcontaining!LB!medium!(50!µg/mL)!at!37!°C!until!reaching!an!OD600!of!~!0.8.!

The!cells!were!then!induced!with!0.4!mM!isopropylBβBgalactopyranoside!(IPTG)!for!18! h!at!25!°C!and!harvested!by!centrifugation!at!6500!RPM!for!10!m.!Collected!cells!were! resuspended!in!50!mM!HEPES,!200!mM!NaCl,!50!mM!imidazole,!pH!8.0!(lysis!buffer)! until! completely! homogenized,! and! disrupted! by! passage! through! a! French! press! at!

1200! PSI.! After! centrifugation! at! 20,000! RPM,! the! resulting! supernatant! was! loaded! onto! a! 5! mL! HisTrap! FF! column! (GE! Heathcare)! and! washed! with! lysis! buffer.! Pure! protein! was! eluted! off! the! column! with! 50! mM! HEPES,! 200! mM! NaCl,! 500! mM! imidazole,! pH! 8.0! (elution! buffer).! Fractions! containing! pure! protein! were! collected,! pooled! and! dialysed! against! three! changes! of! buffer! (1! L! each)! containing! 50! mM!

HEPES,!200!mM!NaCl!at!pH!8.0.!Purity!was!verified!by!SDSBPAGE.!Yield:!8.1!mg/g!wet!

! 44! cell!paste.!Various!orthologs!of!flK!were!cloned!and!expressed!as!described!above,!each! with!some!modification!to!the!procedure.!

The! gene! encoding! MA0038! was! amplified! from! Methanosarcina, acetivorans,

(ATCC! 35395DB5)! genomic! DNA! and! cloned! into! pETB23a(+)! expression! vector.!

Transformed!cells!containing!MA0038!were!grown!in!LB!medium!containing!ampicillin!

(100! µg/mL).! Purification! of! MA0038! was! carried! out! as! described! above.! Yield:! 15! mg/g!wet!cell!paste.!

The!gene!encoding!BVU_1957!was!amplified!from!Bacteroides,vulgatus,genomic!

DNA!(ATCC!8482DB5)!and!cloned!into!pETB23a(+).!Transformed!cells!were!grown!in!LB! medium! containing! ampicillin! (100! µg/mL).! Harvested! cells! were! resuspended! and! lysed! in! 50! mM! MES,! pH! 6.5! (lysis! buffer).! After! centrifugation! at! 20,000! RPM,! the! supernatant!was!loaded!onto!a!10!mL!DEAE!anion!exchange!column!(GE!Healthcare)! and! washed! with! lysis! buffer.! The! protein! was! eluted! using! a! gradient! of! 0B100%! elution! buffer! (50! mM! MES,! 1! M! KCl,! pH! 6.5)! over! 90! m.! SemiBpure! fractions! were! collected,! pooled! and! concentrated! to! a! 4! mL! aliquot,! which! was! then! loaded! onto! a!

HiPrep! 16/60! Sephacryl! SB200! HR! gel! filtration! column! (GE! Healthcare)! and! washed! with! 50! mM! MES,! 100! mM! NaCl,! pH! 6.5! (dialysis! buffer)! until! protein! was! eluted.!

Fractions! containing! pure! protein! were! collected! and! pooled.! Purity! was! verified! by!

SDSBPAGE.!Yield:!14.1!mg/g!wet!cell!paste.!!

The!gene!encoding!TTHA0967!was!amplified!from!Thermus,thermophilus,(ATCC!

BAAB163D)! genomic! DNA! and! cloned! into! pETB28a(+).! Transformed! cells! containing!

TTHA0967!were!grown!in!LB!medium!containing!ampicillin!(100!µg/mL).!Purification! of!TTHA0967!was!carried!out!as!described!above.!Yield:!4.14!mg/g!wet!cell!paste.!

! 45! The! gene! encoding! Galf_1995! was! synthesized! by! GenScript.! NdeI! and! XhoI! restriction!endonucleases!were!used!to!cut!the!gene!out!of!the!supplied!pUC57!vector! and!T4!DNA!ligase!was!used!to!insert!it!into!pETB28a(+)!expression!vector!(NdeI/XhoI! digested).! Expression! and! purification! of! protein! was! performed! as! described! above! using!similar!buffers!at!pH!7.8.!Yield:!19!mg/g!wet!cell!paste.!

(

2.2.4(Determination(of(SteadyDState(Kinetic(Parameters(

Thioesterase!activity!was!measured!using!a!Shimadzu!UV1800!UV!Spectrometer! and!the!5,5’BdithioBbisB2Bnitrobenzoic!acid!(DTNB)!assay.!!Reactions!were!monitored!at!

412!nm!(Δε!=!13.6!mMB1cmB1)!and!carried!out!at!25!°C!in!500!µL!solutions!containing!

DTNB!buffer!(50!mM!HEPES,!100!mM!NaCl,!and!2!mM!DTNB!at!pH!7.5),!enzyme!and! varying! concentrations! of! substrate! ranging! from! 0.5B5x! Km.! Initial! velocity! data,! measured! as! a! function! of! substrate! concentration,! were! analyzed! using! Enzyme!

Kinetics!v1.4!and!equation!(1):!

V!=!Vmax[S]/([S]+Km)!! ! ! ! !!!!(1)!

! where! V! is! the! initial! velocity,! Vmax! is! the! maximum! velocity,! [S]! is! the! substrate! concentration!and!Km!is!the!Michaelis!constant.!kcat!was!calculated!from!Vmax/[E],!where!

[E]!is!the!final!enzyme!concentration.!

(

2.2.5(Crystallization(and(XDray(Structure(Determination(of(MA0038(and(BVU1957(!

MA0038!and!BVU_1957!crystallization!and!overall!XBray!structure!

! 46! determination!was!performed!by!Tianjang!Ji!under!the!advisement!of!Dr.!Karen!Allen!at!

Boston!University![34].!

2.2.6(Bioinformatic(Analysis((

Biological,Range,of,the,fl,Cluster,

, The!biological!range!of!the!fluorinase!cluster!was!determined!using!an!inBhouse! program! called! ContextBLAST,! which! was! written! using! the! Biopython! package! for!

Python!2.7![35].!In!short,!individual!BLAST!searches!were!run!using!the!NCBI!database! with!each!gene!in!the!S.,cattleya,fluorinase!cluster!(including!Iso,!Deh!and!Tran)!as!the! query! sequences.! Sequence! matches! containing! >30%! sequence! identity! over! >70%! query! coverage! were! retained.! Gene! clustering! was! determined! by! running! a! BLAST! search!on!each!neighboring!gene!out!to!10!genes!on!either!side!of!the!query!gene.!Only! neighboring! sequence! matches! in! the! same! species! as! the! query! sequence! and! containing! >30%! sequence! identity! over! >70%! query! coverage! were! retained.! The!

BLAST!results!for!all!of!the!query!sequences!and!neighboring!sequences!were!compiled! together!and!manually!inspected!for!conserved!gene!clusters.!

Ortholog,Biological,Range,

A! sequence! similarity! network! was! generated! with! the! Enzyme! Function!

Initiative! Enzyme! Similarity! Tool! (EFIBEST)! (http://efi.igb.illinois.edu/efiBest/)! using! the! BLASTBbased! method! with! the! S., cattleya, flK! sequence! as! the! BLAST! input,! a! minimum! length! of! 0,! a! maximum! length! of! 50,000! and! an! EBvalue! cutoff! of! 30.! A! multiple! sequence! alignment! was! generated! from! the! curated! list! of! sequences! using!

! 47! Clustal! Omega! from! the! European! Molecular! Biology! Laboratory’s! European!

Bioinformatics! Institute! (EMBLBEBI)! (http://www.ebi.ac.uk/Tools/msa/clustalo/)! where!specific!marker!residues!were!used!to!filter!out!nonBorthologous!sequences.!CDB

HIT! (http://weizhongBlab.ucsd.edu/cdhit_suite/cgiBbin/index.cgi)! was! then! used! to! cluster! the! remaining! sequences! into! representative! groupings! at! a! 90%! sequence! identity!cutoff.!The!representative!sequences!were!then!used!as!a!filter!for!the!original! sequence!similarity!network!to!create!a!90%!representative!node!network.!Sequence! similarity! and! representative! node! networks! were! visualized! using! Cytoscape! 3.2.!

Multiple! alignment! files! were! visualized! using! ESPript! 3.0!

(http://espript.ibcp.fr/ESPript/ESPript/).!!

2.3(Results(and(Discussion(

2.3.1(Biological(Range(of(the(Fluorinase(Cluster((

Given! the! seemingly! limited! biological! range! of! the! fluorinase! cluster,! we! attempted! to! discovered! further! functional! clusters! using! an! inBhouse! program,!

ContextBLAST.! Beyond! the! four! organisms! already! discovered,! our! searches! revealed! only! one! other! potential! cluster! in! Streptomyces, albulus, PDB1.! However,! upon! closer! analysis!of!the!remaining!hit,!alignments!of!the!individual!genes!with!their!respective!S., cattleya, counterparts! showed! very! little! sequence! identity! overall.! Ultimately! this! indicates! that! S., albulus, does! not! actually! contain! a! fluorinase! biosynethetic! cluster,! though!it!is!impossible!to!say!for!sure!given!the!lack!of!experimental!data.!

, !!

(

! 48! 2.3.2(Biological(Range(and(Sequence(Analysis(of(flK(Orthologs(

An! attempt! to! discover! orthologous! flK! sequences! outside! of! fluorometabolite! producers!was!made!by!tracking!the!biological!range!of!the!gene.!To!identify!potential! flK!orthologs!in!other!bacterial!species!as!well!as!other!domains!of!life,!a!BLAST!search! of!the!S.,cattleya,flK!sequence!generated!a!list!of!over!1,500!potential!flK!orthologs.!To! define! a! protein! as! orthologous! with! as! much! accuracy! as! possible,! marker! residues! were! selected! based! on! experimental! evidence! of! their! involvement! in! FAcCoA! hydrolysis!and/or!substrate!specificity.!Based!on!previous!works!(mentioned!earlier),! the!active!site!residues!comprising!the!catalytic!triad!(Thr42,!Glu50!and!His76)!were! selected!as!the!markers.!A!multiple!alignment!file!of!the!BLAST!results!was!generated! and!sequences!lacking!any!of!the!three!marker!residues!were!discarded!from!further! evaluation.! Some! mutational! exceptions! were! made,! including! E50D! (functional! interchangeability)!and!T42S!(based!on!experimental!evidence!that!a!T42S!mutation!in!

S.,cattleya!flK!still!retained!FAcCoA!hydrolysis!activity,!albeit!lower)![24].!Noteworthy! residues!included!Arg120,!which!has!a!proposed!function!in!positioning!the!substrate! fluoro!group![25].!However,!the!lack!of!experimental!evidence!indicating!its!necessity! for! FAcCoA! hydrolysis! or! selectivity! made! it! unwise! to! use! as! a! definitive! marker! residue.!!

After! filtering! nonBorthologous! sequences! based! on! the! three! defined! marker! residues,! over! 1,200! putative! orthologs! from! more! than! 600! different! species! were! identified.!While!the!majority!of!orthologous!sequences!were!found!in!Bacteria,!a!few! are!spread!throughout!Archaea!and!lower!Eukaryotes!as!well.!In!Bacteria,!putative!flK! orthologs! appear! to! span! a! large! biological! range,! with! members! discovered! in! 13!

! 49! different! phyla.! While! flK! orthologs! have! been! identified! throughout! Acidobacteria,!

Chloroflexi,! Cyanobacteria,! DeinococcusBthermus,! Fusobacteria,! Planctomycetes,!

Spirochaetes! and! Verrucomicrobia,! they! only! account! for! roughly! 10%! of! the! total! number! of! identified! orthologs.! The! remaining! 90%! are! found! in! Actinobacteria,!

Bacteroidetes,!Firmicutes!and!Proteobacteria,!indicating!the!true!range!to!be!narrower! that!previously!thought.!

Overall,!despite!sharing!the!same!tertiary!structure!(the!HD!fold),!the!putative! orthologs!share!very!modest!sequence!homology!(Appendix!AB1B1).!Though!given!the! inherent! sequence! plasticity! seen! throughout! all! members! of! the! hot! dogBfold! superfamily,! this! was! not! completely! surprising.! A! large! discrepancy! can! be! see! in! overall! sequence! length,! with! most! sequences! ranging! between! 120! and! 160! amino! acids.!Some!outlying!sequences!even!range!between!200!and!600!amino!acids!in!length,! indicating!the!presence!of!a!fusion!domain.!However,!the!largest!discrepancy!between! the!flK!sequence!and!the!majority!of!putative!orthologs!is!seen!in!the!length!of!the!lid! structure! and! may! possibly! hold! a! clue! in! identifying! a! divergent! function.! Only! a! handful! of! lid! sequences! align! with! the! flk! lid! motif,! though! there! is! still! little! to! no! sequence!homology!throughout!this!motif.!The!remaining!sequences!appear!to!have!a! truncated! lid! sequence! (compared! to! flK),! lacking! on! average! six! residues! in! the! extended! loop.! When! depicted! in! a! representative! node! network! (RNN),! it! is! evident! that!sequence!homology!is!quite!variable!between!phyla!(Figure!2B7).!Within!some,!the! lack!of!any!significant!sequence!homology!can!be!seen!through!the!existence!of!many! separate,! nonBconnecting! clusters.! While! in! others,! larger,! more! interBconnected! clustering!is!evident!of!conserved!sequence!homology.!!

! 50! The!biological!range!in!Actinobacteria,!the!only!phylum!with!fluorometaboliteB producing!organisms,!is!surprisingly!limited!with!putative!orthologs!only!found!in!51! different!species!(8%!of!the!total!number!identified).!Despite!the!fact!that!almost!all!of! the!sequences!exclusively!belong!to!the!order!of!Actinomycetales,!a!very!modest!degree! of! homology! is! shared! throughout,! seen! again! in! the! RNN! by! the! many! distinct!

Actinobacteria! clusters.! Within! this! small! population! of! orthologs,! very! little! conservation!of!sequence!length!is!noticed,!with!monomers!ranging!anywhere!from!119! to!156!amino!acids,!and!even!one!at!197!amino!acids!in!length.!Additionally,!the!same! large!discrepancy!in!the!length!of!the!lid!sequence!can!be!tracked!within!Actinobacteria,! with!the!majority!of!putative!orthologs!containing!a!truncated!lid!structure!(Appendix!

AB1B2).!Unexpectedly,!the!closest!flK!orthologs!(with!the!exception!of!S.,xinghainesis,and!

S., sp, MA37)! are! not! members! of! Actinobacteria,! but! rather! part! of! Proteobacteria.!

Altogether,!it!would!appear!is!if!the!flK!thioesterase!has!not!been!traditionally!evolved! throughout! Actinobacteria.! Rather,! it! would! appear! as! if! S., cattleya, and! the! other! fluorometaboliteBproducing! organisms! acquired! the! TE! from! Proteobacteria! through! horizontal!gene!transfer!(HGT).!As!for!the!remaining!orthologs,!the!limited!range!and! lack!of!sequence!homology!with!any!other!bacterial!members!give!little!clues!as!to!the! biological! roles! they! may! be! playing.! Putative! orthologs! within! the! Bacteroidetes! phylum! are! found! primarily! within! the! Bacteroidales! order,! evenly! distributed! throughout! the! Bacteroidaceae,! Porphyromonada,! Prevotellaceae! and! Rikenellaceae! families.!Orthologs!in!this!phylum!account!for!only!12%!of!the!total!number,!and!while! conservation!of!sequence!length!is!modest!(about!the!same!as!in!Actinobacteria),!the!

RNN!shows!a!large!grouping!of!interBconnected!clusters,!indicating!that!the!overall!

! 51! 2 1

4 8 1

1 42

4 1

1 1

1 1 1 1 1 1 1 1 1

3 68 1

31 3

5 3

1 2 1 1 1 1 5 1 1 1 5

2 4 2 2 3 1 1 1 1 1 1 1 4 1 5 1 1 1 1 1 1 2 1 1 5 1 1 1 1 1 1 1 5 2 1 1 1 1 1 1 1 1 1 1 2 1 1 1 2 1 1 1 1 5 2 3 1 2 1 8 4 1 2 3 1 1 1 1 1 1 1 1 1 2 1 1 1 1 1 3 4 1 1 4 1 1 3 1 1 1 1 1 1 1 2 1 1 1 2 1 1 2 1 1 3 2 1 2 1 1 1 1 9 2 1 1 1 2 1 1 1 2 1 1 2 4 16 1 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 3 2 1 1 1 1 2 1 1 3 1 1 1 3 1 1 1 1 2 1 3 3 1 1 3 1 2 3 1 1 4 1 10 2 1 1 7 1 1 5 3 1 1 2 2 1 4 1 1 2 1 1 1 1 1 1 3 1 1 1 6 2 2 1 1 1 1 1 1 1 1 2 1 2 1 1 2 1 1 1 1 1 1 1 1 2 1 1 2 5 1 2 1 1 2 1 1 1 2 1 1 2 5 1 2 1 1 1 2 1 2 2 2 1 2 1 1 12 2 2 6 4 1 1 1 1 1 2 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 2 1 7 1 4

1 1 1 1 1 2 1 1 1 1 4 1 1 2

1 1 1 4

1 1 1 1

2 1 1 1 1 1 2 1 1 1 1 7 1 1 325 1 1 4 1 2 2 6 1 2 1 2 4 2 1 1

2 1 1 3 2 1 1 2 1 1 3 1 4 2 1 2 1 1 1

1 1 1

1 1 2 1 1 2 1 1 2 2 2 1 1 1 1

1 1 8 1 1 5

1 1 1 1 1 1 1 1 1 1 1

1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 !

Figure( 2D7.( Representative! node! network! of! putative! bacterial! flK! orthologs.!

Representatives! are! colorBcoded! by! phylum:! Acidobacteria! (Dark! Green),!

Actinobacteria! (Orange),! Bacteroidetes! (Blue),! Chloroflexi! (Yellow),! Cyanobacteria!

(Lavender),! DeinococcusBThermus! (Purple),! Firmicutes! (Green),! Fusobacteria! (Pink),!

Planctomycetes!(Salmon),!Proteobacteria!(Red),!Spirochaetes!(Teal),!Verrucomicrobia!

(Grey).!Cloned!orthologs!are!indicated!by!arrows:!(1)!flK,!(2)!BVU_1957,!(3)!cgp_0542,!

(4)!Galf_1995,!(5)!TTHA0967.!

! 52! sequence! homology! within! Bacteroidetes! is! much! higher.! Additionally,! a! multiple! sequence! alignment! indicates! the! majority! of! residue! positions! are! moderatelyBtoB highly!conserved!within!the!phylum!(Appendix!AB1B3).!Furthermore,!the!truncation!of! the!lid!structure!appears!to!be!a!defining!feature!within!Bacteroidetes,!as!every!putative! ortholog! is! lacking! residues! in! the! extended! loop.! Given! the! higher! overall! sequence! homology!within!Bacteroidetes,!it!is!more!likely!that!these!sequences!were!all!derived! from!a!more!common!ancestor,!and!therefore,!a!common!function.!!

In!Proteobacteria,!the!second!largest!grouping!of!flK!orthologs!(25%),!sequences! are! distributed! predominantly! throughout! the! Alpha! and! Beta! classes,! modestly! throughout! the! Delta! and! Gamma! classes! and! weakly! in! Epsilon.! As! seen! in!

Actinobacteria,!there!is!a!large!degree!of!sequence!diversity!within!the!phylum!(Figure!

2B7).!Furthermore,!a!multiple!sequence!alignment!indicates!a!very!low!overall!shared! homology! (Appendix! AB1B4).! However,! further! inspection! shows! the! presence! of! distinct!groupings!that!share!moderate!sequence!identity!within,!and!based!on!the!lid! motif! alone,! three! distinct! groupings! were! identified.! Roughly! 60%! of! the! sequences! within!Proteobacteria!contain!a!truncated!lid!sequence!while!40%!contain!a!fullBlength! lid!as!seen!in!the!flK!sequence.!Interestingly!enough,!the!sequences!containing!the!full! lid! motif! are! further! divided! into! two! distinct! groupings.! While! the! motifs! in! either! group!appear!to!be!similar!in!length,!sequence!homology!is!wholly!unconserved.!The! larger!of!the!two!groupings!shows!fair!conservation!within!its!motif!and!the!sequences! align! more! or! less! with! the! flK! lid.! The! smaller! grouping! indicates! no! conservation! beyond!a!similar!sequence!length.!While!it!would!appear!that!the!majority!of!sequence! homology!within!Proteobacteria!is!classBdependent!(Figure!2B8,!clusters!2!and!4B8),!the!

! 53! 1 2

6 1 5 1 1 1 4 7 1 2 1 1 1 1 1 1 2 1 1 1 1 1 1 1 3 2 1 1 1 1

2 1 1

2 4 1 1

1 6 1 1 2

1 1 1 8

2 4 1 1 5 7 1

1 1 2 1 8

1 1 1

1 2

1 1 1 1 1 2 2 1 1 1 1 1 1

Figure( 2D8.( Representative! node! network! of! putative! bacterial! flK! orthologs! within!

Proteobacteria.! Representatives! are! colored! by! class:! Alpha! (red),! Beta! (blue),! Delta!

(green),!Epsilon!(yellow),!Gamma!(pink).!

! closest! orthologs! to! the! original! flK! sequence! are! spread! throughout! all! 4! classes!

(Figure!2B8,!cluster!1).!

Firmicutes! contains! the! largest! number! of! orthologBcontaining! species! (over!

55%).!Given!that!the!number!of!compared!sequences!(over!350)!is!significantly!larger! than!in!any!other!phylum,!sequence!conservation!within!the!Firmicutes!is!fairly!high.!

While! separate! groupings! of! low! sequence! homology! do! exist! with! the! phylum,! the!

! 54! majority! of! sequences! are! clustered! together,! indicating! a! possible! common! function!

(Figure!2B7).!Within!Firmicutes,!the!majority!of!sequences!belong!to!the!Clostridia!class! with! moderate! groupings! in! Bacilli! and! Negativicutes.! Furthermore,! over! 50%! of! the! sequences! within! Clostridia! are! found! in! the! genus! Clostridium.! Interestingly! enough,! the!majority!of!Firmicutes!sequences!are!clustered!with!the!majority!of!Bacteroidetes,! indicating!a!large!degree!of!interBphylum!homology.!Additionally,!this!could!point!to!a! potential! common! ancestor! in! the! evolution! of! a! divergent! function.! Analysis! of! the! sequences! within! Firmicutes! indicates! that! the! majority! contains! a! truncated! lid! structure,! not! surprising! given! the! close! clustering! of! the! Firmicutes! with! the!

Bacteroidetes!(Appendix!AB1B5).!!

While!the!orthologs!within!DeinococcusBThermus!(DT)!only!account!for!roughly!

3%! of! the! total! number! identified,! they! contain! some! interesting! sequence! modifications! not! seen! across! other! phyla.! Orthologs! within! this! phylum! contain! the! largest!degree!of!sequence!homology,!with!the!majority!of!residue!positions!highly!or! completely!conserved.!This!can!be!seen!in!both!the!RNN!by!the!tight!clustering!of!the! phylum! (Figure! 2B7).! Interestingly! enough,! while! members! of! DT! contain! a! semiB truncated!lid!motif!(as!compared!to!flK),!it!appears!to!be!distinct!from!the!lid!truncation! seen! throughout! Bacteroidetes! and! the! majority! of! orthologs! (Appendix! AB1B6).!

Additionally,!the!most!distinct!sequence!variation!is!in!the!highly!conserved!active!site!

Arg!residue.!Throughout!all!of!the!orthologs!within!DT,!this!residue!is!mutated!to!Gln.!

Given! the! proposed! (though! unverified)! function! of! this! residue! in! flK,! this! mutation! could!have!a!profound!effect!on!the!active!scaffold,!altering!its!chemistry!and!substrate! specificity.!!

! 55! Overall,!sequence!analyses!of!the!flK!orthologs!indicate!three!distinct!sequence! motifs! centered! on! the! helical! lid! structure.! Given! its! proximity! to! the! active! site,! discrepancies! in! its! overall! shape! could! potentially! alter! substrate! specificity! and! overall!functionality,!and!may!provide!a!basis!for!functional!divergence.!The!fullBlength! lid!(flKBlike)!motif!has!been!found!to!be!fairly!limited!within!the!flK!subfamily,!and!may! indicate!that!its!function!is!not!widespread!throughout.!As!well,!the!semiBtruncated!lid! motif! seen! in! DT! is! limited! within! its! phylum,! potentially! a! result! of! specific! functionality!within!this!group!of!bacteria.!Conversely,!the!truncated!lid!motif!seen!in!all!

Bacteroidetes! species! has! a! wide! biological! range,! as! it! is! found! in! the! majority! of! orthologous!sequences.!If!the!truncated!lid!sequence!has!indeed!resulted!in!a!divergent! function,! the! majority! of! flK! orthologs! would! be! performing! a! biological! role! that! is! different!from!the!FAc!resistance!function!performed!by!flK.!

! The! human! gut! microbiome! is! a! complex! community! of! bacterial! species! that! have!been!found!to!play!significant!roles!in!human!health.!Thought!to!be!composed!of! over!1,000!species!of!bacteria,!the!gut!microbiome!is!estimated!to!contain!over!100Bfold! more!genes!than!the!human!genome,!and!is!often!referred!to!as!a!“hidden!organ.”![43]!

Given! the! staggering! number! of! inhabiting! species,! the! biological! range! of! the! gut! microbiome! could! be! considered! fairly! limited,! as! its! inhabitants! (primarily)! are! members!of!Firmicutes!and!Bacteroidetes.!Within!Firmicutes,!the!dominant!species!are! members!of!the!genus!Clostridium,whereas!in!Bacteroidetes,!the!dominant!species!are! members!of!Bacteroides,and,Prevotella,genera![43].!To!a!lesser!degree,!the!gut!is!also! populated! by! species! within! Actinobacteria,! Fusobacteria! and! Verrucomicrobia! [43].!

Overall,! this! biological! range! is! consistent! with! the! biological! range! of! flK! orthologs.!

! 56! Additionally,!as!the!majority!of!sequences!within!these!phyla!contain!the!truncated!lid! motif,! especially! in! Firmicutes! and! Bacteroidetes! (the! two! major! factions! of! gut! microbiota),! it! is! possible! that! the! flK! scaffold! may! have! evolved! throughout! the! gut! microbiome!to!perform!a!role!specific!to!this!environment.!!

( (

2.3.3(Cloning(and(Isolation(of(flK(Orthologs((

( To!test!for!alternative!functionality!within!the!flK!subfamily,!specific!ortholgous! sequences! were! selected! for! cloning,! isolation! and! testing.! In! total,! ten! (previously! uncharacterized)! orthologs! (S., cattleya, flK! included)! were! chosen! (Table! 2B1).!

Unfortunately,! only! six! of! the! ten! selected! (MA0038,! cgp_0542,! flK,! BVU_1957,!

TTHA0967! and! Galf_1995)! were! successfully! isolated.! The! remaining! orthologs! were! successfully!cloned!but!were!unable!to!be!isolated!due!to!stability!issues.!!

2.3.4(Substrate(Specificity(of(flK(Orthologs(

All!of!the!cloned!and!isolated!orthologs!were!assayed!for!thioesterase!activity!by! measuring!individual!steadyBstate!kinetic!parameters!for!the!hydrolysis!of!various!acylB

CoA!substrates!(Table!2B2).!To!start,!each!protein!was!assayed!for!FAcCoA!and!AcCoA! hydrolysis! activity! in! order! to! measure! the! amount! of! substrate! discrimination! exhibited!between!the!two.!HighBlevel!discrimination!is!the!hallmark!of!the!flK!reaction! and!the!basis!for!its!biological!function!in!allowing!normal!TCA!cycle!in!the!presence!of!

FAc.! Discrepancies! in! this! trend! may! indicate! alternative! functionality.! For! accurate! comparison,!FAcCoA!and!AcCoA!hydrolysis!activity!was!also!measured!with!the!original!

S.,cattleya,flK!under!the!same!conditions.!As!expected,!flK!displays!high!activity!towards!!

! 57! Kingdom Phylum Class Order Family Genus Species Gene8ID Archaea Euryarchaeota Methanomicrobia Methanosarcinales Methanosarcinaceae Methanosarcina Methanosarcina3acetivorans MA_0038 Bacteria Actinobacteria Actinobacteria Actinomycetales Corynebacteriaceae Corynebacterium Corynebacterium3glutamicum cgp_0542 Bacteria Actinobacteria Actinobacteria Actinomycetales Streptomycetaceae Streptomyces Streptomyces3cattleya flK Bacteria Bacteroidetes Bacteroidia Bacteroidales Bacteroidaceae Bacteroides Bacteroides3vulgatus BVU_1957 Bacteria DeinococcusJThermus Deinococci Thermales Thermaceae Thermus Thermus3thermophilus TTHA0967 Bacteria Firmicutes Bacilli Bacillales Paenibacillaceae Paenibacillus Paenibacillus3larvae ERIC2_c10050 Bacteria Firmicutes Bacilli Lactobacillales Enterococcaceae Enterococcus Enterococcus3faecalis EFS1_1315 Bacteria Firmicutes Clostridia Clostridiales Peptococcaceae Syntrophobotulus Syntrophobotulus3glycolicus Sgly_2172 Bacteria Proteobacteria Alphaproteobacteria Rhizobiales Bradyrhizobiaceae Bradyrhizobium Bradyrhizobium3japonicum BJ6T_15640 Bacteria Proteobacteria Betaproteobacteria Gallionellales Gallionellaceae Gallionella Gallionella3capsiferriformans Galf_1995 !

Table(2D1.!Taxonomic!information!of!flK!orthologs!selected!for!cloning!and!isolation.(

FAcCoA! while! exhibiting! an! almost! 105Bfold! decrease! in! AcCoA! activity.! This! large! discrepancy! in! activity! between! the! two! substrates! is! mainly! due! to! the! >2000Bfold! decrease! in! kcat! for! AcCoA,! though! Km! is! also! increased! by! >100Bfold.! Overall,! this! activity!measured!for!FAcCoA!and!AcCoA!hydrolysis!by!flK!is!in!good!agreement!with! previously!reported!steadyBstate!parameters![24,!25].!!

While!all!isolated!orthologs!displayed!activity!towards!FAcCoA!hydrolysis,!not!all! were! able! to! discriminate! between! FAcCoA! and! AcCoA! at! a! high! level.! In! fact,! only!

MA0038!and!TTHA0967!displayed!flKBlike!discrimination!(104!and!103Bfold!differences,! respectively)! between! FAcCoA! and! AcCoA! hydrolysis.! Given! the! Arg/Gln! mutation! in!

TTHA0967!(member!of!DeinococcusBThermus),!this!result!indicates!that!Arg120!in!the! flK!active!site!is!not!critical!for!FAcCoA!hydrolysis.!The!loss!of!substrate!discrimination! in! BVU_1957! and! Galf_1995! was! due! exclusively! to! lower! FAcCoA! activity! as! both! showed! AcCoA! activity! at! comparable! or! lower! levels! to! that! of! flK.! While! BVU_1957! displayed!a!Km!value!comparable!to!flK,!the!measured!Km!for!Galf_1995!was!almost!10B fold!lower.!In!both!cases,!the!main!contribution!for!the!decrease!in!activity!was!seen!in!

3 the! measured! kcat! value,! which! was! about! 10 Bfold! lower! that! of! flK,! indicating! that!

! 58! turnover,! not! binding,! is! the! major! factor.! With! cgp_0542,! nearly! a! complete! loss! of!

FAcCoA/AcCoA!discrimination!was!seen,!as!FAcCoA!activity!was!only!increased!2.6Bfold! compared! to! AcCoA.! Both! a! decrease! in! FAcCoA! activity! and! an! increase! in! AcCoA! activity!contributed!to!this!loss.!cpg_0542!displayed!faster!turnover!and!tighter!binding! with! AcCoA! (as! compared! to! flK)! while! FAcCoA! activity! suffered! mainly! due! to! a! decrease!in!turnover,!not!binding!affinity.!The!large!difference!in!flK!and!cgp_0542!was! an!odd!result,!given!that!the!two!proteins!share!a!fairly!large!sequence!identity!for!this! subfamily! (Figure! 2B11).! While! it! seems! unlikely,! it! is! possible! that! slight! structural! variations!have!given!rise!to!this!dramatic!variation!in!substrate!specificity.!

Aside! from! measuring! FAcCoA! and! AcCoA! hydrolysis! activities,! an! expanded! substrate!screening!was!performed!on!flK!and!all!five!orthologs.!With!the!exception!of!

BVU_1957,!all!orthologs!(including!flK)!displayed!moderate!acetoacetylBCoA!(AacCoA)! hydrolysis! activity,! with! kinetic! efficiencies! measuring! in! the! range! of! 102B104! MB1sB1.!

Interestingly!enough,!cgp_0542!displayed!slightly!higher!activity!towards!AacCoA!than!

FAcCoA,!the!only!ortholog!to!do!so.!Also,!all!orthologs!tested!(including!flK)!were!shown! to! be! active! towards! formylBCoA! (HCoA).! As! with! flK,! MA0038! and! TTHA0967,! HCoA! hydrolysis!activity!did!not!exceed!FAcCoA!hydrolysis!BB!their!kinetic!efficiencies!ranged! from! 101B102Bfold! lower.! In! the! case! of! BVU_1957,! cgp_0542! and! Galf_1995,! HCoA! hydrolysis! was! favored! as! they! each! exhibited! the! highest! overall! kinetic! efficiencies! with! this! substrate.! Furthermore,! BVU_1957! exhibited! the! highest! overall! kinetic! efficiency! towards! hydrolysis! of! HCoA! of! any! other! ortholog/substrate! pair.! The!

7 measured!10 !kinetic!efficiency!is!due!exclusively!to!a!nanomolar!Km,!indicating!HCoA! has!very!tight!binding!interactions!in!the!BVU!active!site.!Given!that!BVU_1957!

! 59! -1 -1 -1 -1 -1 -1 Substrate kcat+(s ) Km+(μM) kcat/Km+(M s ) kcat+(s ) Km+(μM) kcat/Km+(M s )

flK BVU_1957

Formyl'CoA (2.4.±.0.08).x.10'1 7.6.±.0.8 3.1.x.104 3.4.±.0.01 <.1.0 5.7.x.107*

Acetyl'CoA (9.0.±.0.5).x.10'3 (8.6.±.1.1).x.102 1.0.x.101 <.0.00001 ' '

Fluoroacetyl'CoA (2.0.±.0.06).x.101 (6.0.±.0.9).x.101 3.3.x.105 (6.0.±.0.1).x.10'2 (9.6.±.0.6).x.101 6.3.x.102

Acetoacetyl'CoA (16.±.0.3).x.10'2 17.±.1.0 9.4.x.103 <.0.00001 ' '

Benzoyl'CoA <.0.00001 ' ' <.0.00001 ' '

Phenylacetyl'CoA <.0.00001 ' ' <.0.00001 ' '

3'hydroxybenzoyl'CoA <.0.00001 ' ' <.0.00001 ' '

Gentisyl'CoA <.0.00001 ' ' <.0.00001 ' '

1,4'DHNA'CoA <.0.00001 ' ' <.0.00001 ' '

cgp_0542 Galf_1995

Formyl'CoA 7.7.±.0.06 14.±.0.4 5.5.x.105 (3.7.±.0.1).x.10'1 (1.5.±.0.2).x.101 2.5.x.104

Acetyl'CoA 0.26.±.0.01 (2.5.±.0.3).x.102 1.0.x.103 (3.5.±.0.03).x.10'2 (2.3.±.0.07).x.102 1.5.x.102

Fluoroacetyl'CoA (6.0.±.0.2).x.10'2 23.±.2.7 2.6.x.103 (4.0.±.0.1).x.10'2 9.5.±.1.1 4.2.x.103

Acetoacetyl'CoA (31.±.0.9).x.10'1 58.±.5.1 5.3.x.104 (9.8.±.0.4).x.10'2 (2.8.±.0.3).x.102 3.6.x.102

Benzoyl'CoA <.0.00001 ' ' <.0.00001 ' '

Phenylacetyl'CoA <.0.00001 ' ' <.0.00001 ' '

3'hydroxybenzoyl'CoA <.0.00001 ' ' <.0.00001 ' '

Gentisyl'CoA <.0.00001 ' ' <.0.00001 ' '

1,4'DHNA'CoA <.0.00001 ' ' <.0.00001 ' '

MA0038 TTHA0967

Formyl'CoA (2.4.±.0.08).x.10'1 7.6.±.0.8 3.1.x.104 (3.0.±.0.1).x.10'2 (6.4.±.0.3).x.101 4.7.x.102

Acetyl'CoA (9.0.±.0.5).x.10'3 (8.6.±.1.1).x.102 1.0.x.101 (2.4.±.0.3).X.10'2 (1.2.±.0.3).x.103 2.0.x.101

Fluoroacetyl'CoA (2.0.±.0.06).x.101 (6.0.±.0.9).x.101 3.0.x.105 (6.2.±.0.2).x.10'1 (1.6.±.0.2).x.101 3.8.x.104

Acetoacetyl'CoA 0.14.±.0.01 90.±.3.9 1.6.x.103 (1.7.±.0.07).X.10'2 (1.4.±.0.2).x.102 1.2.x.102

Benzoyl'CoA (4.3.±.0.1).x.10'3 47.±.2.0 9.1.x.101 (2.1.±.0.02).x.10'3 (9.6.±.0.5).x.101 2.2.x.101

Phenylacetyl'CoA <.0.00001 ' ' (1.7.±.0.2).x.10'1 (8.5.±.0.5).x.102 2.0.x.102

3'hydroxybenzoyl'CoA (7.4.±.0.2).x.10'3 3.1.±.0.3 2.4.x.103 <.0.00001 ' '

Gentisyl'CoA (9.0.±.0.1).x.10'3 41.±.2.5 2.2.x.102 <.0.00001 ' ' 1,4'DHNA'CoA <.0.00001 ' ' <.0.00001 ' ' (

(

Table( 2D2.( Steady!state!kinetic!constants!for!flK!and!orthologBcatalyzed!hydrolysis!of! various!acylBCoA!substrates.!*kcat/Km!calculated!from!an!estimated!Km!of!60!nM.!

! 60! exhibited! the! smallest! substrate! range,! (only! active! with! HCoA! and! FAcCoA),! this! supports! the! notion! that! the! truncated! lid! motif! has! resulted! in! an! altered! substrate! specificity!profile,!and!potentially!a!different!biological!function.!!

! Additionally,! both! MA0038! and! TTHA0967! exhibited! slight! activity! towards! aromatic!acylBCoA!substrates.!While!MA0038!displayed!only!slight!to!moderate!activity! towards! benzoyl,! gentisyl! and! 3BhydroxybenzoylBCoA,! the! measured! Km! values! for! all! three! substrates! were! very! competitive! with! (and! in! some! cases,! lower! than)! the! respective!Km!values!for!formylB,!acetylB,!fluoroB!and!acetoacetylBCoA.!This!indicates!that! while!turnover!is!low,!the!active!site!is!able!to!easily!accommodate!aromatic!substrates.!

TTHA0967!displayed!low!level!activity!for!benzoylBCoA!and!phenylacetylBCoA.!While!he! measured! kcat! for! phenylacetylBCoA! was! reasonable! compared! to! other! substrates,! its! high!Km!value!indicates!that!the!TTHA0967!active!site!is!not!set!up!to!accommodate(this! substrate.!!On!the!other!hand,!the!measured!Km!for!benzoylBCoA!was!competitive!with! other!measured!substrates!while!kcat!was!much!lower,!highlighting!the!effect!that!the! phenylacetylBCoA!methylene!group!plays!in!substrate!binding!and!specificity.!(

(

2.3.5(Structural(Analysis(of(flK(and(Isolated(Orthologs(

With! the! substrate! screening! results! indicating! the! potential! for! divergent! function,!a!structural!analysis!of!available!ortholog!crystal!structures!was!carried!out!to! identify!a!structural!basis!for!these!results.!To!date,!only!the!crystal!structures!of!flK! and!TTHA0967!had!been!solved![24,!25,!39].!However,!attempts!to!crystallize!MA0038! from! M., acetivorans, and! BVU_1957! from! B., vulgatus, were! successful.! Additionally,! a! crystal!structure!of!BVU_1957!with!coenzyme!A!(CoASH)!coBcrystallized!was!also!

! 61! !

Figure( 2D9.( Crystal! structure! overlays! of! flK! (PDB:! 3KV8)! (blue),! MA0038! (pink),!

BVU_1957!(green)!and!TTHA0967!(PDB:2CWZ)(orange).!Insert!shows!conservation!of! key!catalytic!residues.!

! obtained.! Unfortunately,! attempts! to! crystallize! Galf_1995! for! XBray! structure! determination!were!unsuccessful.!!

In! general,! all! four! of! the! crystal! structures! overlay! very! well,! indicating! an! overall!conservation!of!the!core!fold!(Figure!2B9).!Additionally,!the!active!site!marker! residues!(Thr,!Glu!and!His)!are!all!in!relatively!identical!positions,!indicating!the!same! mode! of! catalysis! (Figure! 2B9).! Given! the! conservation! of! catalytic! residues,! the! differences!in!activity!must!be!due!to!changes!in!active!site!topology.!To!further!analyze!

! 62! the!active!site!architectures!of!the!four!orthologs,!active!site!volumes!were!generated! using!the!CASTp!server![40].!Overall,!it!appears!as!if!two!structural!features!define!the! active!site!spaces!of!flK!and!the!other!orthologs:!the!size!and!flexibility!of!the!lid!motif! and! the! positioning! of! the! (mostly! conserved)! arginine! residue.! According! to! its! substrate! specificity! profile,! flK! shows! a! fairly! limited! range,! only! active! towards! substrates!with!relatively!small!acyl!(R)!groups.!Both!the!positioning!of!the!lid!over!the! active! site! and! Arg120! along! the! bottom! restricts! the! available! volume! within.!

Furthermore,! a! number! of! hydrogenBbonding! interactions! between! the! lid! and! core! domains!were!found!to!render!the!lid!fairly!inflexible,!effectively!locking!the!size!and! shape!of!the!active!site![34].!As!discussed!earlier,!the!largest!discrepancy!in!sequence! between!the!flK!thioesterase!and!the!majority!of!putative!orthologs!is!in!the!lid!motif,! with! flK! containing! a! larger! lid! segment! than! most! other! orthologs.! Of! the! available! crystal! structures,! MA0038! shares! the! highest! sequence! homology! to! flK! with! 50%! sequence! identity! overall.! Additionally,! MA0038! appears! to! contain! a! full! lid! motif! similar!to!flK!(Figure!2B10).!The!positioning!of!Arg121!is!in!good!alignment!with!flK!as! well,! resulting! in! a! very! similarly! sized! active! site! (Figure! 2B10).! According! to! CASTp! calculations,!the!size!of!MA0038’s!active!site!is!actually!slightly!larger!than!that!of!flK! and! may! explain! why! it! is! able! to! accommodate! benzoylBCoA! and! a! few! of! its! hydroxylated!derivatives.!From!a!structural!basis,!the!slightly!larger!active!site!could!be! due!to!more!lid!flexibility!as!MA0038!was!found!to!have!fewer!lidBcore!interactions!than! flK![34].!!

Alternatively,! the! BVU_1957! (BVU)! crystal! structure! highlights! the! profound! effect!of!a!truncated!lid!motif!(Figure!2B10).!As!seen!in!the!substrate!screening,!BVU!was!!

! 63! !

! 64! !

Figure( 2D10.( Structural! comparison! of! flK! (teal)! (PDB:! 3KV8),! MA0038! (magenta),!

BVU_1957!(green)!and!TTHA0967!(PDB:!2CWZ)!(red).!The!helical!lid!motif!is!colored!in! yellow.!Active!site!volumes!are!colored!by!electrostatic!potential.!

! not! active! for! any! substrates! larger! than! FAcCoA,! a! direct! contrast! to! the! other! orthologs!tested.!Additionally,!the!high!HCoA!activity!indicated!that!the!active!site!might! be! set! up! to! bind! this! substrate! the! best.! Although! the! core! structure! and! catalytic! residues!align!very!well!with!flK!and!MA0038,!the!BVU!active!site!is!almost!half!the!size! of!either,!as!calculated!by!CASTp.!Given!that!Arg110!is!in!the!same!position!as!R120!and!

R121,!the!lid!motif!is!solely!responsible!for!this!loss!in!active!site!volume.!In!comparison! with!both!flK!and!MA0038,!the!truncated!lid!in!BVU!appears!to!shorten!the!length!of!the! active!site,!limiting!the!size!of!acyl!group!that!could!be!accommodated.!Additionally,!the!

BVU! lid! motif! (despite! its! size)! appears! to! have! more! hydrogen! bonding! interactions! with!the!core,!further!limiting!its!flexibility!and!potentially,!substrate!specificity![34].!

These!structural!features!help!to!explain!why!BVU!was!not!active!with!any!substrates!!

! 65! β1 η1 α1 α2 α3 η2 flK TT 1 10 20 30 40 50 flK M KDG. M RV G ERFTHDF VV PPHK TV RH L YPESP E FAEFPE V FAT GF MV GL ME WA C V R AM A P MA0038 M DSST L KP G LAYEFRFK I PENK TV PY L YPESP E FQVMPK V FAT GF MV GLF E WA C I Q AI N P Galf_1995 M KD.T L KP G IRFEHKY LV PANK TV PA L YPESP E FLAMPE V FAT GF MV GF LE WA C I M AI K P cgp_0542 M KE.T L TT G LTHQMTY IV PANR TV PH L LPEAA E FETMPD V LAT GY MV GI IE WA C M E LL R P TTHA0967 M R..P I PE G YEAVFET VV TPEM TV RF.....E E LGPVHP V YAT Y WMV KH ME LA GRK II L P BVU_1957 M E.....T G LTYTSTV VV SKENV A AT M ..GSG D L....N V FAT P AMV AL ME NA A M S AV AD

β2 β3 β4 flK TT TT TT 60 70 80 90 100 110 flK YL E .PG E G SL G T A I CV TH T A A T P PG LT V TV T A E L RS VEGRRLSW R V SA H D GV.DE I GS G T MA0038 YL D FPA E Q TV G T D V RL SH S A A T P PG LT V TV K I K L EK IEGRKLTF S I IA D D GV.DK I SE G T Galf_1995 HL D WPE E Q SV G T H I NV SH E A A T P PG LE V TA S V E L TI VDGRRLTF A V SA H D GV.DT I AR G T cgp_0542 HL D .DG E I SL G T H V NF SH A A P T V PG ST V TI D V E V TE I NR R A VTF N I TA A D EF.AT I ST G T TTHA0967 FL E .EG E EG IG S Y V EAR H L A SAL PG MR V R V V A RHEKT EG N RV YAR V EA YNELGDL I GV G R BVU_1957 EL P.EGST TV G AM M NT TH IKP S AV G DT V SA T A V L KE VEGRKLTF E V RA Q D SK.GV I GE G T 1

β5 α4 flK ...... 120 130 flK H E R A VI HLE K F NA KV ...RQ....KTPAG MA0038 H E R F II DAA K F NS KA .EAKA....KNANN Galf_1995 H E R Y VI NKE K F DN KL RDKRE....KINQR cgp_0542 H Q R G VV NRE K F VS RL PEAPK....E...N TTHA0967 TEQV IL PKA K VEALFRRLKERWEAERSPS BVU_1957 H V R Y IV DKE K F MS KL ...... S (

Figure(2D11.(Multiple!alignment!of!flK!and!cloned!flKBorthologs.!Completely!conserved! residues!are!highlighted!in!red!and!highly!conserved!residues!are!highlighted!in!yellow.!

Secondary!structure!was!overlaid!using!the!flK!crystal!structure!(PDB:!3KV8_B).!

! larger!than!FAcCoA!and!may!also!help!provide!a!structural!basis!for!the!high!level!of!

HCoA!activity.!!

In! looking! at! the! TTHA0967! (TTHA)! sequence! (Figure! 2B11),! it! would! appear! that!its!lid!motif!falls!somewhere!between!truncated!and!full!length.!A!look!at!its!crystal! structure! (Figure! 2B10)! confirms! this! observation,! as! it! appears! larger! than! BVU! but! smaller!than!flK!or!MA0038.!This!semiBlimitation!on!the!TTHA!active!site!could!explain!

! 66! why! it! was! still! able! to! discriminate! between! FAcCoA! and! AcCoA,! albeit! to! a! lesser! degree!than!flK!or!MA0038.!Furthermore,!while!this!semiBtruncation!should!technically! limit! the! volume! of! the! active! site! (as! seen! in! BVU! with! the! truncated! lid),! CASTp! calculations! indicate! the! active! site! to! be! generally! around! the! same! size! as! flK! and!

MA0038.!The!reasoning!for!this!can!be!found!in!the!bottom!of!the!active!site.!Where!flK,!

MA0038! and! BVU! all! have! identically! positioned! arginine! residues,! TTHA! has! a! glutamine!residue,!positioned!away!from!the!active!site.!This!residue!swap!opens!up!a! large! portion! of! the! lower! TTHA! active! site! and! may! explain! why! it! is! able! to! accommodate!the!aromatic!substrates!phenylacetylBCoA!and!benozylBCoA.!

(

2.3.6(Gene(Context(Analysis(

! With! sequence,! activity! and! structural! analyses! indicating! a! divergence! of! function! in! the! flK! scaffold,! we! turned! to! a! gene! context! analysis! in! the! hopes! that! neighboring! genes! may! provide! further! clues! in! identifying! such! a! function.!

Unfortunately,!compared!to!the!number!of!organisms!with!orthologous!sequences,!the! available! gene! context! data! is! pretty! sparse,! limiting! the! power! of! this! analysis.!

Nonetheless,! we! were! still! able! to! pull! a! few! clues! from! the! data.! Starting! with! the! cloned! and! isolated! orthologs,! MA0038,! TTHA0967,! BVU_1957,! Galf_1995! and! cgp_0542,! we! found! that! they! really! shared! no! conservation! of! gene! context! whatsoever.!Though!with!the!majority!of!genes!in!the!MA0038!cluster!unannotated,!few! clues!were!provided!about!the!putative!function!of!this!particular!ortholog.!Galf_1995! appears!to!be!clustered!with!genes!involved!in!arginine!and!proline!metabolism,!given! its!proximity!to!an!acetylornithine!and!succinylornithine!aminotransferase!(Galf_1990),!!

! 67! TE# FTHFS# FolP# FolB# folK#

2-amino-4-hydroxy- 6-hydroxymethyl- dihydroneopterin dihydropterin pyro- dihydropteroate aldoase (folB) phosphokinase (folK) 2-amino-4-hydroxy synthase (folP) Dihydro- 2-amino-4-hydroxy 7,8-dihydrop- -6-hydroxymethyldihydropterin teroate neopterin -6-hydroxymethyldihydropterin diphosphate

dihydrofolate synthase (folC)

Dihydrofolate (DHF)

formate: dihydrofolate tetrahydrofolate Formyl-THF reductase (folA) ligase (FTHFS) dehydrogenase Dihydrofolate (DHF) Tetrahydrofolate (THF) N10-formyl-THF 5,10-methenyl-THF

Formyl-THF Methylene- dehydrogenase tetrahydrofolate reductase 5-methyl-THF 5,10-methylenete-THF !

Figure(2D12.(Gene!context!within!T.,potens,(Firmicutes)!showing!flK!ortholog!(TE)!coB localization! with! genes! involved! in! folate! biosynthesis! (red! box)! and! the! one! carbon! pool!by!folate!(blue!box).!

(

! an! ornithine! carbamoyltransferase! (Galf_1995)! and! an! arginosuccinate! synthase!

(Galf_1996).! While! arginine! is! considered! one! of! the! most! versatile! amino! acid! as! its! metabolism!provides!precursors!for!a!number!of!processes!such!as!the!biosynthesis!of! proteins,!nitric!oxide,!creatine!and!urea,!it!is!unclear!what!role!a!hot!dog!thioesterase! might!play!in!this!metabolic!pathway![41].!

! The!gene!neighborhood!surrounding!cgp_0542!reveals!to!be!localized!around!a! gene!cluster!for!the!synthesis!of!menaquinone,!or!vitamin!k.!Menaquinone!biosynthesis,!

! 68! known! as! the! men! pathway! is! a! 9Bstep! synthetic! route,! converting! chorismate! to! menaquinone.!The!cluster!is!loosely!localized!as!cgp_0533!is!an!OBsuccinylbenzoylBCoA! ligase! (MenE),! cgp_0548! is! a! naphthoate! synthase! (MenB),! cgp_0551! is! an! OB succinylbenzoylBCoA! synthase! (MenC)! and! cgp_0552! is! a! 2BsuccinylB5BenolpyruvylB6B hydroxyB3BcyclohexeneB1Bcarboxylate!synthase!(MenD).!Interestingly!enough,!the!men! pathway!has!been!shown!to!utilize!a!hot!dogBfold!thioesterase!in!the!hydrolysis!of!1,4B dihydroxyB2BnaphthoylBCoA!(DHNABCoA)!to!its!corresponding!fatty!acid![42].!However,! cgp_0542!did!not!exhibit!any!hydrolysis!activity!towards!DHNABCoA,!making!it!unlikely! to!function!in!this!role.!

! TTHA0967! appears! to! be! clustered! in! an! operon! for! phenylacetic! acid! (PAA)! degradation.! Its! gene! neighbors! include! PaaI:! phenylacetylBCoA! thioesterase!

(TTHA0965),! PaaK:! phenylacetylBCoA! ligase! (TTHA0966),! PaaDCBA:! ringB1,2B phenylacetylBCoA! epoxidase! (TTHA0969BTTHA0972)! and! PaaX:! TetR! transcriptional! regulator! (TTHA0973).! As! a! common! intermediate! in! the! breakdown! of! various! aromatic!compounds,!the!PAA!pathway!is!critical!pathway!in!the!utilization!of!aromatic! carbon!sources![39].!Interestingly!enough,!a!broader!look!at!the!available!gene!context! of!all!putative!flK!orthologs!shows!quite!a!few!instances!of!flK!orthologs!clustered!in! close!proximity!to!a!PAA!degradation!operon.!While!this!appears!to!be!fairly!conserved! throughout! DeinococcusBthermus! (the! phylum! containing! TTHA0967),! various! genes! throughout!Actinobacteria,!Firmicutes!and!Proteobacteria!share!a!similar!gene!context! as!well.!Like!TTHA0967,!PaaI!is!a!hotdogBfold!thioesterase!that!catalyzes!the!conversion! of!phenylacetylBCoA!(PABCoA)!to!phenylacetate!(PA).!While!TTHA0967!did!exhibit!PAB!

! 69! TE# guaB# guaA# guaA# PAICS# purE# purC# purS# purQ# purL#

purF# purM# GART# purH# purD#

purF purD GART purSQL (0922) (0926) (0924) (0919-0921) PRPP Ribosylamine-5-P GAR FGAR FGAM

purM (0923) purH purB purC PAICS (0925) (?) (0918) (0916) FAICAR AICAR SAICAR CAIR AIR

purO purE (?) (0917) guaB guaA 5-carboxyamino-1- (0909) (0910, 0915) (5-phospho-D- IMP XMP GMP ribosyl)-imidazole

AMPD hprT AMP

Hypoxanthine !

Figure(2D13.(Gene!context!within!A.,metalliredigens!(Firmicutes)!showing!flK!ortholog!

(red)!coBlocalization!with!a!gene!operon!for!de,novo,purine!biosynthesis!and!salvage.!

CoA!hydrolysis!activity,!it!was!not!on!a!biologically!relevant!scale,!indicating!its!function! elsewhere.!!

! A! look! at! the! BVU_1957! gene! neighborhood! shows! it! to! be! near! aconitase!

(BVU_1959),! isocitrate! dehydrogenase! (BVU_1960)! and! citrate! synthase! (BVU_1961).!

All! three! of! these! enzymes! play! critical! roles! in! the! citric! acid! cycle,! mediating! the! multistep!conversion!of!oxaloacetate!and!acetylBCoa! to!αBketoglutrate.!Given!the!high! formylBCoA!activity!and!narrow!substrate!range!exhibited!by!BVU_1957,!it!is!uncertain! if! this! gene! context! is! relevant.! Further! downfield! from! BVU_1957! is! a! dihydrofolate!

! 70! reductase!(BVU_1949)!and!thymidylate!synthase!(BVU_1950).!Both!genes!are!involved! in!one!carbon!metabolism!by!folate!(Figure!2B12).!!Folate!and!folate!derivatives!act!as! single!carbon!donors!and!play!critical!roles!in!various!cellular!processes!such!as!DNA! methylation!and!repair,!purine!synthesis!and!carbon!fixation![44].!A!wider!look!at!the! rest!of!the!available!flK!ortholog!gene!context!discovered!a!large!amount!of!orthologs! coBlocated! with! formateBtetrahydrofolate! ligase,! the! enzyme! responsible! for! the! conversion!of!formate!and!tetrahydrofolate!(THF)!to!N10BformylBTHF!in!the!one!carbon! pool.!Additionally,!a!number!of!orthologs!were!found!to!coBlocalize!with!genes!involved! in! de,novo! purine! synthesis! and! salvage! (Figure! 2B13).! Furthermore,! many! orthologs! were!found!in!close!proximity!to!other!genes!involved!in!carbon!fixation!or!one!carbon! metabolism!like!carbon!monoxide!dehydrogenase!and!malate!dehydrogenase.!

2.3.7(Divergence(of(Function(

( The!human!gut!microbiome!plays!a!large!role!in!overall!health!and!wellbeing,!as! disruption! of! gut! flora! has! been! shown! to! have! significant! effects! of! a! variety! of! intestinal! conditions! such! as! obesity,! malnutrition,! diabetes,! ulcerative! colitis! and!

Crohn’s! disease! [43,! 45].! Living! in! a! symbiotic! relationship! with! the! human! body,! organisms!in!the!gut!microbiome!can!essentially!be!broken!down!into!two!major!classes! based! on! their! function:! fermenters! and! hydrogenotrophs! [46,! 47].! The! role! of! fermenters!is!in!the!breakdown!of!undigested!dietary!components,!such!as!proteins!and! carbohydrates.!In!the!process,!a!number!of!fermentation!products!such!as!shortBchain! fatty!acids!(acetate,!propionate,!butyrate)!and!gases!(CO2!and!H2)!are!formed![46,!47].!

Responsible!for!the!removal!of!CO2!and!H2!from!the!intestinal!tract,!hydrogenotrophs!!

! 71! O

2e-, 2H+ CoAS H CO2 H2O AMP BVU-like TE O IMP

O H ATP GMP ADP, Pi, H2O FTHFS AICAR FAICAR Tetrahydrofolate (THF) N H Formyl-THF H H N N N H N 2 dehydrogenase R HN O N N -formyl-THF H 10 O HN R N N ,N -methenyl-THF N 5 10 CH3 CoASH H R H O - + CoIII 2 2e , 2H Formyl-THF Methyl- dehydrogenase transferase CO2 CO

CFeSP Methylene- CODH/ACS N tetrahydrofolate reductase H N H R CoI N N5,N10-methylen-THF O CH3 HN R H C SCoA (6S)-N5-methyl-THF 3 Acetyl-CoA (

Figure( 2D14.( Reaction! scheme! depicting! the! potential! involvement! of! BVUBlike! orthologs! in! the! one! carbon! pool! and! its! downfield! contributions! to! de, novo! purine! biosynthesis! and! salvage! (blue)! and! acetogenic! carbon! fixation! (WoodBLjundahl! pathway)! (red).! FTHFS:! formateBtetrahydrofolate! synthetase.! CODH/ACS:! carbon! monoxide/acetylBCoA!synthase!complex.!

(hydrogen!consumers)!are!capable!of!utilizing!these!gases!as!carbon!sources!through! the! process! of! carbon! fixation! [47].! Given! the! mostly! oxygenBlimiting! environment! of!

! 72! the! gut,! carbon! fixation! is! performed! under! anaerobic! conditions.! One! such! group! of! hydrogenotrophs! is! acetogenic! bacteria,! which! are! capable! of! producing! acetylBCoA! through!carbon!fixation![48].!Primarily!in!Clostridium,species,!acetogens!are!capable!of! utilizing!the!WoodBLjundahl!pathway!to!convert!CO2!and!H2!to!acetylBCoA!in!a!THF!and! cobalaminBdependent!reaction!(Figure!2B14).!In!short,!THF!is!converted!to!N10BformylB

THF!by!the!action!of!formateBtetrahydrofolate!ligase.!In!a!series!of!redox!reactions,!N10B formylBTHF!is!converted!to!(6S)BN5BmethylBTHF,!where!it!acts!as!a!methyl!group!donor.!

Utilizing! the! carbon! monoxide! dehydrogenase/acetylBCoA! synthase! (CODH/ACS)! complex,!the!methyl!group!is!transferred,!along!with!carbon!monoxide!to!coenzyme!A,! forming!acetylBCoA.!Given!the!apparent!role!of!BVU_1957!as!a!formylBCoA!hydrolase,!it! is!possible!that!this!version!of!the!flK!scaffold!(truncated!lid)!within!Clostridium,might! function!within!the!gut!microbiota!in!the!anaerobic!fixation!of!CO2!and!H2!by!providing! an! alternate! source! of! formate! for! conversion! to! N10BformylBTHF.! As! formylatedB derivatives! are! required! for! a! variety! of! other! cellular! processes! (like! de,novo,purine! synthesis! and! salvage),! it! is! possible! that! the! nonBacetogenic! orthologs! may! utilize! formylBCoA! hydrolase! activity! in! a! similar! manner,! providing! formate! for! the! one! carbon!pool!(Figure!2B14).!!

(

2.4(Summary!

( Given! the! highly! specific! role! of! the! flK! thioesterase! in! providing! resistance! to!

FAc!poisoning!in!the!fluorometaboliteBproducer,!S.,cattleya,!it!was!evident!that!the!flK! scaffold!was!highly!evolved,!doubtless!the!result!of!functional!divergence!within!the!HD! superfamily.!In!an!attempt!to!track!this!functional!divergence,!we!initiated!a!combined!

! 73! bioinformatics,!mechanistic!and!structural!analysis!to!uncover!novel!functionality!with! the!flK!subfamily.!

! While! the! fluorinase! cluster! was! found! to! be! extremely! limited! in! biological! range,!the!flK!scaffold!was!found!in!orthologs!through!many!different!phyla,!indicating! an! alternative! function! for! the! species! that! without! the! ability! to! synthesize! fluorometabolites.! Additionally,! the! flK! biological! range! was! similar! to! the! biological! range! of! the! gut! microbiome,! indicating! a! potentially! functionality! within! the! human! and! mammalian! digestive! tract.! Sequence! analyses! of! flK! orthologs! indicated! three! distinct!versions!of!the!helical!lid!structure,!supported!from!the!analysis!of!the!crystal! structures! from! flK,! BVU_1957! and! TTHA0967.! The! alterations! in! the! helical! lid,! resulting!in!distinct!reshaping!of!the!TE!active!site,!was!found!to!play!a!role!in!substrate! specificity!and!activity,!as!BVU_1957!was!highly!active!and!specific!for!formylBCoA.!The! truncated!lid!(BVUBlike)!structure!was!found!to!be!the!most!prevalent!amongst!the!flK! orthologs,!as!all!of!the!Bacteroidetes!and!the!majority!of!Firmicutes!and!other!species! conserved! this! motif,! and! indicated! that! the! majority! of! flK! orthologs! function! more! closely!to!BVU_1957!and!flK.!!

! With!additional!support!from!gene!context!analyses,!a!novel!divergent!function! was!proposed!for!BVU_1957!(and!the!“BVUBlike”!orthologs)!in!providing!formate!for!the! one!carbon!pool.!Additionally,!given!the!prevalence!of!flK!orthologs!coBlocalized!with! the! PAA! degradation! pathway! (especially! in! DeinococcusBThermus),! and! taking! into! account,!the!lowBlevel!PABCoA!hydrolase!activity!seen!in!the!TTHA0967Blike!scaffold,!we! have!proposed!a!potential!evolutionary!path!for!the!flK!subfamily.!!

! 74! ! The!original!flK!thioesterase!(TTHA0967Blike)!may!have!evolved!from!the!PaaI! thioesterase!as!a!result!of!gene!duplication!events.!Through!lateral!gene!transfer,!the! entire! PAA! operon! (TTHA0967Blike! ortholog! included)! was! acquired! by! organisms! within! Proteobacteria,! Firmicutes! and! Actinobacteria.! Over! time,! further! structural! divergence!led!to!the!evolution!of!the!BVUBlike!ortholog!(truncated!lid)!and!the!flKBlike! ortholog!(full!lid).!The!flK!ortholog!was!utilized!for!the!role!of!FAc!detoxification!in!S.! cattleya,! other! fluoroacetate! producers! and! possibly! in! other! bacteria! requiring! this! resistance!pathway.!Alternatively,!the!BVUBlike!thioesterase!evolved!the!role!of!formylB

CoA!thioesterase!within!the!gut!microbiome!and!was!traditionally!evolved!throughout! its!coBhabiting!species.!!

(

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! 83! Chapter(3(

Characterizing(the(Contribution(of(Multiple(Acyl7CoA(Synthetases(Towards(

Enhanced(Virulence(in(Pseudomonas*aeruginosa!

(

3.1(Introduction(

! Cystic! fibrosis,! also! known! as! mucoviscidosis,! is! an! autosomal! recessive! genetic!disorder!primarily!affecting!the!respiratory!and!digestive!systems![1].!The! most! common! lethal! genetic! disease! in! Caucasian! populations,! cystic! fibrosis! (CF)! affects!mainly!children!and!young!adults,!with!more!than!75%!of!people!diagnosed! at!age!two!or!younger![1,!2].!With!an!estimated!30,000!children!and!adults!affected! in!the!United!States!(70,000!worldwide)!and!about!1,000!new!diagnoses!each!year,!

CF!is!characterized!by!mutations!in!the!cystic!fibrosis!transmembrane!conductance! regulator!(CFTR),!a!membraneObound!protein!expressed!in!epithelial!and!blood!cells!

[1O3].!An!ABCOclass!ion!transporter,!CFTR!is!an!ATPOgated!anion!channel!responsible! for! the! soluble! transport! of! chloride,! thiocyanate! and! bicarbonate! ions! across! the! cell!membrane![2].!This!transport!helps!drive!the!osmotic!efflux!of!water!out!of!the! cell!into!the!surrounding!mucus,!maintaining!its!proper!fluidity!to!effectively!protect! organs!from!invading!bacteria!and!foreign!particles.!While!two!copies!of!the!CFTR! gene! are! carried! (one! from! each! parent),! only! one! functioning! gene! is! needed! for! proper! ion! transport.! In! patients! with! CF,! mutationOderived! dysfunction! occurs! in! both!CFTR!genes,!resulting!in!a!total!loss!of!ion!transport![4].!While!over!1,800!CFTR! mutations!have!been!documented,!only!a!few!occur!at!a!frequency!greater!that!0.1%!!

! 84! I" II" III"

Figure( 371.( Comparison! of! wildOtype! (I)! and! mutant! (II/III)! CFTR.! CFOcausing! mutations!in!the!CFTR!gene!can!result!in!dysfunctional!protein!(II)!or!cause!CFTR! breakdown! before! implantation! into! the! plasma! membrane! (III).! Both! types! of! mutation! inhibit! ion! transport! across! the! membrane! and! lead! to! accumulation! of! thick,!dry!mucus.!!(

[5].!The!most!common!mutation!in!patients!with!CF!is!ΔF508,!a!single!amino!acid! deletion!of!phenylalanine!occurring!at!position!508.!Occurring!in!over!70%!of!the!CF! community,!ΔF508!leads!to!CFTR!misfolding!and!results!in!break!down!shortly!after! translation!and!ultimately!failure!to!reach!the!cell!membrane![6].!The!consequence! of! CFOcausing! mutations! in! CFTR! genes! is! an! accumulation! of! thick,! dehydrated! mucus! (sputum)! that! blocks! airways! and! hinders! breathing! [1].! Additionally,! the! warm,!nutrientOrich!environment!of!CF!sputum!leads!to!a!veritable!breeding!ground! for! bacteria! adapted! to! survive! under! such! conditions.! Furthermore,! decreased!

! 85! levels! of! periciliary! fluid! preventing! the! mucociliary! clearance! of! said! bacteria! results! in! the! recruitment! of! inflammatory! mediators,! leading! to! inflammation! on! top!of!chronic!bacterial!infection!and!ultimately,!irreparable!decline!in!pulmonary! function![7].!Although!the!life!expectancy!of!CF!patients!has!risen!dramatically!in!the! past! 30! years! (from! age! 7! to! ~40),! thanks! to! an! enhanced! understanding! of! its! underlying! genetic! causes! and! treatment,! most! patients! eventually! succumb! to! bacterial! airway! infection! after! a! shortened! lifetime! spent! in! hospitals! and! on! aggressive!antibiotic!regimens![7].!

One! of! the! hallmarks! of! cystic! fibrosis! is! the! complex! microbiome! that! inhabits!the!lung!sputum.!This!unique!environment!is!comprised!of!a!wide!range!of! microbes!and!is!in!constant!flux!throughout!the!life!of!the!patient.!In!some!adults! with! CF,! up! to! 37! different! phylotypes! have! been! discovered,! including! several! members! from! Bacillales,! Bacteroidales,! Burkholderiales,! Chlostridiales,!

Lactobacillales,! Methanosarcinales,! Pseudomonadales! and! Xanthomonadales! [2].!!

The! most! prevalent! species! of! bacteria! include! Staphylococcus, aureus,, the!

Burkholderia, cepacia, complex,! Haemophilus, influenzae,, Mycobacterium, sp., and!

Pseudomonas,aeruginosa,[8].!During!infancy!and!early!childhood,!CF!patients!suffer! predominantly! from! S.,aureus,infections! that! damage! epithelial! surfaces! and! pave! the!way!for!dominating!pathogens!such!as!P.,aeruginosa![8].!!

P.,aeruginosa,belongs! to! the! genus! Pseudomonas,! whose! various! groupings! include! nonOpathogenic! species! (e.g.! P., putida,! P., denitrificans,! P., knackmussii)! as! well!as!a!variety!of!other!animal!(P.,oryzihabitans),!plant!(P.,syringae)!and!fish!(P., plecoglossicida)!pathogens.!Found!in!soil,!water,!on!plants!and!in!the!human!gut!and!

! 86! respiratory!system,!P.,aeruginosa,leads!somewhat!of!a!dual!life.!On!the!one!hand,!it! acts!as!a!freeOliving!bacterium,!capable!of!surviving!and!thriving!on!its!own.!More! notably,! however,! is! its! role! as! an! aggressive! opportunistic! pathogen,! infecting! immunoOcompromised! hosts! [9].! Found! on! hospital! equipment! and! implanted! devices,! such! as! catheters,! P., aeruginosa, is! responsible! for! a! myriad! of! deadly! infections,!ranging!from!urinary!tract!infections!(UTIs)!to!sepsis!in!cancer!patients,! and! is! one! of! the! most! common! infections! found! in! patients! with! nosocomial! pneumonia! [10,! 11].! In! CF! airways,! P., aeruginosa! infections! are! intermittent! throughout!childhood!and!early!adolescence.!However,!by!early!adulthood,!chronic! infection! is! established! and! P.,aeruginosa,quickly! becomes! the! dominant! infecting! species!in!the!CF!airway![3].!The!underlying!mechanism!of!this!success!is!the!ability! to! rapidly! adapt! to! a! constantly! fluctuating! environment,! and! despite! aggressive! antibiotic! treatment,! P., aeruginosa, represents! the! leading! cause! of! morbidity! and! mortality!in!adult!CF!patients![12].!

The! key! for! establishing! chronic! infection! and! thriving! in! a! pathogenic! environment!is!the!ability!to!effectively!utilize!host!and!coOhabitantOderived!energy! sources!while!successfully!fighting!off!the!constant!barrage!of!antimicrobial!agents! released! by! both! parties.! To! theses! ends,! P., aeruginosa, employs! an! impressive! arsenal!of!virulence!factors,!signaling!molecules,!xenobiotic!efflux!pumps!and!other! various! chemical! warfare! agents! to! exploit! both! host! and! coOhabitant! alike.!

Additionally,!P.,aeruginosa!is!armed!with!an!extensive!metabolic!suite,!allowing!it! utilize! a! wide! variety! of! carbon! sources! from! many! types! of! environmental! conditions.! Perhaps! the! most! important! concept! in! P., aeruginosa! pathogenesis! is!

! 87! extensive!biofilm!formation.!Biofilms!are!composed!of!a!complex!polymer!matrix!of! polysaccharide,! DNA,! protein! and! other! macromolecules! [13].! Housed! within! are! populations! of! bacteria! that! form! large,! fully! networked! communities.! These! communities!have!significant!survival!advantages!over!single!colonies!in!that!they! are!able!to!communicate!by!quorum!sensing,!a!process!of!emitting!small!molecule! signals! in! response! to! fluctuating! environmental! conditions.! This! system! of! communication! allows! the! organized! population! to! perform! groupOwide! actions! such! as! extending! or! limiting! biofilm! production,! inducing! the! transcription! of! virulence! factors! and! utilizing! communal! energy! sources.! In! P., aeruginosa,! the! primary! polysaccharide! component! of! biolfilm! is! alginate.! Over! the! course! of! intermittent! infection,! the! bacterium! has! been! shown! to! deregulate! enzymes! directly! involved! in! alginate! production! (such! as! mucA! and! algD)! to! undergo! conversion! to! a! mucoid! phenotype! [7].! This! overproduction! of! alginate! leads! to! a! thicker,! stickier! biofilm! which! renders! antibiotic! penetration! much! more! difficult! and! also! increases! resistance! to! phagocytosis! by! host! macrophages! [7,! 14].!

Additionally,! this! enhanced! biofilm! structure! prevents! clearance! of! the! bacterium! from!host!airways!and!generally!marks!the!establishment!of!chronic,infection.!!

In! P., aeruginosa,! the! quorum! sensing! process! is! governed! by! three! interconnected! regulatory! systems.! When! responding! to! environmental! fluctuations,! affected! members! of! the! (biofilmOcontained)! bacterial! community! release! autoinducer! molecules,! which! at! high! enough! concentration,! initiate! communityOwide!transcription!of!genes!involved!in!adaptation!and!virulence!factor! production! [15].! ! The! las, and! Rhl, systems! are! both! twoOcomponent! (LuxI/LuxRO

! 88! type)! regulatory! systems! consisting! of! an! autoinducer! synthase! (LuxIOlike)! and! a! cognate! transcriptional! activator! (LuxROlike).! Both! are! mediated! by! an! NO acylhomoserine! lactone! (HSL)! autoinducer,! specifically! NO3OoxoOdodecanoyl! homoserine! lactone! (3OC12OHSL)! and! NObutyryl! homoserine! lactone! (C4OHSL),! respectively! [16,! 17].! The! third! regulatory! system,! known! as! the! pqs,system,! is! a! biosynthetic!pathway!for!the!production!of!4OheptylO3OhydroxyO4Oquinolone!(PQS).!

An!autoinducer!for!the!pqs,system,!PQS!is!a!member!of!the!4Oquinolone!class!and! has! been! shown! to! enhance! biofilm! formation! through! changes! in! swarming! motility,!inhibition!of!host!TOcell!proliferation!and!production!of!antibacterial!agents!

[18O20].! The! carefully! coordinated! regulatory! cascade! of! the! lasCRhlCpqs, system!

(Figure!3O2)!is!thought!to!control!up!to!11%!of!the!P.,aeruginosa,genome!and!not! only!serves!as!a!form!of!interOregulation!but!also!allows!for!the!redundant!release!of! important! virulence! factors! such! as! elastase,! pyocyanin,! siderophores,! lipopolysaccharides,! rhamnolipids! and! various! proteases! [21O23].! These! virulence! factors!play!a!large!role!in!P.,aeruginosa,pathogenesis!and!are!directly!linked!to!its! survival.!For!example,!P.,aeruginosa,has!been!found!to!modify!the!OOantigen!or!lipid!

A! components! of! lipopolysaccharides! (LPS)! to! either! avoid! immune! detection! or! trigger! inflammation,! cytokine! release! and! toxic! shock,! depending! on! its! environment! [15].! The! nonOribosomal! peptide! synthase! (NRPS)Oproduced! siderophores!pyochelin!and!pyoverdine!allow!P.,aeruginosa,to!survive!under!ironO depleted! conditions! by! chelating! and! transporting! hostOderived! iron! into! the! cell.!

Furthermore,! pyocyanin! is! thought! to! be! able! to! scavenge! oxygen! from! biofilm! boundaries!during!anaerobic!conditions!in!addition!to!inducing!apoptosis!in!host!

! 89! LasR + 3OC12-HSL + LasI 3OC12-HSL Virulence I" Factors

RhlR + + C4-HSL + + RhlI II" C4-HSL - + pqsR PQS + + - + pqsA pqsB pqsC pqsD pqsH III" PQS !

Figure(372.!Quorum!signaling!regulatory!cascade!in!P.,aeruginosa!consisting!of!the! las, (I),! Rhl, (II)! and! pqs, (III), systems.! Black! arrows! denote! autoinducer! synthesis!

(straight)! and! binding! (curved).! Transcriptional! activation! is! denoted! by! blue! arrows!(+)!and!repression!is!denoted!by!red!arrows!(O).!

! and!coOhabitant!cells![15,!24O26].!Additionally,!rhamnolipids!facilitate!in!the!uptake! and! release! of! hydrophobic! compounds,! help! dictate! biofilm! morphology,! inhibit! macrophage!function!and!have!been!shown!to!possess!antimicrobial!activity![27].!!

! Biofilm!formation!in!the!CF!lung!relies!on!highOdensity!cell!(HDC)!replication!

[28].!Like!all!cellular!growth!processes,!HDC!replication!is!dependent!on!a!readily! available!source!of!energy.!In!CF!sputum!as!well!as!in!extensive!biofilm!formations,!

! 90! growth! conditions! are! completely! heterogeneous! and! can! range! from! aerobic! to! oxygenOlimited! to! completely! anaerobic! [29,! 30].! This! steep! oxygen! gradient! provides!a!constantly!changing!environment!that!demands!a!versatile!metabolism! for!energy!acquisition!and!survival.!P.,aeruginosa!is!known!mainly!as!a!facultative! aerobe,! preferring! to! utilize! any! one! of! five! terminal! oxidases! (varying! in! oxygen! affinity,! energy! coupling! and! stress! tolerance)! to! catalyze! the! conversion! of! molecular!oxygen!to!water![31].!In!response!to!constantly!changing!oxygen!levels! during!pathogenesis,!P.,aeruginosa,is!able!to!utilize!nitrate!and!nitrite!as!terminal! electron! acceptors! (denitrification)! when! oxygen! is! limited! [31].! Additionally,! phenazines! like! pyocyanin! can! also! act! as! electron! acceptors! [32].! Under! strictly! anaerobic!conditions,!P.,aeruginosa!has!been!shown!to!utilize!arginine!and!pyruvate! fermentation!pathways!to!produce!ATP,!genes!of!which!are!linked!to!virulence,!longO term!survival!and!biofilm!formation![33O35].!!

Variable! growth! conditions! inevitably! lead! to! a! broad! range! of! energy! sources! and! relative! abundances.! Under! nutrientOpoor! conditions,! the! catabolite! repression!control!(CRC)!system!is!responsible!for!tuning!P.,aeruginosa,metabolism! and!optimizing!nutrient!utilization!in,vivo.!A!signaling!cascade!under!the!control!of! the! Crc, gene,! the! CRC! system! is! intricately! linked! to! P., aeruginosa, pathogenesis,! affecting! over! 360! separate! genes! involved! in! carbohydrate! metabolism,! biofilm! formation,! antibiotic! resistance! and! virulence! [36,! 37].! The! CRC! system! also! regulates! amino! acid! import! and! utilization! with! research! showing! amino! acid! degradation! genes,! proteases! and! peptide! transporters! to! be! upregulated! in! CF! growth!conditions![28].!This!indicates!hostOderived!amino!acids!to!be!an!important!

! 91! energy! source! in, vivo., Another! valuable! source! of! hostOderived! energy! is! lung! surfactant,! composed! primarily! of! dipalmitoylphosphatidylcholine! (DPPC).! P., aeruginosa,contains!lipases!and!phospholipase!C!to!cleave!DPPC!into!its!constituent! components:! palmitate! (x2),! glycerol! and! phosphocholineOOall! of! which! can! be! metabolized! for! energy! [38].! In! fact,! genes! involved! in! longOchain! fatty! acid! activation! and! βOoxidation! are! upregulated! in! CF! models! and! mutant! strains! in! mouse! lungs! displayed! decreased! production! of! virulence! factors! and! retarded! growth! [28,! 39].! This! points! to! a! potentially! significant! role! of! hostOderived! long! chain!fatty!acids!toward!P.,aeruginosa,virulence!and!energy!acquisition.!!

In! addition! to! utilizing! hostOderived! energy! sources,! P., aeruginosa, may! be! able! to! utilize! small! molecule! carbon! sources! excreted! by! coOhabiting! bacterial! species.! In! the! mostly! anaerobic! recesses! of! the! CF! lung,! coOhabiting! bacteria! utilizing! a! variety! of! fermentation! pathways! release! a! myriad! of! small! molecule! fermentation!products!including!shortOchain!fatty!acids,!ethanol!and!CO2![47].!One! such! product,! 2,3Obutanedione,! has! been! shown! to! be! present! in! nearly! all! CF! respiratory! tracts! [44].! Additionally,! studies! have! shown! that! utilization! of! exogenous!2,3Obutanedione!by!P.,aeruginosa,leads!to!increased!biofilm!density!and! virulence!factor!production,!enhanced!levels!of!pyocyanin,!HSL!and!other!quorum! sensing!signals,!and!increased!antimicrobial!activity![24].!With!the!ability!to!exploit! coOhabiting!bacteria!by!feeding!off!of!their!excreted!fermentation!products!and!then! instigating! an! immune! response! and! engaging! in! chemical! warfare! to! become! the! dominant!pathogenic!species,!it!comes!as!no!shock!that!over!a!patient’s!lifetime,!the!

! 92! diversity! of! the! CF! microbiome! dramatically! decreases,! with! P., aeruginosa, consistently!found!as!the!main!pathogenic!species!in!adults![24].!!

, Perhaps! the! main! reason! why! P.,aeruginosa,is! such! a! capable! and! virulent! pathogen! is! the! size! of! its! genome.! At! roughly! 6.3! million! base! pairs! (Mbp),! this! unusually!large!genome!far!exceeds!the!size!of!many!other!notable!pathogens!such! as!E.,coli!(4.7!Mbp),!S.,aureus,(2.8!Mbp),!H.,influenzae!(1.8!Mbp)!and!M.,tuberculosis!!

(4.4! Mbp)! [42,! 43].! Analysis! of! the! P., aeruginosa, genome! reveals! a! much! larger! number! of! distinct! gene! clusters,! implying! that! its! size! is! not! due! to! genomic! duplication!but!rather!due!to!adapted!evolution!for!increased!genomic!complexity.!

In! fact,! with! over! 5,000! open! reading! frames! (ORFs),! the! genetic! complexity! of! P., aeruginosa,is!thought!to!more!closely!resemble!that!of!the!simple!eukaryotic!yeast!S., cerevisae, than! other! actual! bacteria! [43].! With! increased! numbers! of! regulatory! genes,!nutrientOuptake!transporters,!xenobiotic!efflux!pumps!and!secretory!systems,!

P., aeruginosa, appears! to! be! evolutionarily! adapted! to! survive! and! thrive! in! a! number!of!niche!environments.!Of!particular!interest!are!the!large!numbers!of!genes! associated! βOoxidation! and! other! metabolic! pathways! and! indicates! a! potential! ability!to!utilize!a!wideOrange!of!organic!substrates!as!carbon!sources.!,

As!discussed!in!chapter!1,!AcylOCoA!synthetases!are!the!enzymes!responsible! for!the!activation!of!carboxylic!acids!into!their!respective!acylOCoA!substrates.!Best! known! for! their! role! in! longOchain! fatty! acid! activation! in! βOoxidation,! acylOCoA! synthetases!(ligases)!are!critical!components!are!cellular!metabolism!throughout!all! domains! of! life.! In! addition! to! longOchain! fatty! acylOCoA! ligases,! prokaryotic! organisms!(which!grow!under!a!variety!of!conditions)!are!known!to!employ!short!

! 93! and!mediumOchain!as!well!as!various!aromatic!ligases![62,!63].!Given!the!enhanced! size! of! the! P., aeruginosa, genome,! it! is! not! totally! surprising! to! discover! a! large! number!of!standalone!acylOCoA!ligases.!Given!the!importance!of!acylOCoA!ligases!in! energy! utilization,! secondary! metabolite! production! and! virulence! factor! production,! we! hypothesize! that! the! increased! number! of! standalone! ligases! in! P., aeruginosa,contributes!to!its!enhanced!virulence!in!the!CF!lung!environment.!In!this! chapter!we!explore!the!potential!activation!of!host!and!coOhabitantOderived!organic! fatty! acids! by! the! multiple! standalone! acylOCoA! synthetases! expressed! by! P., aeruginosa.! The! work! within! defines! the! parameters! and! results! for! a! novel! highO throughput! substrate! screening! coupled! with! steadyOstate! kinetic! verification! of! activity.!Along!with!inOdepth!bioinformatic!analyses,!the!results!and!the!conclusions! we!draw!from!them!allow!us!to!gain!a!better!understanding!of!the!potential!energy! acquisition! pathways! employed! by! P., aeruginosa, during! pathogenesis! in! the! CF! airway.!!

3.2(Methods(and(Materials(

3.2.1(Materials*

! All!restriction!enzymes,!T4!DNA!ligase,!and!Deep!Vent!DNA!polymerase!were! purchased!from!NEB.!Pfu!Turbo!DNA!polymerase!was!purchased!from!Agilent!and! all!custom!oligonucleotide!primers!were!synthesized!by!Invitrogen.!Genomic!DNA! was!purchased!from!ATCC.!Protein!samples!were!purified!on!an!ÄKTA!FPLC!system!

(GE! Healthcare)! by! monitoring! UV! absorbance! at! 280! nm.! Protein! concentrations! were!determined!using!the!Bradford!method.!Various!carboxylate!substrates!and!all!

! 94! other!chemicals!were!purchased!from!Sigma!or!Fisher!unless!otherwise!specified.!

Mass!spectrometry!analysis!was!performed!by!the!Mass!Spectrometry!Facility!at!the!

University!of!New!Mexico.!!

3.2.2(Bioinformatics(Analysis(

Standalone! ligases! in! P., aeruginosa, were! identified! using! the! SupFam! database! (http://supfam.cs.bris.ac.uk/SUPERFAMILY/index.html).! BLAST! searches! of! individual! standalone! ligases! were! conducted! throughout! all! of! the! sequenced! genomes!deposited!in!the!NCBI!database!using!the!Standard!Protein!BLAST!server!

(http://blast.ncbi.nlm.nih.gov/Blast.cgi?PROGRAM=blastp&PAGE_TYPE=BlastSearc! h&LINK_LOC=blasthome).! Protein! sequences! were! aligned! using! Clustal! Omega!

(http://www.ebi.ac.uk/Tools/msa/clustalo/)! and! visualized! in! CLC! Sequence!

Viewer! 7.! Each! ligase! was! selected! as! the! query! sequence! using! the! default! parameters.! Additionally,! the! search! parameters! were! set! to! filter! regions! of! low! complexity! and! select! for! the! top! 5,000! hits.! Each! ligase! was! subjected! to! three! separate! BLAST! searches:! (1)! within! Pseudomonas! (excluding! Pseudomonas, aeruginosa),! (2)! within! Proteobacteria, (excluding! Pseudomonas)! and! (3)! within!

Bacteria! (excluding! Proteobacteria).! To! distinguish! between! orthologs! of! each! ligase,! pairwise! sequence! identities! were! calculated! for! each! one! and! a! sequence! identity!cutoff!was!selected!based!on!the!results.!Putative!orthologs!were!identified! by!those!sequences!containing!≥50%!sequence!identity!for!≥80%!coverage!with!the! exception!of!PA0996,!PA1617!and!PA2893!where!the!sequence!identity!cutoff!was! lowered!to!≥40%!sequence!identity!for!≥80%!coverage.!Gene!neighborhood!analysis!

! 95! was!performed!by!looking!at!available!gene!context!data!in!the!NCBI!Gene!database!

(http://www.ncbi.nlm.nih.gov/gene/)!and!the!KEGG!Sequence!Similarity!DataBase!

(http://www.kegg.jp/kegg/ssdb/).!

(

3.2.3(Cloning,(Expression,(and(Purification(of(P.*aeruginosa(Ligases(

! The! gene! encoding! PA0887! was! amplified! by! PCR! using! Pseudomonas, aeruginosa! PAO1OLAC! genomic! DNA! (ATCC! 47085DO5),! custom! oligonucleotide! primers!and!Pfu!Turbo!DNA!polymerase.!The!gene!product!was!digested!with!NdeI! and!XhoI!restriction!endonucleases!and!ligated!into!pETO23a(+)!expression!vector! using!T4!DNA!Ligase.!The!resulting!ligation!product!was!then!used!to!transform!E., coli!BL21(DE3)!pLysE!chemically!competent!cells!and!the!transformation!was!plated! on! LB! media! containing! ampicillin! (100! μg/mL).! A! single! colony! containing! overexpressed! protein! was! used! to! inoculate! 2! L! TB! media! and! incubated! at! 37!

°C/200! RPM! until! reaching! an! OD600! of! approximately! 0.6.! Cells! were! induced! by! addition! of! IPTG! (0.4! mM)! and! allowed! to! incubate! overnight! at! 18! °C/200! RPM.!

Cells!were!harvested!by!centrifugation!at!4!°C/6500!RPM;!cell!paste!was!collected! and! resuspended! in! iceOcold! lysis! buffer! (50! mM! HEPES,! 200! mM! NaCl,! 50! mM! imidazole,!pH!7.5).!Cells!were!lysed!by!passage!through!a!French!press!at!1200!PSI! and! then! centrifuged! at! 4! °C/20,000! RPM.! The! collected! supernatant! was! then! loaded! onto! a! 5! mL! HisTrap! FF! column! (GE! Healthcare)! and! washed! with! lysis! buffer.!Pure!protein!was!eluted!off!the!column!with!elution!buffer!(50!mM!HEPES,!

200! mM! NaCl,! 500! mM! imidazole,! pH! 7.5)! and! fractions! containing! pure! protein! were!collected,!pooled!and!dialyzed!against!three!changes!of!dialysis!buffer!(50!mM!

! 96! HEPES,!200!mM!NaCl,!pH!7.5).!Purity!was!verified!by!SDSOPAGE.!Yield:!4.5!mg/g!wet! cell!paste.!

! The!gene!encoding!PA0996!was!cloned,!expressed!and!purified!as!described! above! with! some! modification! to! the! procedure.! The! PCROamplified! gene! product! was!digested!with!NdeI!and!BamHI!restriction!endonucleases!and!ligated!into!pETO

28a(+)! expression! vector.! The! ligation! product! was! used! to! transform! E., coli, T7!

Express! Iq! chemically! competent! cells! and! the! transformation! was! plated! on! LB! media!containing!kanamycin!(40!μg/mL).!A!single!colony!containing!overexpressed! protein!was!used!to!inoculate!1!L!TB!media!and!incubated!at!37!°C/200!RPM!until! reaching!an!OD600!of!approximately!0.8.!Yield:!5.5!mg/g!wet!cell!paste.!

! The!gene!encoding!PA1997!was!cloned,!expressed,!and!purified!as!described! above! with! some! modification! to! the! procedure.! The! PCROamplified! gene! product! was!digested!with!NdeI!and!EcoRI!restriction!endonucleases!and!ligated!into!pETO

28a(+)!expression!vector.!The!resulting!ligation!product!was!then!used!to!transform!

E., coli! BL21(DE3)! pLysE! chemically! competent! cells! and! the! transformation! was! plated!on!LB!media!containing!kanamycin!(40!μg/mL).!A!single!colony!containing! overexpressed! protein! was! used! to! inoculate! 1! L! TB! media! and! incubated! at! 37!

°C/200!RPM!until!reaching!an!OD600!of!approximately!0.8.!Yield:!10.4!mg/g!wet!cell! paste.!

! The!gene!encoding!PA2555!was!cloned,!expressed,!and!purified!as!described! above! with! some! modification! to! the! procedure.! The! PCROamplified! gene! product! was!digested!with!NdeI!and!HindIII!restriction!endonucleases!and!ligated!into!pETO

28a(+)! expression! vector.! The! ligation! product! was! used! to! transform! E., coli, T7!

! 97! Express! Iq! chemically! competent! cells! and! the! transformation! was! plated! on! LB! media!containing!kanamycin!(40!μg/mL).!A!single!colony!containing!overexpressed! protein!was!used!to!inoculate!1!L!TB!media!and!incubated!at!37!°C/200!RPM!until! reaching!an!OD600!of!approximately!0.8.!Pure!protein!was!eluted!off!the!column!with! elution! buffer! (50! mM! HEPES,! 200! mM! NaCl,! 500! mM! imidazole)! and! fractions! containing!pure!protein!were!collected!and!pooled.!Protein!was!dialyzed!by!loading! onto!a!HiPrep!16/60!Sephacryl!SO200!HR!gel!filtration!column!(GE!Healthcare)!and! washing!with!dialysis!buffer!(50!mM!HEPES,!200!mM!NaCl).!Yield:!7.3!mg/g!wet!cell! paste.!!

! The!gene!encoding!PA2557!was!cloned,!expressed,!and!purified!as!described! above! with! some! modification! to! the! procedure.! Cells! were! harvested! by! centrifugation! at! 4! °C/6500! RPM! and! the! resulting! cell! paste! was! collected! and! resuspended!in!ice!cold!lysis!buffer!(50!mM!BisOTris!Propane,!200!mM!NaCl,!50!mM! imidazole,!pH!7.0).!Pure!protein!was!eluted!off!the!column!with!elution!buffer!(50! mM! BisOTris! Propane,! 200! mM! NaCl,! 500! mM! imidazole,! pH! 7.0)! and! fractions! containing!pure!protein!were!collected,!pooled,!and!dialyzed!against!three!changes! of!dialysis!buffer!(50!mM!BisOTris!Propane,!200!mM!NaCl,!pH!7.0).!Yield:!!11!mg/g! wet!cell!paste!

! The!gene!encoding!PA3568!was!cloned,!expressed,!and!purified!as!described! above!with!some!modifications!to!the!procedure.!!The!PCROamplified!gene!product! was!digested!with!NdeI!and!BamHI!restriction!endonucleases!and!ligated!into!pETO

28a(+)! expression! vector.! The! ligation! product! was! used! to! transform! E., coli, T7!

Express! Iq! chemically! competent! cells! and! the! transformation! was! plated! on! LB!

! 98! media!containing!kanamycin!(40!μg/mL).!A!single!colony!containing!overexpressed! protein!was!then!used!to!inoculate!1!L!TB!media!and!incubated!at!37!°C/200!RPM! until!reaching!an!OD600!of!approximately!0.8.!After!dialysis,!pure!protein!was!loaded! onto!a!HiPrep!16/60!Sephacryl!SO200!HR!gel!filtration!column!(GE!Healthcare)!and! washed! with! dialysis! buffer! (50! mM! HEPES,! 200! mM! NaCl)! to! remove! impurities.!

Yield:!2.9!mg/g!wet!cell!paste.!

! The!gene!encoding!PA3860!was!cloned,!expressed,!and!purified!as!described! above!with!some!modifications!to!the!procedure.!The!PCROamplified!gene!product! was! digested! with! NdeI! and! XhoI! restriction! endonucleases! and! ligated! into! pETO

28a(+)!expression!vector.!The!ligation!product!was!used!to!transform!E.,coli!BL21O

ArcticExpress!chemically!competent!cells!and!the!transformation!was!plated!on!LB! media!containing!kanamycin!(40!μg/mL).!A!single!colony!containing!overexpressed! protein!was!then!used!to!inoculate!1!L!TB!media!and!incubated!at!37!°C/200!RPM! until! reaching! an! OD600! of! approximately! 0.8.! Cells! were! then! induced! by! the! addition! of! IPTG! (0.4! mM)! and! allowed! to! incubate! overnight! at! 12! °C/200! RPM.!

Cells! were! harvested! by! centrifugation! at! 4! °C/6500! RPM! and! the! resulting! cell! paste!was!collected!and!resuspended!in!70!mL!iceOcold!lysis!buffer!(50!mM!HEPES,!

200! mM! NaCl,! 50! mM! imidazole,! pH! 7.5)! containing! 500! μL! protease! inhibitor! cocktail!P8849!(Sigma!072M4052).!The!collected!supernatant!was!loaded!onto!a!1! mL!HisTrap!FF!column!(GE!Healthcare).!Yield:!1.6!mg/g!wet!cell!paste.!

! The!gene!encoding!PA3924!was!cloned,!expressed,!and!purified!as!described! above! with! some! modifications! to! the! procedure.! The! PCROamplified! product! was! digested!with!NdeI!and!XhoI!restriction!endonucleases!and!ligated!into!pETO28a(+)!

! 99! expression!vector.!The!ligation!product!was!then!used!to!transform!T7!Express!Iq! competent!cells.!A!culture!containing!overexpressed!protein!was!used!to!inoculate!1!

L!TB!media!containing!kanamycin!(50!mg/mL)!at!37!°C/200!RPM!until!reaching!an!

OD600!of!approximately!1.0.!Yield:!4.6!mg/g!wet!cell!paste.!

! The!gene!encoding!PA4198!was!cloned,!expressed,!and!purified!as!described! above! with! some! modifications! to! the! procedure.! The! PCROamplified! product! was! digested!with!NdeI!and!XhoI!restriction!endonucleases!and!ligated!into!pETO28a(+)! expression!vector.!The!ligation!product!was!then!used!to!transform!T7!Express!Iq! competent!cells.!A!culture!containing!overexpressed!protein!was!used!to!inoculate!1!

L!TB!media!containing!kanamycin!(50!mg/mL)!at!37!°C/200!RPM!until!reaching!an!

OD600!of!approximately!1.0.!Yield:!1.4!mg/g!wet!cell!paste.!

(

3.2.4(High7throughput(Substrate(Screening(

HighOthroughput! substrate! screening! was! performed! using! the! DTNB! method.! Reactions! were! carried! out! in! a! 96Owell! plate! and! monitored! at! 412! nm! using!a!Molecular!Devices!SpectraMax!i3!multiplate!reader.!Each!reaction!contained! buffer!consisting!of!50!mM!HEPES,!5!mM!MgCl2,!2!mM!ATP,!750!μM!CoASH,!and!10!U! commercial! pyrophosphatase.! Individual! substrates! were! added! to! achieve! a! final! concentration!of!500!μM.!Reactions!were!initiated!by!the!addition!of!ligase!(5!μM! final)!or!H2O!in!the!sample!and!control!reactions,!respectively.!After!incubation!at!

25! °C! for! 30! m,! reactions! were! quenched! by! the! addition! of! 2! mM! DTNB.! The! decrease! in! absorbance! attributed! to! the! formation! of! an! acylOCoA! product! was! measured! by! subtracting! each! sample! absorbance! from! its! corresponding! control!

! 100! absorbance.! Activity! was! measured! as! the! percent! change! of! the! sample! from! the! control!absorbance!and!all!reactions!were!carried!out!in!duplicate.!The!total!relative! activity! was! recorded! as! an! average! of! the! two! trials.! Note:! Any! duplicate! sample! trials!with!a!deviation!of!activity!greater!than!10%!were!discarded!and!retested.!!

(

3.2.5(Determination(of(Steady7State(Kinetic(Constants!

! Ligase! activity! was! measured! using! a! Shimadzu! UV1800! UV! Spectrometer! and! a! coupled! assay! involving! adenylate! kinase! (AK),! pyruvate! kinase! (PK)! and! lactate!dehydrogenase!(LDH).!!Reactions!monitored!the!decrease!in!absorbance!at!

340!nm!(Δε!=!6.2!mMO1cmO1)!as!a!result!of!the!oxidation!of!NADH!and!were!carried! out! at! 25! °C! in! 500! µL! solutions! containing! assay! buffer! (50! mM! HEPES,! 5! mM!

MgCl2,!3.5!mM!ATP,!1!mM!CoASH,!200!μM!NADH,!3!mM!PEP,!5!mM!KCl,!11!U!AK,!9!U!

PK!and!9!U!LDH!at!pH!7.5),!enzyme!and!varying!concentrations!of!substrate!ranging! from! 0.5O5x! Km.! Initial! velocity! data,! measured! as! a! function! of! substrate! concentration,!were!analyzed!using!Enzyme!Kinetics!v1.4!and!equation!(1):!

V!=!Vmax[S]/([S]+Km)!! ! ! ! !!!!(1)!

! where! V! is! the! initial! velocity,! Vmax! is! the! maximum! velocity,! [S]! is! the! substrate! concentration! and! Km! is! the! Michaelis! constant.! kcat! was! calculated! from! Vmax/[E],! where![E]!is!the!total!enzyme!concentration.!

(

! 101! 3.3(Results(and(Discussion!

3.3.1(Selection(of(Standalone(Ligases(

In! order! to! select! standalone! ligases! for! biochemical! characterization,! we! searched!for!all!of!the!AMPOadenylateOforming!domains!in!P.,aeruginosa!PAO1.!We! identified!27!ligase!domains,!10!of!which!appeared!to!be!part!of!multiOdomain!NRPS! or!PKS!clusters!due!to!their!large!sequence!length!(>!800!amino!acids)!(Table!3O1).!

PA1215,! PA1221! and! PA4228! (pchD)! were! also! classified! as! part! of! NRPS! or! PKS! clusters!based!on!previous!literature![40,!41].!After!filtering,!14!standOalone!ligase! domains! remained! as! potential! acylOCoA! synthetases! involved! in! fatty! acid! and! organic! acid! metabolism.! The! characterization! of! some! P., aeruginosa! standalone! ligases!(PA1617,!PA3299!and!PA3300)!have!been!described!previously![39,!42]!and! thus!allowed!us!to!exclude!them!from!our!investigation.!Of!the!ten!remaining!genes! that! were! targeted,! PA2893! proved! too! unstable! to! obtain! pure! protein! for! highO throughput!screening!or!kinetic!testing.!!

(

3.3.2(Determination(of(Ligase(Substrate(Specificity(Profiles!

The! substrate! specificity! profiles! of! the! purified! P.,aeruginosa,ligases! were! determined! via! highOthroughput! screening! (HTS)! and! verified! by! measuring! the! steadyOstate!kinetic!parameters!of!each!reaction.!Given!the!solubility!limits!of!long! chain!fatty!acids!and!the!inherent!risk!of!micelle!formation,!substrates!with!carbon! chains!longer!than!C14!(myristate)!were!not!tested!under!the!HTS!assay!conditions.(

However,! ligases! indicating! potential! long! chain! activity! from! the! HTS! were! individually!tested!with!longer!chain!fatty!acids!(>!C14)!by!measuring!their!steadyO!(

! 102! GI#Number Accession#Number Gene#ID Alias Gene#Length Domain#Length 15596084 NP_249578.1 PA0887 acsA 651 17264233 15596193 NP_249687.1 PA0996 pqsA 517 14250533 15596412 NP_249906.1 PA1215 428 2242433 15596418 NP_249912.1 PA1221 618 23253633 15596814 NP_250308.1 PA1617 555 8254033 15597193 NP_250687.1 PA1997 651 19264333 15597498 NP_250992.1 PA2302 ambE 2124 4142906,11922126933 15597501 NP_250995.1 PA2305 ambB 1249 208275233 15597595 NP_251089.1 PA2399 pvdD 2448 4652103833 110645305 NP_251090.2 PA2400 pvdJ 2157 14992207133 15597598 NP_251092.1 PA2402 5149 4742103133 15597620 NP_251114.1 PA2424 pvdL 4342 10942165333 15597751 NP_251245.1 PA2555 555 28254333 15597753 NP_251247.1 PA2557 564 16255233 15598089 NP_251583.1 PA2893 AtuH 608 18257233 15598495 NP_251989.1 PA3299 fadD1 562 18255733 15598496 NP_251990.1 PA3300 fadD2 562 21256133 15598523 NP_252017.1 PA3227 2352 413296933 15598764 NP_252258.1 PA3568 628 3262333 15599055 NP_252549.1 PA3860 632 22257033 15599119 NP_252613.1 PA3924 560 29254633 15599273 NP_252767.1 PA4078 991 6252233 15599393 NP_252887.1 PA4198 540 13253833 15599421 NP_252915.1 PA4225 pchF 1809 5062968,13452142533 15599422 NP_252916.1 PA4226 pchE 1438 5432991,12862133333 15599424 NP_252918.1 PA4228 pchD 547 21254133 15599927 NP_253421.1 PA4733 acsB 645 11263733 (

Table(371.(Ligase!domains!in!Pseudomonas,aeruginosa!PAO1.!PKS/NRPS!complexes! are!highlighted!in!red!and!previously!characterized!genes!are!highlighted!in!gray.(

! state! kinetic! parameters.! The! various! ligases! produced! by! P.,aeruginosa! exhibit! a! broad! spectrum! of! substrate! ranges! and! specificities! (Figure! 3O3).! While! some! appear! to! be! specific! for! just! shortOchain! fatty! acid! or! aromatic! acid! substrates,! others!appear!to!exhibit!more!promiscuous!behavior,!activating!a!range!of!fatty!acid! substrates!from!shorterOchain!to!longOchain.!The!results!described!within!imply!that! chain!length!is!the!major!contributing!factor!to!specificity!and!activity!while!the!

! 103! 20"

16" Long%Chain*

12" Medium%Chain*

Short%Chain*

8" Carbon*Chain*Length*

0" PA0887* PA1997* PA2555* PA2557* PA3568* PA3860* PA3924* PA4198*

Figure( 373.( Substrate!specificity!of!alkyl!fatty!acidOactivating!ligases.!Bars!indicate! range! of! biologically! relevant! activity! of! each! ligase! in! terms! of! fatty! acid! carbon! length.! Colored! regions! are! defined! by! carbon! length! as! short! (green),! medium!

(blue)!and!long!(red)!chain!fatty!acids.!!

! addition!of!substituent!groups!(hydroxyl,!methyl,!unsaturated!bonds)!OO!for!the!most! part!OO!have!only!modest!effects!on!overall!activity.!

AcetylCCoA,Synthetase,Activity,,

Annotated! as! an! acetylOCoA! synthetase! (acs),! PA0887! (acsA)! displays! high! sequence! identity! to! other! previously! characterized! acs! enzymes! in! other! species,! including!E.,coli!and!S.,enterica,[40,!43].!Crystal!structures!obtained!from!S.,enterica,

! 104! acs!show!a!very!small!binding!pocket,!defined!by!W414,!which!blocks!the!rear!of!the! active!site!and!restricts!substrate!accommodation!to!short!chain!fatty!acids!up!to!C3! in! length! [40].! This! is! consistent! with! our! HTS! data! (Table! 3O2)! as! it! revealed!

PA0887!to!display!narrow!specificity,!preferring!parent!chain!lengths!of!only!C2OC3.!

The!measured!steadyOstate!kinetic!parameters!verified!this!activity,!indicating!that!

PA0887! could! only! activate! acetate! and! propionate! with! any! biological! relevance.!

While! the! turnover! numbers! (kcat)! measured! for! PA0887Ocatalyzed! hydrolysis! of! acetate! and! propionate! were! essentially! identical! (Table! 3O3),! the! Km! value! for! acetate!is!roughly!3.5Ofold!lower!than!the!Km!measured!for!propionate,!indicating!a! more!ideal!fit!in!the!acsA!active!site.!This!discrepancy!in!substrate!binding!leads!to!a!

5Ofold!higher!overall!kinetic!efficiency!(kcat/Km)!with!acetate,!indicating!it!to!be!the! physiological!substrate!of!acsA.!!

ShortCChain,Activity,

From! the! HTS! and! kinetic! data,! five! ligases! (PA1997,! PA2555,! PA2557,!

PA3568!and!PA4198)!were!shown!to!exhibit!preferences!for!shortOchain!fatty!acid! substrates.! Interestingly,! while! all! indicate! a! similar! overall! trend! in! activity,! each! ligase! displays! a! different! substrate! specificity! range! and! physiological! substrate,! indicating! each! one’s! potential! involvement! in! distinct! biological! roles.! PA3568! appears! to! exhibit! a! shortOchain! preference! similar! to! acsA,! albeit! with! a! slightly! wider! range.! Able! to! activate! acetate! and! propionate,! the! HTS! also! indicated! moderate!to!high!activity!with!a!variety!of!butyrate!derivatives,!as!well!as!valerate!

(Table!3O2).!However,!steadyOstat!kinetics!verified!physiologically!relevant!activity!

! 105! only! with! acetate,! propionate,! butyrate! and! isobutyrate! (Table! 3O3).! The! greatest! discrepancies! between! substrates! were! seen! in! their! respective! Km! values,! with! a!

10Ofold!difference!between!the!lowest!(isobutyrate)!and!the!highest!(acetate).!The! trend! in! Km! value! would! indicate! that! the! PA3568! active! site! is! set! up! to! best! accommodate!fatty!acids!with!a!C3!parent!chain,!ideally!with!branched!substituents.!

This! result! is! not! entirely! surprising! given! that! PA3568! shares! 44%! sequence! identity! with! the! propionylOCoA! synthetase! (prpE)! from! S., typhimurium,! a! ligase! shown! to! function! in! the! catabolism! of! propionate! [44].! Slight! discrepancies! in! turnover!number!appear!to!be!proportional!to!the!significantly!differing!Km!values,! leading! to! only! slight! differences! in! overall! kinetic! efficiency! for! each! substrate.!

While! PA3568! is! able! to! activate! propionate! and! butyrate! with! high! efficiency! as! well,!the!results!suggest!that!isobutyrate!is!its!physiological!substrate.!HTS!analysis! of! PA1997! indicated! it! to! be! moderately! promiscuous,! as! it! appeared! to! be! active! with! acetoacetate,! butyrate! and! other! C4! derivatives! ((D/L)OβO! hydroxybutyrate,! crotonoate,! butenoate)! (Table! 3O2).! Outside! of! C4! substrates,! some! moderate! activity! was! also! seen! with! the! saturated! C5! and! C6! fatty! acids,! valerate! and! hexanoate.!Annotated!as!an!acetoacetylOCoA!synthetase,!kinetic!analysis!showed!the! highest! overall! efficiency! with! acetoacetate,! verifying! it! to! be! the! physiological! substrate! for! PA1997.! Furthermore,! the! data! reveals! that! only! the! C4! derivatives,! acetoacetate! and! (L)OβOhydroxybutyrate,! can! be! activated! by! PA1997! with! any! biological!relevance,!as!they!displayed!a!102Ofold!higher!overall!efficiency!than!all!of! the! other! substrates! tested.! While! all! of! the! measured! kcat! values! (except! (D)OβO hydroxybutyrate)!!appear!to!be!competitive!with!each!other,!the!major!contributing!

! 106! Ligase8High;Throughput8Screening Substrate PA0887 PA0996 PA1997 PA2555 PA2557 PA3568 PA3860 PA3924 PA4198 Formic'Acid 0.052 0.000 0.000 0.000 0.026 0.000 0.090 0.001 0.000 Acetic'Acid 1.000 0.057 0.000 0.000 0.342 0.949 0.079 0.009 0.547 Propionic'Acid 1.000 0.001 0.000 0.089 0.986 0.914 0.087 0.396 0.943 Butyric'Acid 0.133 0.000 0.683 0.550 1.000 0.963 0.090 1.000 0.980 Valeric'Acid 0.047 0.000 0.424 0.499 1.000 0.579 0.107 1.000 0.966 Hexanoic'Acid 0.067 0.000 0.400 0.000 0.878 0.000 0.296 1.000 0.127 Octanoic'Acid 0.073 0.000 0.025 0.000 0.033 0.000 0.677 1.000 0.004 Decanoic'Acid 0.163 0.000 0.000 0.025 0.071 0.036 0.765 0.508 0.091 Lauric'Acid 0.214 0.018 0.000 0.020 0.125 0.052 0.372 0.814 0.058 Myristic'Acid 0.154 0.021 0.034 0.016 0.152 0.040 n/a 0.033 0.131 Isobutyric'Acid 0.615 0.000 0.000 0.998 0.841 0.900 0.078 0.006 0.865 Isovaleric'Acid 0.085 0.000 0.000 0.021 0.956 0.173 0.068 0.057 0.134 Citronellic'Acid 0.085 0.000 0.138 0.010 0.075 0.046 0.098 0.972 0.003 Crotonoic'Acid 0.130 0.033 0.263 0.149 0.995 0.858 0.063 0.856 0.934 3Lbutenoic'Acid 0.217 0.000 0.146 0.366 1.000 0.995 0.066 0.588 0.965 Hexenoic'Acid'(transL3) 0.146 0.000 0.152 0.000 0.104 0.094 0.050 1.000 0.100 Octenoic'Acid'(transL2) 0.137 0.006 0.004 0.010 0.101 0.001 0.092 1.000 0.051 Decenoic'Acid'(transL2) 0.108 0.027 0.018 0.000 0.112 0.000 0.090 0.881 0.102 αLhydroxybutyric'Acid 0.094 0.044 0.003 0.000 0.137 0.014 0.097 0.109 0.363 (D)LβLhydroxybutyric'Acid 0.138 0.012 0.684 0.000 0.646 0.177 0.105 0.689 0.973 (L)LβLhydroxybutyric'Acid 0.077 0.000 0.496 0.000 0.638 0.139 0.058 0.018 0.642 Gluconic'Acid 0.164 0.004 0.075 0.000 0.068 0.000 0.085 0.039 0.042 Glyoxylic'Acid 0.157 0.025 0.053 0.033 0.049 0.022 0.094 0.046 0.023 Pyruvic'Acid 0.099 0.021 0.019 0.010 0.089 0.005 0.082 0.066 0.045 αLketobutyric'Acid 0.089 0.061 0.000 0.000 0.052 0.017 0.051 0.025 0.049 Acetoacetic'Acid 0.117 0.018 0.790 0.040 0.075 0.019 0.051 0.015 0.546 Malic'Acid 0.059 0.000 0.000 0.057 0.015 0.000 0.095 0.035 0.005 Fumaric'Acid 0.048 0.000 0.000 0.000 0.035 0.000 0.096 0.045 0.000 Glutaconic'Acid 0.095 0.000 0.000 0.000 0.036 0.000 0.096 0.000 0.000 Malonic'Acid 0.013 0.000 0.000 0.000 0.016 0.000 0.078 0.026 0.024 Methylmalonic'Acid 0.123 0.000 0.000 0.000 0.041 0.000 0.098 0.133 0.004 Succinic'Acid 0.138 0.041 0.054 0.000 0.058 0.000 0.101 0.023 0.016 Oxaloacetic'Acid 0.054 0.000 0.016 0.000 0.023 0.000 0.067 0.060 0.003 Oxalic'Acid 0.109 0.001 0.045 0.000 0.048 0.000 0.059 0.026 0.013 Glutaric'Acid 0.118 0.013 0.000 0.005 0.027 0.000 0.082 0.057 0.036 αLketoglutaric'Acid 0.076 0.011 0.000 0.000 0.028 0.000 0.040 0.016 0.017 Citric'Acid 0.032 0.000 0.000 0.000 0.029 0.000 n/a 0.040 0.031 Aconitic'Acid 0.111 0.022 0.000 0.036 0.038 0.008 0.061 0.007 0.009 Isocitric'Acid 0.110 0.028 0.023 0.012 0.054 0.007 0.084 0.034 0.053 Benzoic'Acid 0.057 0.851 0.060 0.000 0.277 0.010 0.095 0.034 0.034 1Lnaphthoic'Acid 0.086 0.181 0.031 0.000 0.029 0.000 0.110 0.023 0.000 2Lnaphthoic'Acid 0.046 0.000 0.058 0.000 0.016 0.000 0.071 0.062 0.000 Phenylacetic'Acid 0.083 0.010 0.000 0.000 0.043 0.000 0.099 0.301 0.010 Phthalic'Acid 0.057 0.040 0.020 0.000 0.022 0.000 0.081 0.073 0.027 2Lmethylbenzoic'Acid 0.058 0.734 0.024 0.000 0.018 0.000 0.040 0.077 0.000 3Lmethylbenzoic'Acid 0.096 0.798 0.000 0.051 0.063 0.000 0.087 0.065 0.037 4Lmethylbenzoic'Acid 0.089 0.043 0.014 0.016 0.032 0.000 0.066 0.081 0.078 4Lethylbenzoic'Acid 0.104 0.000 0.004 0.000 0.072 0.000 0.109 0.300 0.097 2Lhydroxybenzoic'Acid 0.027 0.027 0.011 0.043 0.000 0.000 n/a 0.082 0.023 3Lhydroxybenzoic'Acid 0.094 0.871 0.000 0.001 0.015 0.012 0.088 0.000 0.006 4Lhydroxybenzoic'Acid 0.123 0.000 0.019 0.000 0.022 0.008 0.054 0.024 0.040 2,3Ldihydroxybenzoic'Acid 0.047 0.012 0.033 0.000 0.035 0.000 0.144 0.052 0.031 2,4Ldihydroxybenzoic'Acid 0.045 0.009 0.000 0.000 0.012 0.000 0.074 0.000 0.000 2,5Ldihydroxybenzoic'Acid 0.026 0.018 0.048 0.000 0.000 0.000 0.087 0.035 0.001 2Lmethoxybenzoic'Acid 0.146 0.038 0.000 0.000 0.071 0.000 0.128 0.135 0.008 3Lmethoxybenzoic'Acid 0.052 0.578 0.024 0.000 0.035 0.000 0.080 0.143 0.018 4Lmethoxybenzoic'Acid 0.085 0.026 0.040 0.000 0.000 0.000 0.084 0.074 0.036 Anthranilic'Acid 0.151 0.690 0.048 0.000 0.069 0.001 0.099 0.032 0.067 3Lnitrobenzoic'Acid 0.048 0.008 0.000 0.003 0.035 0.003 0.079 0.047 0.050 2Lcyanobenzoic'Acid 0.065 0.016 0.000 0.000 0.063 0.041 0.096 0.047 0.040 3Lcyanobenzoic'Acid 0.017 0.000 0.000 0.014 0.000 0.000 0.065 0.035 0.023 4Lcyanobenzoic'Acid 0.127 0.002 0.000 0.000 0.032 0.000 0.063 0.006 0.192 !

! 107! Ligase8High;Throughput8Screening8(Con't) Substrate PA0887 PA0996 PA1997 PA2555 PA2557 PA3568 PA3860 PA3924 PA4198 2"bromobenzoic,Acid 0.110 0.202 0.000 0.004 0.054 0.008 0.106 0.029 0.028 3"bromobenzoic,Acid 0.020 0.166 0.019 0.000 0.023 0.000 0.093 0.068 0.054 4"bromobenzoic,Acid 0.013 0.000 0.000 0.000 0.020 0.000 0.097 0.092 0.027 2"chlorobenzoic,Acid 0.037 0.447 0.000 0.000 0.046 0.000 0.105 0.047 0.009 3"chlorobenzoic,Acid 0.052 0.542 0.040 0.011 0.038 0.000 0.109 0.292 0.000 4"chlorobenzoic,Acid 0.066 0.000 0.007 0.000 0.008 0.014 0.107 0.140 0.043 2,4"dichlorobenzoic,Acid 0.043 0.000 0.010 0.000 0.000 0.000 0.073 0.179 0.013 2,5"dichlorobenzoic,Acid 0.071 0.018 0.000 0.000 0.042 0.000 0.090 0.002 0.008 2,6"dichlorobenzoic,Acid 0.062 0.000 0.034 0.000 0.046 0.000 0.091 0.015 0.061 3,4"dichlorobenzoic,Acid 0.018 0.000 0.000 0.000 0.017 0.000 0.077 0.053 0.057 2,4,6"trichlorobenzoic,Acid 0.010 0.000 0.000 0.009 0.005 0.000 0.085 0.039 0.002 4"fluorobenzoic,Acid 0.020 0.905 0.000 0.000 0.378 0.000 0.082 0.015 0.111 4"(trifluoromethyl)"benzoic,Acid 0.118 0.048 0.048 0.027 0.033 0.000 0.089 0.131 0.225 2"iodobenzoic,Acid 0.010 0.000 0.021 0.000 0.018 0.000 0.103 0.090 0.005 3"iodobenzoic,Acid 0.022 0.047 0.041 0.000 0.003 0.000 0.113 0.039 0.000 Glycine 0.034 0.039 0.052 0.013 0.005 0.000 0.095 0.020 0.009 Alanine 0.040 0.030 0.000 0.017 0.015 0.009 0.097 0.246 0.017 Serine 0.045 0.049 0.000 0.002 0.000 0.000 0.079 0.089 0.000 Threonine 0.069 0.000 0.000 0.000 0.009 0.000 0.414 0.203 0.014 Cysteine 0.091 0.000 0.000 0.000 0.053 0.006 0.174 0.040 0.043 Valine 0.075 0.000 0.000 0.021 0.030 0.004 0.074 0.007 0.584 Leucine 0.014 0.000 0.000 0.000 0.000 0.000 0.029 0.060 0.002 Isoleucine 0.018 0.005 0.000 0.000 0.004 0.000 0.073 0.043 0.000 Methionine 0.051 0.000 0.000 0.007 0.020 0.000 0.097 0.019 0.019 Proline 0.012 0.018 0.000 0.000 0.003 0.000 0.065 0.126 0.042 Phenylalanine 0.040 0.000 0.000 0.000 0.023 0.000 0.393 0.148 0.013 Aspartic,Acid 0.096 0.000 0.132 0.000 0.023 0.000 0.064 0.060 0.044 Glutamic,Acid 0.049 0.007 0.088 0.035 0.004 0.000 0.107 0.020 0.008 Asparagine 0.091 0.000 0.063 0.000 0.008 0.000 0.099 0.024 0.017 Glutamine 0.000 0.000 0.137 0.000 0.000 0.000 0.387 0.000 0.000 Histidine 0.070 0.000 0.012 0.000 0.000 0.000 0.499 0.028 0.000 Lysine 0.097 0.000 0.042 0.021 0.000 0.000 0.682 0.005 0.000 Arginine 0.103 0.000 0.045 0.075 0.000 0.000 0.542 0.010 0.000 (

Table( 372.! HighOthroughput! screening! of! P., aeruginosa, ligases! with! various! fatty! acid!and!amino!acid!substrates.!Activity!is!colored!on!a!gradient!scale!from!red!(high! activity)!to!white!(no!activity).!Untested!substrates!are!denoted!by!“n/a.”!!

! factor!comes!from!the!difference!in!substrate!binding,!with!acetoacetate!and!(L)OβO hydroxybutyrate! displaying! Km! values! 18O70Ofold! lower! than! the! other! substrates! tested!(Table!3O3).!This!result!indicates!the!potential!importance!of!a!βOpositioned! oxygen!substituent!(hydroxy!or!keto)!for!tight!substrate!binding.!Additionally,!the!

32Ofold! difference! in! Km! between! (D)! and! (L)OβOhydroxybutyrate! is! indicative! of! a!

! 108! highly!stereospecific!active!site!arrangement.!Overall,!the!results!defining!PA1997! as! an! acetoacetylOCoA! synthetase! (AACS)! are! in! good! agreement! with! previously! characterized! AACS! enzymes! in! other! organisms,! including! the! stereospecific! preference!for!(L)OβOhydroxybutyrate,!which!has!been!described!previously!in!the! nitrogenOfixing!bacterium,!S.,meliloti![45].!

! Located! only! two! genes! apart,! PA2555! and! PA2557! exhibit! similar! yet! remarkably! different! substrate! specificity! profiles.! The! HTS! results! indicated! a! narrow!substrate!range!for!PA2555,!showing!it!to!prefer!saturated!and,!to!a!lesser! extent,!unsaturated!C4!and!C5!derivatives!(Table!3O2).!Unfortunately,!due!to!protein! stability! issues,! we! were! not! able! to! obtain! enough! pure! protein! to! measure! any! steadyOstate!kinetic!parameters!and!can!not!accurately!speculate!on!which!activities! may!or!may!not!be!biologically!relevant.!However,!given!the!overall!reliability!of!the!

HTS,!we!feel!confident!in!the!conclusions!we!can!draw!from!it.!By!far,!it!appears!that! isobutyrate! is! the! physiological! substrate,! as! PA2555! exhibited! twice! as! much! activity! in! the! HTS! as! butyrate! and! valerate.! This! is! interesting! considering! the! negligible! activity! seen! with! (the! structurally! similar)! propionate! and! hints! at! the! significance! of! the! branched! methyl! group! for! substrate! accommodation! and! binding.!Additionally,!the!low!level!activity!seen!with!crotonoate!and!3Obutenoate,! mostly! likely! attributed! to! large! Km! values,! indicate! the! binding! of! unsaturated! substrates!to!be!unfavorable!and!suggests!the!importance!of!substrate!flexibility!as! well! in! the! PA2555! active! site.! An! opposing! trend! was! seen! with! PA2557,! as! it! displayed! a! large! degree! of! promiscuity! for! a! variety! of! short! chain! fatty! acids.! In! general,!PA2557!appeared!to!be!highly!active!with!both!linear!and!branched!

! 109! PA0887 PA2557

-1 -1 -1 -1 -1 -1 Substrate kcat+ (s ) Km+(μM) kcat/Km+(M s ) Substrate kcat+(s ) Km+(μM) kcat/Km+(M s ) Acetate 4.1+±+0.1 26+±+1.9 1.6+x+105 Acetate 2.0)±)0.06 (3.0)±)0.4))x)10 4 6.6)x)10 1 Propionate 4.1)±)0.2 90)±)11 4.6)x)10 4 Propionate 12.7)±)0.7 (1.5)±)0.3))x)10 3 8.5)x)10 3 Butyrate 0.5)±)0.01 (2.4)±)0.1))x)10 3 2.2)x)10 2 Butyrate 19)±)0.5 216)±)17 8.7)x)10 4 Isobutyrate 1.1)±)0.1 (4.7)±)0.9))x)10 3 2.4)x)10 2 Isobutyrate 17)±)0.4 787)±)74 2.1)x)10 4 3Bbutenoate 0.2)±)0.05 (1.0)±)0.1))x)10 4 2.2)x)10 1 Crotonoate 22)±)0.5 549)±)38 3.9)x)10 4 3Bbutenoate 18.7)±)0.7 (1.6)±)0.2))x)10 3 1.2)x)10 4 DBβBHB 3.1)±)0.1 (1.1)±)0.1))x)10 4) 2.8)x)10 2) PA0996 LBβBHB 4.5)±)0.13 (4.3)±)0.4))x)10 3 1.0)x)10 3) -1 -1 -1 B2 3 1 Substrate kcat+ (s ) Km+(μM) kcat/Km+(M s ) αBHB (6.0)±)0.2))x)10 (1.3)±)0.1))x)10 4.7)x)10 Benzoate 2.7)±)0.1 41)±)5.3 6.5)x)10 4 Acetoacetate (1.4)±)0.1))x)10 B1 (6.2)±)0.8))x)10 3 2.3)x)10 1 2Bmethylbenzoate 1.4)±)0.1 78)±)14 1.8)x)10 4 Valerate 18)±)0.8 190)±)27 9.4)x)10 4) 3Bhydroxybenzoate 2.6)±)0.1 51)±)3.1 5.1)x)10 4 Isovalerate 19.4+±+0.3 23+±+1.2 8.4+x+105 3Bmethoxybenzoate 2.0)±)0.1 114)±)12 1.8)x)10 4 Hexanoate 1.3)±)0.06 547)±)72 2.4)x)10 3 Anthranilate 1.7+±+0.1 16+±+1.6 1.0+x+105 Hexenoate (6.3)±)0.2))x)10 B2 539)±)53 1.2)x)10 2) 2Bbromobenzoate (1.2)±)0.03))x)10B1 181)±)12 6.4)x)10 2 Myristate >)0.0001 n/a n/a 2Bchlorobenzoate (5.1)±)0.1))x)10 B1 315)±)26 1.6)x)10 3 Palmitate >)0.0001 n/a n/a 4Bfluorobenzoate 2.9)±)0.1 280)±)15) 1.1)x)10 4 Oleate >)0.0001 n/a n/a Linoleate >)0.0001 n/a n/a Linolenate >)0.0001 n/a n/a PA1997 Arachindonate >)0.0001 n/a n/a -1 -1 -1 3 Substrate kcat+ (s ) Km+(μM) kcat/Km+(M s ) Benzoate 0.16)±)0.01 140)±)11 1.1)x)10 Butyrate 2.5)±)0.2 (5.0)±)1.0))x)10 3 4.9)x)10 2 4Bfluorobenzoate 0.23)±)0.03 58)±)24 3.9)x)10 3 (L)BβBHB 2.0)±)0.1 100)±)12 2.0)x)10 4 Crotonate 0.2)±)0.01 (1.4)±)0.1))x)10 3 1.0)x)10 2 PA3924 (D)BβBHB 0.3)±)0.01 (3.2)±)0.4))x)10 3 1.0)x)10 2 4 -1 -1 -1 Acetoacetate 2.4+±+0.1 75+±+12 3.1+x+10 Substrate kcat+ (s ) Km+(μM) kcat/Km+(M s ) Valerate 3.0)±)0.2 (5.3)±)0.8))x)10 3 5.6)x)10 2 Propionate >)0.0001 n/a n/a Hexanoate 2.7)±)0.2 (2.4)±)0.5))x)10 3 1.1)x)10 3 Butyrate 11)±)0.3 (3.7)±)0.3))x)10 3 2.9)x)10 3 Hexenoate >)0.0001 n/a n/a 3Bbutenoate >)0.0001 n/a n/a (D)BβBHB 7.7)±)0.05 (3.1)±)0.1))x)10 4 2.5)x)10 2 (L)BβBHB >)0.0001 n/a n/a PA3860 Valerate 30)±)1.4 150)±)20 1.9)x)10 5 -1 -1 -1 5 Substrate kcat+(s ) Km+(μM) kcat/Km+(M s ) Hexanoate 16)±)1.0 22)±)3.7) 7.0)x)10 Octanoate (4.0)±)0.07))x)10B1 522)±)30 7.6)x)10 2 Hexenoate 34)±)2.1 63)±)11 5.4)x)10 5 Decanoate 1.8)±)0.03 61)±)3.5 2.9)x)10 4 Octanoate 45)±)1.5 22)±)2.7 2.0)x)10 6 Laurate 3.4)±)0.08 26)±)2.1 1.3)x)10 5 Octenoate 51)±)1.5 20)±)2.1 2.5)x)10 6 Myristate 1.1)±)0.02 5.5)±)0.3 1.9)x)10 5 Decanoate 21)±)1.0 5.1)±)0.8 4.1)x)10 6 Myristoleate 3.2)±)0.1 14)±)1.3 2.3)x)10 5 Laurate 7.1+±+0.1 <+1.0 1.0+x+107 Palmitate (4.3+±+0.05)+x+10 -1 (3.4+±+0.2)+x+10 -1 1.3+x+106 Myristate 10)±)0.5 45)±)5.8 2.3)x)10 5 Palmitoleate (9.9)±)0.1))x)10 B1 1.0)±)0.04 9.9)x)10 5 Myristoleate 5.5)±)0.2 20)±)2.2 2.7)x)10 5 Oleate (5.4)±)0.1))x)10 B1 1.7)±)0.14 3.1)x)10 5 Palmitate 6.4)±)0.2 30)±)3.0 2.2)x)10 5 Linoleate 1.1)±)0.04 3.4)±)0.4 3.1)x)10 5 Palmitoleate 3.4)±)0.05 12.5)±)0.7 2.7)x)10 5 Linolenate (6.7)±)0.1))x)10 B1 3.5)±)0.2 1.9)x)10 5) Oleate 4.6)±)0.1 17)±)1.2 2.8)x)10 5) Arachidonate 1.4)±)0.04 7.1)±)0.6 1.9)x)10 5 Linoleate 6.2)±)0.3 16.3)±)2.3 3.8)x)10 5 Linolenate 4.0)±)0.1 42)±)2.7 9.5)x)10 4 Arachidonate >)0.0001 n/a n/a PA4198 Phenylacetate (5.1)±)0.3))x)10 B1 (5.3)±)0.9))x)10 3 9.5)x)10 1 -1 -1 -1 Substrate kcat+ (s ) Km+(μM) kcat/Km+(M s ) Acetate 6.3)±)0.07 (4.5)±)0.16))x)104 1.4)x)10 2 PA3568 Propionate 3.4)±)0.04 120)±)10 3.0)x)10 4 5 -1 -1 -1 Butyrate 19+±+1.0 87+±+7.0 2.2+x+10 Substrate kcat+(s ) Km+(μM) kcat/Km+(M s ) Isobutyrate 2.2)±)0.08 (3.0)±)0.3))x)10 3 7.4)x)10 2 Acetate 1.3)±)0.1 530)±)85 2.5)x)10 3 Crotonate 4.0)±)0.2 100)±)10 4.0)x)10 4 Propionate 3.2)±)0.1 130)±)10 2.4)x)10 4 3Bbutenoate 18)±)0.5 57)±)5.4 3.1)x)10 5 Butyrate 3.5)±)0.2 200)±)20 1.8)x)10 4 (D)BβBHB 3.9)±)0.1 (3.2)±)0.3))x)10 3 1.2)x)10 3 Isobutyrate 2.6+±+0.1 54+±+7.4 4.9+x+104 (L)BβBHB 4.3)±)0.1 (3.3)±).3))x)103 1.8)x)10 3 Crotonate 3.0)±)0.3 (2.1)±)0.5))x)103 1.4)x)10 3 Acetoacetate 2.0)±)0.1 (8.3)±)0.9))x)10 3 2.4)x)10 2 3Bbutenoate 8.4)±)0.5 (1.2)±)0.2))x)103 6.8)x)10 3 Valerate 11)±)0.6 108)±)16 1.0)x)10 5 Valerate 1.0)±)0.01 (2.2)±)0.1))x)10 3 4.6)x)10 2 Hexanoate (4.9)±)0.03))x)10B2 890)±)20 5.5)x)10 1 Valine >)0.0001 n/a n/a !

Table( 373.! SteadyOstate! kinetic! constants! for! P., aeruginosa! ligaseOcatalyzed! activation!of!various!fatty,!aromatic!and!amino!acids.!Substrates!highlighted!in!grey!

! 110! indicate!Km!values!too!high!for!biologically!relevant!activity.!Substrates!highlighted! in!red!represent!the!highest!overall!kinetic!efficiency.!!(HB):!hydroxybutyrate.!

! saturated!fatty!acids!ranging!from!propionate!to!hexanoate,!as!well!as!with!a!variety! of!other!C4!derivatives!including!crotonoate,!3Obutenoate,!(D/L)OβOhydroxybutyrate,!

αOhydroxybutyrate! and! acetoacetate.! Slight! activity! was! also! seen! with! acetate,! hexenoate! and! the! medium! and! long! chain! fatty! acids,! laurate! and! myristate,! respectively.!The!steadyOstate!kinetic!parameters!displayed!a!much!narrower!range! of! biologically! relevant! activity! for! PA2557,! indicating! only! highOlevel! activity! for! butyrate,! isobutyrate,! crotonoate,! valerate,! isovalerate! and! hexanoate! (Table! 3O3).!

While!all!of!the!substrates!tested!once!again!exhibit!competitive!turnover!numbers,! acetate,!propionate!and!the!other!C4!derivatives!display!millimolarOlevel!Km!values.!

Isovalerate! appears! to! be! the! physiological! substrate,! with! its! 105! overall! kinetic! efficiency!at!least!10Ofold!higher!than!any!other!substrate!tested.!The!higher!degree! of!efficiency!is!directly!attributed!to!the!measured!Km!value!for!isovalerate,!as!it!is!8O fold! lower! than! the! Km! for! valerate.! As! with! PA2555,! this! again! indicates! the! significant! role! of! a! branched! methyl! group! in! substrate! binding! and! orientation.!

Interestingly! enough,! PA2557! also! displayed! activity! for! benzoate! and! 4O fluorobenzoate,!the!only!ligase!found!to!be!active!with!both!aliphatic!and!aromatic! acid!substrates.!While!the!kinetic!efficiencies!measured!for!both!are!102Ofold!lower! than!that!of!isovalerate,!the!lower!Km!values!indicate!favorable!binding!interactions! in! the! PA2557! active! site,! possibly! arising! from! πOstacking! (both)! and/or!

! 111! electrostatic! (4Ofluorobenzoate)! interactions! from! the! aromatic! ring! and! paraO substituted!fluoro!group,!respectively.!

! According!to!the!HTS,!PA4198!displayed!a!range!of!activity!(modest!to!high)! with! all! of! the! C4! derivatives! tested! (Table! 3O2).! High! activity! was! also! seen! with! valerate,!along!with!moderate!activity!towards!acetate!and!the!amino!acid,!valine.!

Some!lowOlevel!activity!was!indicated!for!hexanoate!as!well.!The!steadyOstate!kinetic! parameters!revealed!the!range!of!physiological!activity!for!PA4198!to!be!specific!for! linear!substrates!from!C3OC5!in!length!(Table!3O3).!Substrates!active!in!the!HTS!such! as! (D/L)OβOhydroxybutyrate,! αOhydroxybutyrate,! acetoacetate! and! isobutyrate! all! displayed!millimolar!Km!values,!indicating!the!presence!of!a!small,!narrow!binding! pocket! in! which! any! interactions! from! branching! substituent! groups! are! unfavorable.!While!PA4198!exhibited!the!highest!turnover!rate!with!acetate,!it!also!

4 exhibited!the!least!favored!binding!interactions!with!a!Km!in!the!10 !range.!Only!C3O

C5! derivatives! had! measured! Km! values! within! a! physiological! range.! Overall,! 3O butenoate!has!the!most!favorable!binding!interactions!and!paired!with!a!fairly!high! kcat!value,!has!the!highest!kinetic!efficiency!with!PA4198.!

MediumCChain,Activity!

, PA3924! exhibited! the! largest! range! of! HTS! activity,! activating! linear! fatty! acids! from! propionate! to! laurate! (C3OC12).! PA3924! also! displayed! high! activity! toward! a! variety! of! C4! derivatives! as! well! as! the! medium! chain! derivatives,! citronellate,! hexenoate,! octenoate! and! decenoate.! From! the! steadyOstate! kinetic! parameters,!we!found!that!the!lowOlevel!propionate!activity!seen!in!the!HTS!was!just!

! 112! an! artifact,! as! PA3924! was! unable! to! activate! propionate! with! any! physiological! relevance.! Of! the! C4! derivatives,! only! butyrate! and! (D)OβOhydroxybutyrate! were! activated! at! all,! and! with! lowOlevel! kinetic! efficiency! due! to! millimolar! Km! values.!

After!testing!long!chain!substrates,!we!found!that!PA3924!can!activate!saturated!and! unsaturated! fatty! acids! from! valerate! (C5)! to! oleate! (C18)! with! physiological! relevance.! We! noticed! an! emerging! trend! of! increasing! kinetic! efficiency! with! increasing! chain! length! that! maxed! out! with! laurate,! As! chain! length! increased! further,!the!kinetic!efficiency!decreased!and!remained!relatively!the!same.!For!the! shortOmedium!chain!fatty!acids!leading!up!to!laurate,!this!trend!is!described!mainly! through!substrate!binding!and!accommodation.!While!the!calculated!kcat!values!are! relatively!competitive!with!each!other,!the!Km!decreases!over!150Ofold!from!valerate! to! laurate,! where! PA3924! exhibits! a! subOmicromolar! value.! Beyond! laurate,! the! largest! discrepancies! in! Km! arise! from! comparing! saturated! and! unsaturated! derivatives.! This! can! be! explained! from! the! decreased! flexibility! of! the! long! chain! substrates! that! most! likely! need! to! wind! around! in! the! active! site! in! order! to! be! properly! accommodated.! Overall,! the! data! points! to! PA3924! as! the! most! promiscuous! of! the! P.,aeruginosa,ligases! with! optimal! efficiency! toward! mediumO chain!substrates.!!

LongCChain,Activity,

The! only! ligase! characterized! in! this! study! to! be! active! for! longOchain! substrates!was!PA3860.!While!previous!works!have!characterized!PA3299,!PA3300! and!PA1617!as!fellow!longOchain!ligases,!the!substrate!specificity!profile!of!PA3860,!

! 113! to! date,! has! yet! to! be! determined! [39,! 42].! From! the! HTS,! PA3860! exhibited! moderate!activity!with!medium!to!long!chain!fatty!acids!from!hexanoate!to!laurate!

(C6OC12).!Interestingly!enough,!derivatives!between!C6!and!C12!in!length!such!as! citronellate,! hexenoate,! octenoate! and! decenoate! displayed! no! activity,! indicating! the! specific! nature! of! the! PA3860! active! site! for! linear,! saturated! fatty! acid! substrates.!Upon!determination!of!the!steadyOstate!kinetic!parameters,!PA3860!was! found! to! be! even! more! active! for! longOchain! fatty! acids,! catalyzing! thioester! formation!of!substrates!all!the!way!up!to!C20!in!length.!As!with!the!trend!displayed! by!PA3924,!a!similar!trend!was!also!noticed!for!PA3860!activity.!The!measured!kcat! values!remain!fairly!constant!and!competitive!between!all!substrates!tested!while!

Km!values!steadily!decreased!by!over!1500Ofold!from!hexanoate!to!palmitate.!While! the!Km!increased!with!increasing!carbon!length!over!C16!(albeit!very!slow),!it!was! noticed! that! unsaturated! derivatives! of! longOchain! fatty! acids! were! also! accommodated! by! the! PA3860! active! site.! The! subOmicromolar! Km! measured! for! palmitate! resulted! in! the! highest! overall! kinetic! efficiency,! indicating! it! to! be! the! physiological!substrate!for!PA3860.,

Aromatic,Activity,

PA0996,! annotated! as! pqsA,! was! the! only! standalone! ligase! found! to! be! associated! with! aromatic! carboxylate! derivatives.! The! HTS! results! indicated! high! activity! with! benzoate! and! a! variety! of! derivatives! including! anthranilate! and! methylO,! hydroxyO! and! methoxybenzoate! moieties,! as! well! as! various! halogenated! derivatives! (Table! 3O2).! SteadyOstate! kinetic! data! indicates! anthranilate! to! be! the!

! 114! physiological! substrate,! due! mainly! to! a! 3O20Ofold! lower! Km! value! than! other! substrates!tested!(Table!3O3).!Besides!anthranilate,!PA0996!displayed!the!higher!Km! values! (and! lower! efficiencies)! with! all! benzoate! substituents! when! compared! to! just! benzoate.! This! indicates! that! the! active! site! is! setup! specifically! for! an! orthoO positioned!amine!substituent.!Additionally,!the!halogenated!derivatives!2Obromo,!2O chloro!and!4Ofluorobenzoate!displayed!the!highest!respective!Km!values!and!lowest! overall! efficiencies.! These! results! are! in! good! agreement! with! previous! characterization!of!PA0996!as!an!anthraniloylOCoA!ligase!where!the!highest!activity! was!observed!with!anthranilate!and!benzoate!in!both!cases![46].!

(

3.3.3(Biological(Roles(of(P.*aeruginosa*Ligases!

Utilization,of,Fermentation,Products,,

, Fermentation!is!the!metabolic!process!utilized!by!all!anaerobic!bacteria!(and! some!aerobic!bacteria)!to!generate!energy!in!the!absence!of!oxygen!and!represents! the! primary! means! of! ATP! production! under! these! conditions.! The! principal! byproducts! produced! by! carbohydrate! and! amino! acid! fermentation! are! acetate,! propionate,!and!butyrate!but!ethanol,!malonate,!2,3Obutanediol,!valerate,!hexanoate,! isobutyrate,! and! isovalerate! are! also! produced! in! varying! amounts! [47].! SCFA! fermentation!has!largely!been!studied!in!the!human!colon!and!butyrate!has!recently! received! much! attention! as! a! product! of! microbial! metabolism! and! mucosal! immunity!modulator.!However,!as!more!sophisticated!techniques!become!available! to!profile!bacterial!populations,!it!has!become!clear!that!many!fermenting!species! also!colonize!the!CF!lung.!,

! 115! In!bacterial!systems,!the!Crc,regulatory!system!is!responsible!for!controlling! the! uptake! and! utilization! of! various! shortOchain! carbon! sources.! This! is! accomplished! by! binding! the! mRNA! of! certain! genes! necessary! for! shortOchain! carbon! utilization! and! repressing! their! translation.! Under! anaerobic! conditions! when! the! utilization! of! poor! carbon! sources! is! necessary! for! survival,! a! twoO component!regulatory!system,!CbrAB,!is!activated!and!induces!the!transcription!of!

CrcZ,! an! sRNA.! CrcZOmediated! repression! of! Crc,leads! to! the! deOrepression! of! the! target!genes,!allowing!them!to!function!in!the!uptake!and!utilization!of!poor!carbon! sources.! Many! target! genes! have! been! identified,! including! estA, (esterase),! acsA,

(acetylOCoA!synthetase)!and!aroP2,(aromatic!amino!acid!uptake!protein)![37].!

! PA0887,!shown!to!be!an!acetylOCoA!synthetase!(acsA),!has!been!the!focus!of! extensive! study.! Its! role! was! originally! described! in! the! utilization! of! acetate,! whereby!acetate!is!activated!by!acsA!and!inactivated!by!sequential!phosphorylation! and!dephosphorylation!(AckCPta!pathway)![48].!Further!studies!revealed!that!acsA! is! critical! for! activation! of! acetate! at! low! cellular! concentrations! (nutrientOpoor! conditions),!similar!to!conditions!found!in!the!CF!sputum!environment![49].!!

Located!in!the!middle!of!a!large!open!reading!frame,!PA0887!is!sandwiched! between! two! distinct! metabolic! operons! (Figure! 3O4A).! Downstream! from! acsA! is! small!gene!cluster!responsible!for!the!degradation!of!itaconate!(Figure!3O4C)![50].!

Produced!by!activated!macrophages!in!response!to!infection,!itaconate!is!a!potent! inhibitor!of!isocitrate!lyase,!the!key!enzyme!of!the!glyxoylate!bypass!(Figure!3O4B).!

This! diverted! pathway! converts! isocitrate! to! succinate! by! way! of! glyoxylate! and! allows!bacteria!to!synthesize!succinate!under!nutrientOpoor!conditions!when!only!!

! 116! A C CH2 O 0878$ 0882$ 0883$ acsA$ aotJ$ aotQ$ aotM$ HO ! ! ! ! ! //! ! OH ! ! ! ! ! ! O Itaconate aotP$ argR$ aruC$ aruF$ aruG$ aruD$ aruB$ aruE$ ! ! ! ! ! ! ! ! PA0882 ! ! ! ! ! ! ! ! CH O Acetyl+CoA 2 HO SCoA

O Itaconyl*CoA B Oxaloacetate Citrate PA0878

O Acetyl+CoA H C OH Malate Isocitrate 3 HO ICL$ SCoA

O Glyoxylate (3S)*Citramalyl*CoA

Fumarate Ketoglutarate PA0883 Succinate O O HO CH3 H3C SCoA Succinate Succinyl+CoA O Pyruvate Acetyl*CoA !

Figure(374.!(A)!Gene!neighborhood!of!PA0887!(yellow)!showing!the!proximity!of!an! itaconate!degradation!operon!(blue)!as!well!as!arginine!uptake!(teal)!and!utilization!

(red)!operons.!(B)!The!citric!acid!cycle!(black)!showing!the!glyoxylate!bypass!(red).!

The! key! enzyme! Isocitrate! lyase! (ICL)! is! shown! in! blue.! (C)! P., aeruginosa, genes! involved!in!itaconate!degradation.!

! simple! (poor)! carbon! sources! like! acetate! are! available! [51].! Various! pathogenic! species!have!been!shown!to!degrade!itaconate!to!pyruvate!and!acetylOCoA,!not!only! helping! the! bacteria! survive! phagocytosis! but! also! producing! useful! metabolites!

[50].!Near!acsA!is!the!aot/argR!operon!(aotJQM)!responsible!for!arginine!uptake!and!

! 117! regulation.!Along!with!acsA,!the!aotCargR!operon!is!under!the!control!of!the!CbrAB! regulatory!system,!likely!explaining!their!proximity!and!indicating!the!importance!of! arginine! uptake! and! utilization! during! nutrientOpoor! conditions! [49].! While! it! remains!unknown!if!the!pathway!for!itaconate!degradation!is!under!the!control!of! the! CbrAB! regulatory! system! as! well,! it! would! not! be! surprising! considering! its! function!to!protect!a!metabolic!cycle!that!is!used!during!nutrientOpoor!conditions.!

While! its! paralog,! acsA,! has! been! extensively! studied,! research! into! the! individual!role!of!acsB!(encoded!by!PA4733)!has!been!largely!ignored.!Both!genes! share!high!sequence!similarity!to!verified!acetylOCoA!synthetases!in!other!organisms! but!searches!through!the!current!literature!reveal!little!about!the!individual!role!of! acsB,[43].! Due! to! the! high! pairwise! similarity! of! acsB! to! acsA! (63%),! it! is! easy! to! postulate! that! the! two! are! acting! in! a! similar! manner.! Additionally,! the! acsB! gene! appears!to!be!just!upstream!from!and!in!the!same!reading!frame!as!CbrA!and!CbrB,! and! while! it! has! not! been! determined! if! acsB! is! a! target! for! crc, transcriptional! regulation,!this!further!supports!an!acsB!functional!role!similar!to!that!of!acsA.!

PA2555! and! PA2557! are! located! only! two! genes! apart,! and! while! their! specificity!profiles!show!significant!overlap,!they!share!very!low!sequence!identity! with! each! other.! Interestingly! enough,! PA2557! has! a! moderate! biological! range,! highly! conserved! throughout! Pseudomonas,! Gammaproteobacteria! and! within! the!

Actinomycetales!class!of!Actinobacteria.!PA2555,!on!the!other!hand,!seems!to!have!a! very! limited! biological! range,! as! putative! orthologs! were! found! only! within!

Proteobacteria.! This! supports! of! model! of! convergent! evolution,! and! may! also! indicate! a! specialized! role! for! PA2555! pathogenesis! given! its! limited! range!

! 118! throughout! Bacteria.! Analysis! of! the! PA2555/PA2557! gene! context! implies! their! involvement!in!a!fatty!acid!βOoxidation!operon,!given!their!proximity!to!the!putative!

βOoxidation! operon! fadBA3,(PA2553! and! PA2554)! [38].! Additionally,! PA2550! and!

PA2552! encode! putative! acylOCoA! dehydrogenases! while! PA2554! encodes! a! putative! shortOchain! dehydrogenase! and! PA2553! encodes! an! acylOCoA! thiolase.!

Given!these!findings!along!with!the!substrate!specificity!profiles,!it!is!proposed!that!

PA2555!and!PA2557!may!be!able!to!scavenge!mediumOchain!C4!and!C5!fatty!acid! derivatives!for!use!in!a!shortOchain!βOoxidation!pathway.!!

Similar! to! PA2555/PA2557,! PA3568! is! also! located! near! a! putative! βO oxidation! operon! (fadBA2:! PA3589OPA3590)! involved! in! short! chain! fatty! acid! oxidation![38].!PA3589OPA3591!and!PA3593!encode!an!acetylOCOacetyltransferase,! hydroxybutyrylOCoA! dehydrogenase,! enoylOCoA! hydratase! and! an! acylOCoA! dehydrogenase,!respectively.!Adjacent!to!PA3568!are!two!genes!involved!in!valine! degradation,! mmsB, and! mmsA., Encoded! by! PA3569,! mmsB, functions! as! a! 3O hydroxyisobutyrate! dehydrogenase! while! mmsA, (encoded! by! PA3570)! is! a! methylmalonate! semialdehyde! dehydrogenase.! During! valine! degradation,! isobutyrylOCoA! is! converted! to! (S)O3Ohydroxybutyrate! by! way! of! methacrylylOCoA! and!(S)O3OhydroxybutyrylOCoA.!Catalyzing!the!subsequent!step,!mmsB,converts!(S)O

3Ohydroxybutyrate!to!(S)Omethylmalonate!semialdehyde,!which!is!then!converted!to! propionylOCoA! by! mmsA., Given! its! activity! with! propionate,! it! is! entirely! possible! that!PA3568!could!be!functioning!in!propionate!metabolism!as!well,!activating!free! propionate!for!degradation.!!

! 119! Additionally,! the! activated! mediumO! and! branchedOacylOCoAs! formed! from!

SCFA!fermentation!products!can!be!incorporated!into!PHA!polymers,!degraded!via!

βOoxidation!or!amino!acid!degradation!pathways,!or!feed!into!secondary!metabolite! synthesis! (discussed! below).! ButyrylOCoA,! in! particular! (produced! by! PA4198),! could! provide! a! precursor! for! synthesis! of! the! NObutanoylOLOhomoserine! lactone! quorum! signal! [51].! In! addition,! activated! SCFAs! may! provide! building! blocks! for! rhamnolipid!synthesis.!Rhamnolipids!are!composed!of!a!rhamnose!sugar!linked!to!a!

3O(3Ohydroxyalkanoyloxy)Oalkanoic! acid! moeity! [27].! RhlA! is! responsible! for! synthesizing!the!fatty!acid!group!and!utilizes!3OhydroxyacylOCoA!or!3OhydroxyacylO

ACP!intermediates!drawn!from!fatty!acid!synthesis!and!βOoxidation!pathways![27,!

52].!!

Utilization,of,BranchedCChain,Amino,Acids,

Amino!acids!can!provide!an!important!source!of!carbon!and!nitrogen!for!P., aeruginosa!during!pathogenesis.!In!fact,!some!reports!have!shown!higher!levels!of! amino!acids!OO!particularly!leucine!and!isoleucine!OO!in!CF!sputum!as!compared!to! nonOCF! sputum,! attributed! to! the! evolution! of! auxotrophic! strains! in! the! CF! lung!

[53].! In! P., aeruginosa,! leucine! is! degraded! via! the! liu! pathway! (Figure! 3O5B).!

Additionally,! the! degradation! products! of! acyclic! terpenes! (like! citronellol! and! geraniol)!and!the!branchedOchain!fatty!acid!isovalerate!can!feed!into!the!liu!pathway! as! well.! liuA! has! been! characterized! as! an! isovalerylOCoA! dehydrogenase! and! has! been!shown!to!be!required!for!growth!on!isovalerate,!and!leucine![54].!Given!the!!

! 120!

A" ! !

PA1993% ! PA1997% dhcR% dhcA% dhcB% atoB% atoE%

! ! ! ! !

! !

! ! ! !

maiA% fahA% hmgA% liuE% liuD% liuC% liuB% liuA% liuR! %

! ! ! ! !

PA2016% O

B" O O NH3 L"Leucine O O NH3 HO PA2557 L"Tyrosine O Isovalerate O

SCoA O Isovaleryl"CoA HO O liuA

OH O Homogentisate SCoA hmgA 3"methylcrotonyl"CoA

liuB/liuD O O O O O O O O 4"maleylacetoacetate O SCoA 3"methylglutaconyl"CoA maiA

liuC atoE O O O O O O O OH O 4"fumarylacetoacetate O SCoA 3"hydroxy"3"methylglutaryl"CoA TCA Cycle/ O O Glyoxylate fahA Bypass O Acetoacetate O liuE O O PA1997 O Fumarate O O O O N atoB O SCoA SCoA Betaine Acetoacetyl"CoA Acetyl"CoA

OH O cdhA/cdhB O O dhcAB O O atoB O N N N N O O SCoA SCoA Carnitine 3"dehydrocarnitine 3"dehydrocarnityl"CoA Glycyl"bentainyl"CoA (

Figure( 375.( (A)!Gene!neighborhood!surrounding!PA1997!(red)!showing!proximity! to! genes! involved! leucine! (green),! tyrosine! (orange)! and! carnitine! (blue)!

! 121! degradation.! Genes! involved! in! shortOchain! fatty! acid! utilization! are! shown! in! purple.! (B)! Reaction! diagram! showing! lateOstage! steps! of! leucine! and! tyrosine! degradation!as!well!as!carnitine!metabolism.!All!pathways!utilize!atoB!to!produce! the!critical!TCA!cycle!metabolite!acetylOCoA!(yellow).!!

! specificity!of!both!PA2557!and!PA3568!for!isovalerate,!it!could!also!be!involved!in! the! catabolism! of! isovalerate,! leucine,! and! other! methylObranched! compounds!

Figure!3O5B).!

Acetoacetate,Utilization,

, !Analysis! of! the! PA1997! gene! neighborhood! further! supports! its! role! as! an! acetoacetylOCoA!synthetase.!PA1997!is!found!near!four!separate!gene!clusters!that! all!function!in!the!utilization!of!shortOchain!fatty!acid!derivatives!(Figure!3O5A).!Just! upfield! from! PA1997! are! two! genes! involved! in! acetoacetate! utilization:! atoE,and! atoB, (PA2001! and! PA2002,! respectively).! The! protein! encoded! by! atoE, is! an! uncharacterized! shortOfatty! acid! transporter! potentially! involved! in! the! uptake! of! exogenous! acetoacetate.! Once! in! the! cell,! PA1997Oactivated! acetoacetate!

(acetoacetylOCoA)!can!then!be!converted!to!two!molecules!of!acetylOCoA!by!atoB,!an! acetylOCoA! COacetyltransferase.! AcetylOCoA! can! then! feed! into! the! TCA! cycle! or! glyoxylate! bypass,! depending! on! cellular! growth! conditions.! Additionally,! PA1997! may! also! be! involved! in! the! utilization! of! intracellular! acetoacetate,! a! product! of! both!tyrosine!and!leucine!degradation!pathways.!Found!further!upfield!from!atoB!

! 122! and! atoE! are! gene! clusters! for! both! of! these! pathways.! The! genes! encoding! maiA,, fahA!and!hmgA!(PA2007OPA2009,!respectively)!represent!the!final!steps!of!tyrosine! metabolism.!In!the!last!step,!fahAOmediated!conversion!of!4Ofumarylacetoacetate!to! fumarate! produces! acetoacetate,! which! can! then! be! activated! by! PA1997.! The! liu, gene!cluster!(PA2011OPA2016)!OO!just!upfield!from!hmgA!OO!catalyzes!the!final!steps! of!leucine!metabolism.!The!terminal!step!produces!acetylOCoA!and!acetoacetate,!the! latter!of!which!can!be!activated!by!PA1997.!Additionally,!sitting!adjacent!to!PA1997! are! three! genes! directly! involved! in! carnitine! metabolism.! A! quaternary! amine! essential!for!βOoxidation!in!animals,!carnitine!is!required!for!the!soluble!transport!of! longOchain!fatty!acids!into!the!mitochondrial!matrix!(the!carnitine!shuttle).!Due!to! the!physiological!importance!of!the!carnitine!shuttle,!high!cellular!concentrations!of! carnitine! are! found! in! fatOmetabolizing! tissues! as! well! as! in! circulating! and! extracellular! fluids! [55].! While! many! bacteria! can! utilize! carnitine! as! an! osmoprotectant! during! times! of! osmotic! stress,! some! pathogenic! species! like! P., aeruginosa,are!able!to!metabolize!the!small!molecule,!utilizing!it!as!both!a!carbon! and!nitrogen!source!during!pathogenesis![56].!The!genes!encoding!dhcA,and!dhcB,

(PA1999! and! PA2000,! respectively)! are! the! α! and! β! subunits! of! a! 3OketoacidOCoA! transferase! and! have! been! shown! to! catalyze! the! activation! of! dehydrocarnitine!

[55].! The! gene! directly! adjacent! to! PA997,! dhcR! (PA1998),! is! the! divergently! transcribed!transcriptional!regulator!of!dhcAB.!The!reaction!step!following!dhcAB,is! the! formation! of! glycylObetainylOCoA! by! atoB,! forming! acetylOCoA! in! the! process.!

Further! catabolism! results! in! glycineObetaine! and! ultimately! glycine.! While! this!

! 123! pathway!is!not!directly!related!to!PA1997!functionality,!the!common!reliance!of!all! three!pathways!on!atoB!further!supports!the!role!of!PA1997.!!

In!nature,!orthologous!sequences!of!PA1997!are!mainly!concentrated!within! the! alpha! and! gamma! classes! of! Proteobacteria! with! the! majority! of! putative! orthologs!contained!within!the!genus!Pseudomonas.,Outside!of!Proteobacteria,!a!few! sequences! were! identified! with! only! modest! sequence! identity! (50O58%)! within!

Clostridia!(Firmicutes)!as!well!as!Spirochaetes.!!

Polyhydroxyalkanoate,(PHA),Cycling,

Given! their! relative! substrate! activity! profiles! and! relative! gene! neighbors,! many! of! the! P.,aeruginosa,ligases! may! play! a! role! in! polyhydroxyalkanoate! (PHA)! cycling.! PHA! polymers! are! linear! polyester! chains! made! up! of! hydroxyalkyl! monomers.! Found! to! play! a! critical! role! in! carbon! and! energy! storage,! PHAs! are! produced! as! a! result! of! bacterial! fermentation! under! conditions! of! cellular! deficiency! (N,! O,! POlimiting)! or! during! times! of! excess! carbon! (energy)! sources.!

Interestingly!enough,!PHA!synthesis!has!been!found!to!be!under!the!control!of!the!

Crc,regulator!in!P.,putida.!Repressed!when!carbon/nitrogen!levels!are!balanced,!this! indicates! a! potential! role! of! PHA! polymers! in! providing! carbon! sources! under! nutrientOpoor!conditions![57].!PHA!polymers!can!be!composed!of!shortOchain!(C4O

C5,!PHASCL)!or!mediumOchain!(C6OC14,!PHAMCL)!fatty!acid!derivatives!depending!on! the!bacterial!environment.!PHA!polymers!are!quite!diverse,!with!over!150!types!of! hydroxyacids!identified!as!potential!PHA!building!blocks,!including!hydroxyvalerate,! hydroxybutyrate!and!hydroxyisovalerate![58].!Granules!are!synthesized!using!!

! 124! AA/C3 degradation SCFA Acetate Acetoacetate PA2003 PA3568 PA0887 PA2555 PA1997 (D)-3-hydroxyacids PA2557

PA2001 Acetyl-CoA Acetoacetyl-CoA (D)-3-hydroxybutyrate acyl-CoA phaZ phaJ FabG phaZ

phaC phaC (D)-3-hydroxybutyryl-CoA PHB PHASCL (D)-3-hydroxyacyl-CoA TCA Cycle phaC

PA4198 Butyrate Butyryl-CoA Crotonyl-CoA

PA3924 MCFA/LCFA fatty acyl-CoA ketoacyl-CoA

β-oxidation FabG enoyl-CoA (L)-Hydroxyacyl-CoA

phaJ phaC (D)-3-hydroxyacyl-CoA PHAMCL

phaZ PA3924

(D)-3-hydroxyacid (

Figure( 376.( Potential! involvement! of! P., aeruginosa! acylOCoA! ligases! in! polyhydroxyalkanoate!(PHA)!cycling.!!

! hydroxyacylOCoA!monomers,!and!depolymerized!to!hydroxycarboxylic!acids!for!use!

(Figure!3O6).!When!cells!are!lysed,!PHA!polymers!are!released!and!members!of!the! community!can!utilize!the!stored!fatty!acids!by!secreted!PHA!depolymerases.!There! is! evidence! that! PHA! granules! in,vivo! are! partially! or! completed! surrounded! by! a! layer!of!PHAOassociated!proteins!(PGAPs)![59].!Not!surprising,!proteins!detected!on!

! 125! granules!include!phaC!and!phaZ!as!well!as!numerous!other!proteins!involved!in!PHA! synthesis!and!breakdown.!

The! most! common! PHA! polymer! produced! is! polyhydroxybutyrate! (PHB).!

Under! starvation! conditions,! PHB! depolymerases! cleave! off, individual! (D)O3O hydroxbutyrate! subunits,! which! are! then! converted! to! acetoacetate! by! (D)O3O hydroxbutyrate! dehydrogenase! (encoded! by! PA2003).!!From! here,! PA1997! may! function! in! the! activation! of! acetoacetate! for! utilization! as! a! carbon! source.! This! function!is!consistent!with!an!acetoacetylOCoA!synthetase!from!S.,meliloti,!which!was! shown! to! activate! acetoacetate! released! from! PHB! [45].! Additionally,! the! specific!

(L)O3Ohydroxybutyrate!activity!measured!for!PA1997!indicates!that!it!may!also!be! able!to!activate!(L)O3Ohydroxybutyrate!as!a!carbon!source.!

The!synthesis!and!catabolism!of!PHASCL!require!acylOCoA!synthetase!activity! with! C4! and! C5! branched! compounds.! Through! βOoxidation! cycling,! an! acylOCoA! precursor! can! be! sequentially! converted! to! (L)O3OhydroxyacylOCoA,! enoylOCoA! and! then! 3OketoacylOCoA.! When! PHAMCL! synthesis! is! required,! the! stereospecific! hydratase!(PhaJ)!and!reductase!(FabG)!can!convert!enoylOCoA!and!3OketoacylOCoA! to! (D)OhydroxyacylOCoA,! respectively! (Figure! 3O6)! [57].! Given! the! proximity! of!

PA2555,! PA2557,! PA3568,! and! PA4198! to! putative! βOoxidation! genes! and! the! presence!of!their!physiological!substrates!in!PHA!polymers,!it!is!possible!that!these! enzymes!are!involved!in!activation!of!branchedOchain!carboxylic!acids!during!PHA! polymerization! and! breakdown.! Conversely,! PHA! breakdown! products! like! isovalerate!and!isobutyrate!may!feed!into!amino!acid!degradation!pathways.!!!

! 126! PHAMCL!polymers!consist!of!monomer!units!C6OC14!in!length.!Rare!in!nature,!

PHAMCL!polymers!are!produced!mainly!by!fluorescent!Pseudomonads![60].!As!one!of! the! few! capable! bacteria,! P., aeruginosa! uses! PHA! synthases! phaC1! and! phaC2.! 3O hydroxyalkanoates!for!PHAMCL!synthesis!are!derived!from!βOoxidation!or!fatty!acid! synthesis,! depending! on! growth! conditions.! PHAMCL! plays! an! important! role! in! pathogenesis!as!it!helps!bacteria!survive!during!starvation!conditions!and!regulate! carbon!flow.!Although!never!characterized!in!P.,aeruginosa,!PA3924!has!previously! been!described!as!an!(R)O3Ohydroxyalkanoate!ligase!capable!of!synthesizing!PHAMCL! polymers!when!coOexpressed!in!E.,coli!with!PHA!synthase!genes![61].!PA3924!was! shown! to! enable! the! production! of! PHAMCL! polymers! with! C8,! C10,! and! C12! hydroxyalkanoate!constituents.!

! !

Fatty,Acid,Degradation,

! LongOchain! fatty! acylOCoA! synthetases! are! the! primary! activators! of! fatty! acids! for! degradation! via! the! βOoxidation! pathway! (Figure! 1O1)! and! have! been! characterized!in!many!species,!including!E.,coli,!P.,putida!and!P.,aeruginosa![39,!62,!

63].! PA3299! (fadD1)! and! PA3300! (fadD2)! are! associated! with! the! fadBA5, βO oxidation!operon!and!are!the!major!contributors!to!long!chain!fatty!acid!degradation!

[39,! 64].! FadD1! and! fadD2! are! also! induced! by! the! presence! of! palmitate,! their! preferred! fatty! acid! substrate! [48].! In! addition,! fadD2! knockout! strains! have! decreased! virulence! factor! production! and! decreased! survival! in! mouse! lungs,! implying!longOchain!ligase!involvement!in!pathogenesis[38].!However,!P.,aeruginosa! contains!multiple!fadD!homologs!within!the!genome,!the!functions!of!which!have!yet!

! 127! to!be!determined.!PA1617!(fadD4)!has!been!proposed!to!be!not!only!the!third!major! contributor! (after! fadD1! and! fadD2)! to! fatty! acid! degradation! but! also! solely! responsible! for! acyclic! terpene! degradation! [64].! Previous! characterization! of!

PA1617!shows!specificity!for!longOchain!substrates,!in!good!agreement!with!these! proposed!functions![42].!!Adjacent!to!PA1617!is!a!thioesterase!(PA1618),!and!while! it!preferentially!hydrolyzes!aroylOCoA!substrates,!its!predicted!to!function!as!a!CoA! scavenger!so!PA1617!can!synthesize!necessary!acylOCoA!intermediates![42].!!!

! PA3860! (fadD3), and! PA3924! (fadD6)! were! previously! identified! as! fadD! homologs! but! were! shown! to! play! minimal,! redundant! roles! in! fatty! acid! degradation!as!neither!was!able!to!support!growth!on!longOchain!fatty!acids![64].!!

! The!final!fadD!homolog,!PA2893,!was!identified!as!atuH,(fadD5).,Similar!to!

PA3860!and!PA3924,!it!was!found!to!play!a!minimal,!redundant!role!in!longOchain! fatty!acid!degradation![64].!Furthermore,!given!its!proximity!to!the!atu,gene!cluster!

(PA2886OPA2892),!PA2893!has!been!hypothesized!to!be!involved!in!acyclic!terpene! utilization!as!a!citronellylOCoA!synthetase![65].!Terpenes!are!common!in!plant!oils! and!pigments.!The!atu!pathway!degrades!citronellol,!geraniol,!and!their!derivatives! to! 7OmethylO3OoxoOoctenoylOCoA,! which! is! capable! of! cycling! through! βOoxidation!

[49,! 50].! However,! in! contradiction! to! this! classification,! mutant! strains! lacking!

PA2893!were!still!able!to!survive!on!acyclic!terpene!carbon!sources![64,!66].!As!we! were!unable!to!obtain!stable!protein!samples!of!PA2893!for!screening!and!kinetic! analyses,!its!biological!function!remains!to!be!elucidated.!!

! 128! Membrane,Lipid,Synthesis,

! While! most! bacterial! membranes! contain! phosphatidylethanolamine,! phosphatidylglycerol,! and! cardiolipid,! only! roughly! 10%! of! bacterial! species! also! produce! phosphatidylcholine! (PC)! [67].! PCOproducing! bacteria! are! confined! primarily!to!AlphaO!and!Gammaproteobacteria,!and!include!P.,aeruginosa.!PC!can!be! made! by! methylating! phosphatidyethanolamine! (SAMOdependent! methylation! pathway)! or! by! condensing! free! choline! directly! with! CDPOdiacylglycerol!

(phosphatidylcholine!synthase).!P.,aeruginosa!exclusively!uses!phosphatidylcholine! synthase! (pcs), to! produce! PC! and! is! thought! to! rely! on! the! host! for! a! supply! of! choline! (Figure! 3O7)! [68,! 69].! Interestingly! enough,! mutants! lacking! genes! to! synthesize! PC! show! a! wide! range! of! phenotypes! including! decreased! virulence,! decreased!swarming!motility,!inability!to!escape!macrophages,!growthOimpairment! under!microOaerobic!conditions!and!reduced!survival!upon!freezing![69].!!

! While! the! importance! of! PC! for! P.,aeruginosa! remains! to! be! determined,,P., aeruginosa! is! the! only! PCOcontaining! bacteria! reported! to! have! a! PCOspecific! phospholipase!(pldA)!that!localizes!to!the!periplasmic!space![68,!70].!Considering!its! localization,! pldA! may! be! involved! in! lipid! maintenance! and! selectively! cleave! PC! into! lipid! signaling! molecules! (analogous! to! eukaryotic! signaling! processes)! to! respond!to!environmental!cues![68].!Conversely,!another!phospholipase!D!from!P., aeruginosa! (pldB),! has! recently! been! characterized! as! a! type! IV! secretion! system! effector.!Secreted!from!P.,aeruginosa!along!with!three!cognate!proteins,!it!has!been! shown! to! invade! both! prokaryotic! and! eukaryotic! cells! and! exhibit! antibacterial! activity![71].!PldA!may!be!involved!in!similar!pathways.!!

! 129!

! ! ! !

(A) IpxA cdsA plsB 3853 pcs 3859 3860! ! ! ! ! !

! ! ! !

(A) IpxA//! //! cdsA //! plsB//! 3853 ! pcs 3859 3860

! ! ! ! //! //! //! //! ! (B) Extracellular Medium Bacterial Cytosol (B)Extracellular)Medium) fattyBacterial)Cytosol) acid Phosphatidylcholine Extracellular Medium PA3860 Bacterial Cytosol glycerol-3-P acyltransferase fatty acid LCFA acyl-CoA O-acyltransferase Lysophospholipid fatty acidGlycerol-3-phosphate PA3860 PhosphatidylcholinePhosphatidylcholine (PC)

PA3859 (?) fatty acid 2-acyl-glycerol-3-P PA3860

! ! ! !

acyltransferase(A) IpxA cdsA plsB 3853 pcs 3859 3860! glycerol-3-PO-acyltransferase

fatty acid acyl-CoA O-acyltransferase ! ! ! ! glycero-3-phosphocholine ! pldA //! //!LCFA //! //! Fatty acyl-CoA phosphodiesterase glycerol-3-phosphate 1,2-diacylglycerol-3-Pacyl-CoA O-acyltransferase Lysophospholipid 2-acyl-glycerol-3-P cdsA/PA2536 (B)Lysophospholipid choline CTP PA3859Extracellular (?) Medium Bacterial Cytosol 2-acyl-glycerol-3-P fatty acid CDP-diacylglycerol fatty acid O-acyltransferase PA3859? LCFA Phosphatidylcholineglycero-3-phosphocholine choline pcs acyl-CoA O-acyltransferase PA3860 acyltransferase glycerol-3-P fatty acid Phosphatidylcholine1,2-diacylglycerol-3-P phosphodiesterase acyl-CoA O-acyltransferase 1,2-diacylglycerol-3-P Glycero-3-phosphocholineLysophospholipid glycerol-3-phosphate cdsA (PA2536)

PA3859 (?) 2-acyl-glycerol-3-P cdsA/PA2536

fatty acid CTP CTP ! ! ! ! phosphocholine !

(A) IpxA cdsA plsB 3853 pcs 3859 3860

diesteraseglycero-3-phosphocholine Choline acyl-CoA O-acyltransferase ! ! ! ! //! //! //! //! ! CDP-diacylglycerol-3-PCDP-diacylglycerol phosphodiesterase glycerol-3-phosphate 1,2-diacylglycerol-3-P Glycerol-3-phosphate cdsA/PA2536 (B) choline CTP choline pcs Extracellular Medium Bacterial Cytosol pcs CholineCDP-diacylglycerol fatty acid Phosphatidylcholine Phosphatidylcholine choline pcs PA3860 Phosphatidylcholine acyltransferase glycerol-3-P fatty acid Phosphatidylcholine acyl-CoA O-acyltransferase Lysophospholipid

PA3859 (?) fatty acid 2-acyl-glycerol-3-P

( glycero-3-phosphocholine acyl-CoA O-acyltransferase

phosphodiesterase 1,2-diacylglycerol-3-P Figure( 377.! Potential!glycerol-3-phosphatePA3860! involvement! in! phosphatidylcholine! (PC)! synthesis.! cdsA/PA2536 choline CTP Gene! context! shows! proximity! of! PA3860!CDP-diacylglycerol to! pcs,! PA3859! (carboxylesterase),!

choline pcs PA3853!(putative!acyltransferase),!and!downstream!genes!involved!in!PC!synthesis.!! Phosphatidylcholine !

! The! gene! encoding! pcs! (PA3857)! falls! three! genes! downstream! of! PA3860!

and! catalyzes! the! condensation! of! CDPOdiacylglycerol! and! choline! to! form!

! 130! phosphatidylcholine! (Figure! 3O7).! CDPOdiacylglycerol! is! formed! by! the! addition! of! two!acylOCoA!molecules!to!glycerolO3P!followed!by!conjugation!of!the!diacylglycerol! to! CDP.! The! fatty! acids! present! in! phosphatidylcholine! in! P., aeruginosa, are!

(primarily)!palmitate,!palmitoleate,!stearate,!and!oleate,!all!substrates!that!PA3860! can!utilize.!OOacyltransferases!specific!for!glycerolO3Ophosphate!and!2OacylOglycerolO

3Ophosphate!add!the!acylOCoA!units!to!the!glycerol!backbone!followed!by!addition!of!

CDP!by!phosphatidate!cytidyltransferase.!While!PA3860!does!not!appear!to!cluster! with! any! OOacyltransferases! or! cytidyltransferases,! the! gene! context! and! previous! studies!suggest!a!possible!role!for!PA3860!in!providing!acylOCoA!substrates!for!1,2O diacylglycerol!synthesis!in!phosphatidylcholine!synthesis.!

! PA3859! encodes! a! characterized! carboxylic! ester! hydrolase! that! has! been! reported!to!have!highest!activity!with!C8!and!C10!carboxylic!esters!in,vitro!but!there! is!some!evidence!that!it!prefers!C16!and!C18!chainOlengths!in,vivo!and!can!free!fatty! acids! from! lysophospholipids! [72].! Additional! research! is! needed! to! clarify! the! physiological!role!of!PA3859.!If!PA3859!indeed!cleaves!lysophospholipids,!PA3860! may!scavenge!the!longOchain!fatty!acids!released!and!may!point!to!the!involvement! of! both! enzymes! in! membrane! maintenance.! Furthermore,! if! P., aeruginosa! uses! choline!derived!from!host!PC!for!biosynthesis!of!membrane!molecules,!PA3859!may! participate!in!host!PC!breakdown.!!!

! PA3860!appears!to!have!a!narrow!biological!range!as!putative!orthologs!with! moderate! to! high! sequence! identity! (50O100%)! are! only! found! in! Pseudomonas.,

Within! proteobacteria,! the! majority! of! putative! orthologs! are! in! the! genus!

Burkholderia! genus.! ! Outside! of! proteobacteria,! PA3860! is! only! identified! in! three!

! 131! other!species.!Overall,!this!gene!context!is!consistent!with!PA3860!functioning!in!a! specialized!role,!such!as!PC!synthesis.!

, Additionally,! PA3860! may! play! a! role! in! the! synthesis! of! unsaturated! fatty! acids,! given! its! substrate! specificity! profile.! Producing! unsaturated! fatty! acids! to! incorporate! into! phosphatidylcholine,! phosphatidylethanolamine,! and! phosphatidylglycerol! is! essential! for! regulating! membrane! fluidity! in! response! to! changes! in! environment! and! temperature.! All! bacteria! produce! unsaturated! fatty! acids! anaerobically! using! the! fabAB! dehydrogenase/isomerase! and! fatty! acid! synthesis! intermediate! βOhydroxydecanoylOACP! as! part! of! type! II! fatty! acid! biosynthesis![73].!In!addition,!some!bacteria!contain!oxygenOdependent!desaturases! that!use!saturated!phospholipid!substrates.!P.,aeruginosa!has!two!desaturases,!desA! and! desB,! and! while! desA! functions! similar! to! other! bacterial! desaturases! (using! phospholipid!substrates),!desB!is!unique.!Unlike!any!other!bacterial!desaturase,!desB! selectively! converts! palmitoylOCoA! and! stearoylOCoA! to! their! monounsaturated! counterparts! and! provides! a! way! for! P.,aeruginosa! to! quickly! alter! its! membrane! fluidity!when!longOchain!fatty!acids!are!available![73].!While!fadD1!may!be!the!sole! provider!of!fatty!acylOCoA!to!desB,!as!it!is!the!major!contributor!to!fatty!acid!and!PC! degradation,!it!is!possible!that!PA3860!could!moonlight!in!this!role.!

Regulation,of,Virulence,Factor,Production,,

Along!with!the!NOacylhomoserine!lactone!class!(HSL),!the!4Oquinolone!class! of!signaling!molecules!is!one!of!the!largest!groups!of!chemically!distinct!regulators! of! biofilm! formation! and! virulence! factor! production.! With! over! 50! known!

! 132! quinolones! produced! by! Pseudomonas, aeruginosa! alone! [74],! many! of! these! secondary! metabolites! have! been! shown! to! exhibit! broadOspectrum! antibiotic! properties! as! well! as! quorum! sensing! abilities! [75O77].! Perhaps! the! most! prolific! example! of! the! latter! is! PQS.! As! discussed! earlier,! PQS! plays! a! major! role! in! enhancing! P., aeruginosa, virulence! by! functioning! in! the! lasCRhlCpqs, regulatory! signaling!cascade!(Figure!3O2).!Besides!playing!a!major!role!in!quorum!sensing,!PQS! has! been! shown! to! exhibit! both! pro! and! antiOoxidative! properties! during! times! of! oxidative! stress! and! is! thought! to! play! a! further! role! in! enhancing! P., aeruginosa, virulence!by!breaking!down!weaker!members!of!the!bacterial!community!to!form! smaller,! separate! populations! that! are! able! to! better! cope! with! and! ultimately! overcome!antibiotic!and!oxidative!stresses!in!a!“survival!of!the!fittest”!manner![78].!

Interestingly! enough,! PQS! synthesis! has! been! shown! to! be! upregulated! in! P., aeruginosa, isolates! taken! from! CFOinfected! infants,! indicating! the! additional! importance!of!PQS!during!the!early!stages!of!bacterial!pathogenesis!in!the!CF!lung! environment![79].!

Analysis!of!the!PA0996!gene!context!reveals!it!to!be!the!first!gene!of!a!pqs! operon,! sitting! adjacent! to! pqsB/C,! both! βOketoOacylOACP,! pqsD,! a! transacetylase! homologous! to! FabH1! and! pqsE,! a! response! effector! that! is! not! thought! to! be! involved!with!actual!PQS!biosynthesis![80].!!Found!directly!upfield!and!in!the!same! reading! frame! are! the! genes! encoding! phnA! and! B,! the! two! components! of! the! anthranilate!synthase!complex!(Figure!3O8A).!Along!with!pqsH!(found!much!farther! upfield),! this! gene! cluster! is! known! to! be! responsible! for! the! biosynthesis! of! PQS!

(Figure!3O8B)![80].!As!previously!stated,!the!starting!material!for!the!pqs!pathway!is!

! 133! pqsA% pqsB% pqsC% pqsD% pqsE% phnA% phnB% A% (PA0996)% (PA0997)% (PA0998)% (PA0999)% (PA1000)% (PA1001)% (PA1002)%

NH2 NH2 B% NH2 COOH COOH COOH kynB% kynA% O O

CHO N NH2 H Kynurenine% Formyl%kynurenine% Tryptophan%

kynU%

O O COOH OH pqsABCD% pqsH%

NH2 N N H H Anthanilic%acid% 2HheptylH4Hquionolone% PQS%

phnAB%

COOH

CH2

O COOH OH Chorismic%acid% (

Figure( 378.! PQS! biosynthetic! pathway! in! P., aeruginosa.! (A)! Organization! of! the!

PA0996! gene! cluster! in! P., aeruginosa, PAO1! to! show! coOlocalization! of! pqs!

(red/black)!and!phn!pathway!genes!(blue).!(B)!Reaction!steps!of!the!two!routes!for!

PQS!biosynthesis.!The!“tryptophan”!route!is!catalyzed!by!the!kynurenine!pathway!

(green)!while!the!“chorismate”!route!is!catalyzed!by!the!phn!pathway!(blue).!Both! pathways!converge!to!utilize!the!pqs!pathway!(red/black).!

! anthranilate,! which! can! be! produced! from! two! separate! sources.! The! first! source! comes!as!a!byproduct!of!tryptophan!metabolism!by!way!of!the!kynurenine!pathway.!

! 134! Previous! works! have! shown! that! the! majority! of! anthranilate! needed! for! PQS! synthesis! is! produced! through! this! pathway! [81].! However,! when! tryptophan! is! unavailable,!anthranilate!can!also!be!produced!from!chorismate!through!the!action! of!phnA/B.!In!direct!support!of!these!findings,!a!variety!of!knockout!growth!studies! have!verified!the!role!of!PA0996!in!PQS!biosynthesis![80].!!

In!nature,!PA0996!appears!to!be!quite!rare.!In!fact,!outside!of!various!strains! of! P., aeruginosa,! we! detected! no! orthologous! sequences! within! the! genus!

Pseudomonas! or! outside! the! Proteobacteria! phylum.! Within! Proteobacteria,! only! nine! putative! orthologs! have! been! revealed,! found! in! the! Alpha! (1),! Beta! (5)! and!

Gamma! (3)! classes.! Within! Betaproteobacteria! are! species! from! the! genus!

Burkholderia.!This!is!not!completely!surprising!given!that!it!was!once!considered!to! be!part!of!the!genus!Pseudomonas,!and!in!fact,!is!known!to!contain!plant,!human!and! animal! pathogenic! species,! including! B., cepacia,! a! member! of! the! CFOinfecting!

Burkholderia, cepacia! complex! [82].! The! pqs/phn! cluster! appears! to! be! highly! conserved! throughout! various! strains! of! P., aeruginosa, and! moderately! conserved! throughout! just! a! few! species! of! Burkholderia.! This! coincides! with! recent! findings! that! some! pathogenic! species! of! Burkholderia,have! been! found! to! utilize! the! PQS! signaling!pathway!in!a!similar!manner!as!P.,aeruginosa![83].!Given!the!very!limited! biological! range! and! overall! necessity! of! PA0996! for! PQS! biosynthesis,! this! data! would!support!a!model!in!which!P.,aeruginosa,and/or!another!related!pathogenic! species!have!adapted!and!evolved!the!pqsA!gene!specifically!to!enhance!virulence! for!a!greater!survival!advantage!within!a!host!environment.!!

! 135! 3.4(Summary(

! In! this! chapter,! we! attempted! to! the! characterize! the! contributions! of! nine! standalone!acylOCoA!ligases!towards!enhanced!virulence!in!P.,aeruginosa.,Through! highOthroughput! substrate! screening! and! specificity! profiling! via! steadyOstate! kinetics,! we! discovered! a! myriad! of! acylOCoA! synthetase! activities,! ranging! from! aromatic! to! shortO,! mediumO! and! long! chain! fatty! acid! activation.! As! a! result,! it! appears!as!if!the!individual!ligases!function!in!a!variety!of!metabolic!pathways,!some! specific!to!certain!functions!while!others!share!an!overlapping!functionality.!!

! PA0996! (already! characterized! as! pqsA)! was! verified! in! its! activity! as! an! anthraniloylOCoa! ligase,! functioning! in! the! synthesis! of! PQS,! a! critical! quorum! sensing!molecule.!PA0996!appears!to!be!the!only!ligase!functioning!in!the!activation! of!aromatic!carboxylate!substrates,!as!the!remaining!eight!ligases!target!alkyl!fatty! acids! of! varying! length! and! saturation.! Overall,! four! ligases! (PA0887,! PA1997,!

PA2555,!PA2557!and!PA3568)!were!characterized!as!shortOchain!ligases,!capable!of! activating!a!carboxylate!derivatives!in!the!range!of!C2OC4,!including!branched!and! unsaturated!substituents!as!well!as!acetoacetate.!This!is!indicative!of!a!high!degree! of!scavenging!ability,!as!P.,aeruginosa,may!be!capable!of!utilizing!the!byproducts!of! various!fermentation!and!amino!acid!degradation!pathways,!and!really!highlights!a! potential!basis!for!enhanced!virulence.!

! Alternatively,!a!number!of!longOchain!fatty!acids!(PA1617,!PA2893,!PA3299,!

PA3300,!PA3860!and!PA3924)!are!found!in!the!P.,aeruginosa,genome!as!well.!While!

PA3299!and!PA3300!appear!to!operate!in!typical!βOoxidation!pathways,!PA1617!and!

PA2893!have!been!implicated!in!the!utilization!of!acyclic!terpenes.!Additionally,!the!

! 136! results! of! our! activity! assays! indicate! that! PA3860! and! PA3924! may! be! involved,! respectively,!in!phosphatidylcholine!(PC)!and!PHAMCL!synthesis.!Both!pathways,!rare! in!bacterial!species,!are!likely!to!enhance!P.,aeruginosa,virulence!as!well.!

! Altogether,!the!results!taken!from!this!work!give!us!a!broader!understanding! of! the! potential! energy! acquisition! pathways! employed! by! P., aeruginosa, during! pathogenesis! in! CF! airways.! While! antibiotic! treatments! are! somewhat! effective! during!intermittent!infection!early!on,!they!are!rendered!all!but!useless!once!chronic! infection! is! established.! It! is! our! hope! that! by! better! understanding! the! diverse! metabolic! pathways! available! to! P., aeruginosa,! differential! treatments! targeting! these! survival! mechanisms! may! one! day! be! developed! and! will! provide! a! more! effective!treatment!for!the!thousands!affected!by!this!terrible!disease.!!

(

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(

! 152! Appendix(

(

A)1.(Multiple(Sequence(Alignments(

A)1)1.(Alignment!of!flK!(F8JPF9)!ortholog!representatives!based!on!CDAHIT!

Clustering.!

(

A)1)2.(Alignment!of!flK!(F8JPF9)!with!Actinobacteria!representative!orthologs.!

(

A)1)3.(Alignment!of!flK!(F8JPF9)!with!Bacteroidetes!representative!orthologs.(

(

A)1)4.(Alignment!of!flK!(F8JPF9)!with!Proteobacteria!representative!orthologs.(

(

A)1)5.(Alignment!of!flK!(F8JPF9)!with!Firmicutes!representative!orthologs.(

(

A)1)6.(Alignment!of!flK!(F8JPF9)!and!BVU_1957!(A6L1R5)!with!DeinococcusA

Thermus!representative!orthologs.!

Note:!All!sequences!are!listed!by!UniProt!ID.!Amino!acid!positions!highlighted!in!red! denote!100%!conservation.!Positions!highlighted!in!yellow!denote!highly!conserved! regions.!Structural!overlay!is!based!on!flK!crystal!structure!(PDB:!3KV8).!

! 153! A)1)1(

β1 η1 α1 flK TT 1 10 20 flK ...... MKDG M RV G ERFTHDFV V PPHK TV RH L ..... E3BLC4 ...... MVFNNLIGHLKIMKK L EV G MCYSYTYQ V PLNK TV PYQ..... W5WVP2 ...... PQD.....RLEPLPNDPTPPQEPTV L NP G DTFTKHYT I HPGH TV RD I ..... Q8FQC6 ...... MTINLIDNGP I AV G DTHEFRYT V PGNKA V PD L ..... M1UKN7 ...... MTLNLAENGP I KI G DTFTFEYV V PDNKA V PD L ..... F4GGF8 ...... MNHESISQISFDEVYVV P SHQTA.....RSLFTQL...... C5CWG3 ...... MNQQGVSEICFEETYAVSHDQTA.....RSLF A RL...... Q2JHG3 ...... MKP V PL G TLGTWQGI L GPEQ TA EA L ..... W9CWY1 ...... MTR....TLAATVTKE V SPAD SA ERW..... D2SED2 ...... MTP V PV G ASAVLDVV V TPEM TV RFD..... I4EWF8 ...... MIP V PV G ATAELVVV V TPEM TV RFG..... F0RKI4 ...... MRP I PENHSETLTIT V TDDM TV NFT..... H8GSG4 ...... MRT M PE G FQQQLVIT V TDEM TV QF A ..... Q9RR87 ...... MQT I PE G FTQTLEVT V TDEM TV DF A ..... K9ZXJ5 ...... MKE I PA G YTQTLTIT V THEM TV NFD..... E8U6U0 ...... MLRALMRP I PDAFTQTLTVT V TDDM TV HFE..... Q1IWD4 ...... MRP I PA G FTQILTVT V TDEM TV NFG..... C1CY65 ...... MRP I PA G FTQTLTVN V TEDM TV DFG..... D1C846 ...... MRT I PI G HAATFTFV V TPEM TV DFE..... D7CQM9 ...... MNP I PE G YRATFELT V TDAM TV DFE..... D3PRJ9 ...... MKP I PP G YQASFETT V TEEM TV DFE..... D7BIK7 ...... MQA I PI G YRAEFETV V TAEM TV DFE..... F2NK44 ...... MKP I PI G YQAVFETV V TPEM TV DFE..... B7A757 ...... MRP I PI G YEAVFETV V TEAM TV DFE..... H5SBP7 ...... MA A SLD..... E6SLA6 ...... MKPGFVP G LRAEVEAT V DAGM QA AFD..... F8I237 ...... PRL.....DSRRLSK..EEPPMREG L VP G TKATVTTT V TEQMV A RLG..... Q8EMG8 ...... MKEG L NV G YQSTLEIE V TEDMF A QFG..... T0JGA7 ...... MKEG L CI G DTKTITVE V TREMF A QFG..... U5L8R5 ...... MKPG L KE G AAASIHLT V GSEMF A QFG..... U6BAI1 ...... MKLG L AV G DTAEIQAV V SEDMF A RFE..... K0A8N8 ...... MKNG L SL G DTAVIEAI V TEDMF A QFE..... W7LB64 ...... MKPG L QI G NRAVIRAV V SPEMF A QFE..... S2XPF3 ...... MKRG L EL G REESIEII V TEDMY A AFE..... W4RK43 ...... MLDG L IA G RTASIGFI V TPDMF A RFE..... I8UG48 ...... MKPG I KI G DEAVITAT V TPEMF A QFE..... A6CP21 ...... MVFK..RRVNMKSG M KV G QTASVTVT V TPDMF A QFD..... E3IAI3 ...... MKPG M KV G DTAVVEAV V TPDMF A QFE..... C0Z567 ...... MIDR L QP G TTEEFTVT V TKDMLPVFE..... F5SL03 ...... MKPG L KP G HREQMQVT V TGEM TA SFG..... D5WXP8 ...... MQE..GVEGVKPG L NV G HRETMSIV V ASDMCPAFG..... C7QDW7 ...... MTEAPAEGA P LQAQTTLN V TDAD TA RA L ..... M4ZY06 ...... MT V ET G MRGEATLV V TEAD TA CA L ..... D2AZ27 ...... MLTPMT L TP G LRAQLLIM V EKED TA TK V ..... W2ELW3 ...... MS L AP G LRAEVLIM V ERGD TA IR V ..... D6Y6X3 ...... MT L AP G LRATVSVT V GQPD TA IQ V ..... Q47SH7 ...... MA I DP G LTGTAELT V TTAD TA EA L ..... R4L8I1 ...... MELPEFAP G LSAKVELT V TDAD TA QS L ..... U5VXT8 ...... MELPEFSP G LTARVELT V TDAD TA QA L ..... G8SD04 ...... MELPEFAP G LSARVELT V TDAD TA QS L ..... A4X9A9 .....MDVPGGSAQAWPT..RCPTPRLVTMQEPFSP G STARVELV V TVAD TA QA I ..... C4RK94 ...... MQEQSEQP L AP G RSARVELT V TEAD TA LA A ..... W7W6W1 ...... MQEQPESPFTP G LTARVELT V TDAD TA QA V ..... I0L862 ...... MQEQPEPA L AP G LTAQVELT V TDTD TA QA V ..... F4FFD5 ...... MAMQEQPDRV L TP G LAARVEMT V TDDD TA QA V ..... E6VPY1 ...... MNEFKECLP L EV G IGARESVT V APFH TV PE L ..... I4JL97 ...... MNNPTCTS L SI G LRHSATLT V AARH SV PE V ..... N6ZAE8 ...... MPSHPPI L TA G LRHSEQFT V EPRH TV PE V ..... V2TPW7 ...... MKE L TI G LKYTKEIF V DKSL TV PN V ..... Q8RS32 ...... MKDAMAINHP I PL G LRHSQTLR V DDSL TV PA V ..... D3PYM5 ...... MTEIA L TP G LRHSARLT V DESL TV PA V ..... V5PXQ4 ...... MPSPE L VP G LTFEWTYR V PRKAV V PE L ..... Q476Z6 ...... MSPE L RP G LTFTWEYP V PPKA TV PR L ..... V2GN25 ...... MSPD L KP G LAFTWRYT V PPKA TV PN L ..... H5XC58 ...... MKAS L RP G LTAQLEYV V PAER TV PY L ..... A7HFD1 ...... MKST L AP G VSLTFRYQ V PETK TV PH V ..... M1SXE6 ...... MKTS L KPEIKYEYKFM V TDAQ TV PA M ..... D8FEX3 ...... MKKT L KP G IEHTFSFG I TASKI V PA L ..... D9SHK4 ...... MKDT L KP G IRFEHKYL V PANK TV PA L ..... D5CM24 ...... MKDT L KP G IRYEHRFL V PSSK TV PA L ..... S7VEV0 ...... MKET L KP G LTFEFHYK V PEDK TV PR L ..... W6M343 ...... MRES L QP G LTGEFEFV V PPSK TV PH L ..... G7W6G7 ...... MKST L QS G LDYEFKFT V PDNK TV PY L ..... F2NC79 ...... MKDA L KP G LAYEFRYQ V PLER TV PH L ..... I4CEW2 ...... MKSS L QP G LTFRFRFM V PDNK TV PH L ..... W5XXT0 ...... MKDT L TP G LTHTMTYQ V TPER TV PN L ..... E8WN56 ...... MKDT L AA G IGTTLKFS V PVEK TV PC L ..... Q2RWF7 ...... MRDV L KV G MGETLSFE V PREK TV PF L ..... V5SDR4 ...... MKPT L EA G ATARLTFT V TAEK TV PN L ..... N0B387 ...... MRTAAVKPS L QA G ARTQFSFR V PATK TV PH L ..... D6V9R7 ...... MKPS L TA G STHRFTYR V PENK TV PH L ..... J5PI50 ...... MP G LTHRFSYT V PETK TV PC L ..... D3IB68 ...... M I KE G LEHSAHII V EPEH SA LS V ..... F3ZNZ3 ...... M L KE G LDYSTERI V EKSNL A VT M ..... F3QUN7 ...... M EI G LTHTSTLT V ENKHL A IQ A ..... D1W8U0 ...... MM L KE G LTHTSRLT V TEAQ TA QV I ..... U2MMW3 ...... M L RE G LSHTSRLM V CNDN SA IT L ..... E0NSD9 ...... M L KE G LKHVSRLT V SNAQ SA IAQ..... S8FGJ3 ...... M L EE G LTHTSTLV V AREH TA VA L ..... D5ET49 ...... M I EV G LKHTSTLV V TDDV TA VK I ..... R5GHX3 ...... M KTKLYYTSTST V TAEN TA IA L ..... R6ZRJ4 ...... M EK G LSYTSKTI V NRNN TA IA M ..... R6C452 ...... M EK G LTYTSQTT V CKGN TA LT L ..... F0QZ92 ...... M EK G LTYTSKVK V TQAN TA LA L ..... R6CGH3 ...... M EK G LTYTSVAE V TAAN TA IA L ..... B7BGY9 NK.FIIFG...VGFVRLRERITEYGTINNEVTM L EK G LKHTSRTV V LPDNF A LA M .....

( ( ( ( (

! 154! R5W6M0 ...... MPEK G LKYVARTT V GADN TA LA M ..... R6WTD5 ...... MDMTMTQK L EK G LSATATTT V TAAN TA LV M ..... F2BXC3 ...... MKE.EKMN L KT G LNATSTFK I KSTE TA KF I ..... R7L727 ...... MRI...FHSKNLKG V PMQISHTSTCK V NENN TA ER M ..... E6S7M9 ...... MRQFQHT V TPDD TA AK L ..... W9GKR0 ...... MPMRTFQHT V TPAD TA TA V ..... W9GAD5 ...... MRQFQHT V TPDN TA AS V ..... D3QAD8 ...... MRD...LTELD.PT A LQDVSGRVETI V TQAD TA QS L ..... W5THV3 ...... MK L QP G LEAEFSIE V TAAD TA SA L ..... X0PXW9 ...... M.AGPRLAYRRAVRYE V GESD TA TA L ..... D6KEI0 ...... MHYE V TEAD TA EA L ..... V6KXP8 ...... MRHE V TDAD TA VA V ..... R4Z4Z8 ...... MG I SP G LRGEVEFT V ADAD TA RS L ..... E2SCL7 ...... MTTLTKT V RADD TA RA V ..... A3THU5 ...... MD.NS A AQATSVEATFT V TEDD TA AA L ..... R7XZ68 ...... MSEQQATLTFT V TESD TA IA V ..... A1SHD6 ...... M DAAPTATLTFT V TDDD TA AT L ..... B9DSI3 ...... MTIFSKIFDTEEKH SA SQ M ..... G5KAY0 ...... MAIYSKVYETSTEH SA KY I ..... F5ZJ64 ...... MTKYTHQFDTKSEH SA KH L ..... I7MYH5 ...... MTIYSHIYETNAQH SA KA M ..... V6QBM2 ...... MSVFNYQ A EAQH TA SA I ..... H3NJZ3 ...... MTAIYTKNYE V TPEQA A SA V ..... R9LQS4 ...... MMNTKVEFERV V EEDGL A SS M ..... F2I7N4 ...... MDTYFT V LPHQL A NQ V ..... U2QSW7 ...... MN I KLNKLFEKNFL V EKQH SA KN M ..... E5V1L6 ...... MN I ELNKIFETHYT I KGEN SA KS M ..... F3A7K0 ...... MN I QLDKVFEESYV V KNEN SA EN M ..... E4T2L8 ...... MIHSTFTQSTK V SDHQ TA AY L ..... T2NPG1 ...... MESKKDFL V DLQD TA AV L ..... C8ZYY8 ...... MIEKEFL V TEED TA LA V ..... R2SKA9 ...... MDKQILSYD V AENT TA KN V ..... R3WMJ9 ...... MKKFIQHFV V AENQ TA YA L ..... R2TEP8 ...... MEEFSKTFV V GSKD TA KE M ..... S0KCS8 ...... MIQKDFI V TKEL TA AT I ..... I3TU50 ...... MSEIGDLIA....TL.PG.. L TP G LEGRMTHL V RSQDL A DAW..... U2HCP0 ...... MLDFSFLIP....KE.SALT I QP G LRARATHR V DTISL A DQW..... L8F1C4 ...... MST....TS.PDLTPLI G ASATLDHT V APED SA MNW..... G2NQF4 ...... MTAPSAPEP....LV.AGIDSLV G TSAQLVHR V TERDA A TNW..... C3GXB1 ...... MNFQVDSLGKYEYV V QEKDL A NNW..... J8I2Q3 ...... ME L IA G LKSKFTHE I SENEL A TNW..... C8PLZ4 ...... M KI G IKGDTSFT V TKEML A SQDG.... D4J4P3 ...... ME M KA G MKFETEII V NEGML A NHNDP... U2L2J7 ...... M L KE G MKYREEVE V NREM TA ER L G.... U5RWS7 ...... MD.FSKLFKV G STYVSEYI V KPED TA NF I ..... U3QHT6 ...... MKNG L QP G ATCSRTLT V DRQR TI DF L ..... J3H1U4 ...... M.NFST L HP G SATERPVI V DKQR TI SF L ..... R4X4Z9 ...... M.INDS L RV G AHYSRTIE V DVDR TI SF L ..... Q13AZ6 ...... MRPT L IA G LSASHRIE I DPAR SI DF L ..... H6SJ72 ...... MKTT L HE G LCLTRHLS V DAAR TI TF L ..... M2Y638 ...... MEKNV.MKES L VV G QSATRRWV I DEGR TI GI L ..... M2ZCB4 ...... MKST L VA G LTATRRYE V DKGR TI GF M ..... W0SEE6 ...... MSET L QA G LSATRRVE I DRDR TI SF M ..... N6YXR0 ...... MSDT L QP G LSLTRKYD I DAAR TI AF M ..... W8U886 ...... M.KDV..KP G VKASVQRV V SFED TA AA L ..... F8I584 ...... MT.QEFLPHV G MEGQWSWV V GHEH SA DA L ..... S6H1G1 ...... MSTPII G HRHHCAFVPGLQQ TA AA I ..... X7FEJ4 ...... MKMSVT G YPHTCVFTPGPHQ TA AA I ..... Q13BL0 ...... MTTTHALIVEIAEALMD.ARDV L TV G MSAERQIT V TPEQ TV QHF.V... Q6NAK8 ...... MD.ARDF I TV G MSAERLTT V TQDI TV QHF.V... U1HEJ6 ...... MD.ARDF I KI G MSAERMLV V PPER TV GHF.V... H0TP42 ...... MD.ARDI I KI G LRAERILV V PPER TV GHF.L... K8P616 ...... MN.VLEK V AV G ITGETVVT V THDM TV QHF.L... I0G1C1 ...... MN.PLEK V TT G TTAEKLVT V TPEM TV GH V .V... A6CFP8 ...... M.QDQPPRI G ALHSISFE V ESSQ TI .TFSC... L0DK57 ....QVWG..GRLGDWPEEARLTTSILE.MKSIPRT G MSAEEHLV V DDSNR I .RFAD... M2WUQ8 ...... MSLTGSLSQT V SDVD TA AMF..... G7D213 ...... MRQ I PL G AKGTSTLR V QPEHL A NRFKD... B1M8N7 ...... MQD I PL G AKGSFAML V GPSHL A SQFKD... K8NJU9 ...... MKD I PI G AKGRFGLE V TSQHL A SQFKD... F7ZKG2 ...... MKQD L QI G QSHTLTST V DAAR TA EA L .A... K0DQR3 ...... MNPNFLNPISTLIF.SMTT L GK G FTTSLTVT V TEEHL A SR L DI... I5CHG7 ...... M I EP G QAATLTHT V AAADL A SAHAH... G8MI72 ...... M I EK G RAATLTHT V TSDDL A SAHAR... J7J7X3 ...... M I NS G ATATLKRR V AVQDL A SAFGN... G2LGZ5 ...... M L AV G QTETVTYV V TKHD TA HG L SAEWK D8NFT6 ...... M L TI G DTACATTI V TGAEL A DV L SQ... A8ET68 ...... MS L EI G TKATIDYK V LNKDL A AN L QI... D5V0F7 ...... ME L EI G TKDSIEFK V EDKDL A KN L QI... R5P9P0 ...... M L ET G ITYKQSIT V QPKD TA AVY..... R8VSU0 ...... M EL G IKGYKELT V TEDLL A KN V ..... R7BPJ2 ...... M L SA G TTNTEEIM V SDSN TA LS M ..... R6KH82 ...... M I KV G IIGEVKDK V SENN TA KT L ..... R6GT38 ...... M EV G IKGKRERI V PYEQ TA EY I ..... J4TCP2 ...... M L EV G IKGQREII V TKQN TA AG I ..... H1LX05 ...... M L EV G IKGQRETV V TKEK TA TG I ..... R6P4I1 ...... M L NT G IKGSASEI V TNEK TA VA V ..... U2PAA7 ...... MMNFRGGHKM L KA G IKGEQSVE V VYEN TA TA V ..... F7V6W4 ...... M I ET G IKGTLTDT V TKEK TA AA V ..... R5BBK6 ...... M V EI G IKGRQEEI V TEDL TA SH I ..... R6E7S6 ...... M L EK G IKGQEEIT V NENN TA EA L ..... R5SHR8 ...... M L KK G IKGTGRQV V TEKL TA KE M ..... R6DLF5 ...... M L EK G IRGEQTVV V TKEL SA KE M ..... W7UZ64 ...... MKD I TI G LTGTSELT V SENEL A KN V ..... E8RIW0 ...... MDIQ I PT G IKGKEELV V TFEE TA AKY..... R5RJN6 ...... M V KK G IKGHGEIV V TAAL SA EK M .....

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! 155! R7JYS9 ...... M L EV G LKGHQEMT V TDQF TA KT M ..... R5HR96 ...... M L TP G TALELTFP V TETL TA KT V ..... E0E4V9 ...... M L EV G LKGVEKTV V DKTN TA AA I ..... B1C982 ...... MNSK L TV G LKNERELT V EEKHL A SSK..... F4X8V5 ...... MS V EV G IKGRAEDV V SEKN TA QA A ..... R7B458 ...... M L EA G ITNTETTM V TEQN TA LA L ..... U2BAI1 ...... MGNCEGR.E...PLQKGKKRGTRM L EA G TEGYQELV V TMEE TA LA A ..... U2SW53 ...... M L QK G IKNQIERM V TETI TA EH I ..... R9KEX9 ...... M L EA G IKGYQELT V TEGD TA KAH..... R9IHN6 ...... M L ET G MKGYQEMT V TTAD TA KVY..... D4J9J7 ...... M EA G IKNEKSIV V TEDV TA SK V ..... D4J2H1 ...... M L EI G MKGNAETV V TIDN TA KAF..... N2BI76 ...... M L TV G ISKELNRS V TKET TA AA L ..... F7K912 ...... MLLEEREKV L KT G IKGFRELM V TGEN TA SA L ..... R5KDY4 ...... M EI G VKNVIETT V TENN TA VA M ..... R5CE45 ...... M EI G IKNVIEIK V TKEN TA AA L ..... E2ZHD1 WC..KMWYLIKVFKNKMR...HTARKRRGKYMT L ET G IRGEQSVL V TAAN TA KT M ..... R6WHR1 ...... M ES G IKFTLEKV V TNEM TA RN M ..... R7C7Z2 ...... MS L EV G IKGTCTVT A DESN SA KT M ..... C0CNB6 ...... MYD..ES...TENELLTGGFSM L KA G IKGTQEVL V TEAN TA KT M ..... R6IH13 ...... M EI G IKGKVETV V DGSN TA KF V ..... R5CSL0 ...... M EI G IKGRAETV V NHEN TA KF V ..... I5AU99 ...... M L EP G IKGCVERV V TEEM SA LR M ..... R6BVQ8 ...... M L EA G IKGQHTII V EKEN TA AV M ..... G2T231 ...... M EA G ITGTQTIT V TEEK TA QA M ..... R5Q2H0 ...... M EA G IKGRKETL V TEEN SA QA M ..... D7GQ38 ...... M L KT G IKGRQEVV V SDAN SA KT M ..... R6ZN81 ...... M L EA G IKGREEVM V TEAN SA AA M ..... U2D4S0 ...... MRKEVEKM L KA G IQGRQEVM V TEDN SA SA L ..... R7B681 ...... M L EA G IKGRQEVM V TDEN SA AA V ..... C9R9F2 ...... MS L SV G LRGTATWV V REED TA IVQ..... R5ICW6 ...... MATND I KV G ISGTSTTK V CKENC A SL M ..... C0GGI3 ...... M L KQ G LTGEAAMK V AKGN TA ME V ..... A6NV90 ...... MLTQPE...PQFFREDEVSMS I EI G MTGRAECA V TEQN TA LA V ..... R5B634 ...... MS I SV G AVGTASTV V TPEN TA AA V ..... U2BG97 ...... MGLF...QKKLERRNQFMS V TV G SKGRAACV V NDQN TA AA V ..... R5EG72 ...... MG I TV G MKGVAETLCERED TA KE V ..... D4LA09 ...... MKEFAL G TKGEAETQ V DESRL A CT V ..... U2M1B8 ...... MEP I TI G SKGTASVQ V DETNL A VA V ..... A0LP78 ...... MNDRNK L QP G IKGTSRTS V DAGI TA LS M ..... U2D856 ...... MEFN L KE G LSASSEVT V GGNN TA SFY..... Q3A9K1 ...... MAES L TE G LEGTAKTT V SLEK TA KA V ..... E5UZI6 ...... M M ET G LTYTSTVV V SKENV A AT M ..... D4IPX1 ...... M L EK G LSARSAAT V AAGN TA AA M ..... R7D0I0 ...... M L EK G LTFTAHTT V TENN TA AT L ..... R5LPE1 ...... M L ET G LTHTSSLT V TEAL TA KA M ..... V4RI11 ...... MDQ L KP G LTGRAKMV V GTND TA PR V ..... B2A5Z4 ...... M QV G DRNEIVKE V SKEF SA KEF..... V5SDQ6 ...... MTASERSTS L IP G LEGRAETV V THAL TA AA L ..... U5Q519 ...... M ET G KKLIIEKL V KETD TA EYY..... F5YR67 LH..IIWLKGTGLGLKAV...LWYSSAMELNTF L SA G LRAEKTEQ V TDRN TA KSW..... B0TBP0 ...... MEN I KI G LKGFKEEK V TADN TA IKY..... D5X9M4 ...... MDIN L QT G IKGESKVV V SENN TA IAY..... Q67JY1 ...... MS L AP G LTATAETV V TPENC A RA V ..... D6TKN4 ...... MP L AP G MTAEISTL V TPEN TA AA V ..... F6DMU1 ...... MTTE L KP G LTREVETI V DETN TA IAY..... K8DZX3 ...... ME L TV G MCHETSTT V TENQ TA IAY..... A4J0S5 ...... ME L KT G LIHETSIM V EESN TA IAY..... A5D1P6 ...... MARE L SF G LEGEARTT V DENN TA IAH..... F6CP13 ...... MAGE L KI G LRGEAVTR V TEDN TA LAY..... R6M9E2 ...... MLT L KT G IKGQAQVK V VEKN TA KI M ..... D6KHB2 ...... M V SA G QTATATVT V TESNI A KT M ..... F0T2R2 ...... M EI G AKGKSETV V TEKV TA KT L ..... W0EBY5 ...... MD L KV G LKGRAENV V YEAN TA KS M ..... L0FAB3 ...... MGE L TL G AKGRAEKL V DQTN TA KT M ..... I4D8E6 ...... MA I SI G MRGYAETN V TFSN TA KS M ..... G2G1T3 ...... MN L QL G VRGQAQTI V TSLN TA KA M ..... J7IWS0 ...... MD L KV G RKGRAQTT V STLN TA KT M ..... G7WFQ0 ...... MN L IV G MKGEAKTT V TLSN TA KA M ..... H5XVU6 ...... MT L NV G IKGQAQTT V TVSN TA KA M ..... R6J6I5 ...... MKVE L RP G LQGRAEAI V DTTNI A AV M ..... R6I9I9 ...... MLQE L KP G LTGTAETV V RETN TA LA M ..... E4QCS6 ...... MKKPE L QV G LKFEIEEV V SDNML A SHF..... F4A004 ...... MDQEFH I QI G LCAAVETI V AEKD TA ITF..... G8M073 ...... MENIN L KV G LTASIETK V FNKN SA EE M ..... B8I4C0 ...... ME L KV G LTGNAEVL V SESN TA KK M ..... G1UVA4 ...... MKTA L QV G LKGQSETV V GKELL A SE V ..... B6WPN7 LDRKAVPV....QEQEIS...VSATGRDIMETMPKA G MMGLWETT V EEGML A AA V ..... H1HSX5 ...... M L NP G IKGHQEIT V EEKDL A IH V ..... S3MB87 ...... M EV G IKGDCSFV V TKEML A TA V ..... R7K521 ...... MN I EK G LKNTEQFE V TENL TA AS L ..... U2F0D7 ...... MEQENRSYTATEE V TPEK TA KA V ..... G4KR18 ...... M V MI G AKCQMEQV V TDKL TA AV V ..... U2RDD7 ...... M I LI G TKCRLEQT V TDAL TA AN V ..... R9LX73 ...... M I LI G TKCQLEQT V TQEL TA AA V ..... R6H0F9 ...... M L EI G KSYTIEQV V TPEM TA RA I ..... R6D0Y4 ...... M I EV G MTYTAEKT V TPDM TA KA I ..... R6BIU7 ...... M L QV G LKGKKTFV V TTEN TA EH M ..... R6G283 ...... MS L NT G MKMIKTET V TVENA A KT L ..... B0MBZ2 ...... MK I EI G DKITLSEE V TDEL TA AHW..... R5YWL8 ...... MN I HV G DEIELTEE V VPEN TA EKW..... H1D405 ...... M L EV G LKGEVSKV V QAED TA AR V ..... R6F6N6 ...... M L ET G KSYTQEII V GPKD TA LAH..... R5UUV1 ...... M V QV G KSFTQKMT V QLKD TA AVY..... R7G5H6 ...... MKE I KINSAFTQEMI V SKEQL A SH V ..... E4LXV6 ...... MKHKKEGTFMKE I KLHTKLTKKIT V EESML A SH V .....

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! 156! H1BNL1 ...... MKE V QLHAKCQEEVI V DEAKL A CN V ..... A1HRR7 ...... MALAQA L TV G LRGEVTIK V TPDN TA ERF..... F7NH57 ...... MGQEQTVKP L SV G LRGEAESK V TSEL TA ERF..... M1E923 ...... M I EV G LNAEIETI V TDND TA AFY..... C6Q0E0 ...... MEFN V RE G MASTREIY V TENDV A SK L ..... T0N2A7 ...... MGNK L RE G ISAIIEKV V SENEA A SKY..... I7K9K8 ...... MKEELFDK I KV G LNSIIETT V QGKD SA ANY..... R7RUE8 ...... MIKVKE I RV G KRHIVERV A EPKD SA INY..... N1ZJT1 ...... MEFN L SV G MSMEEEYV V TEAD TA VHF..... A6TLR1 ...... MEFN L KE G MAAQVEIT V AQKD TA QAF..... R4KGW9 ...... MSLTKQ I TL G LTGSAQVE V NDQN TA IAY..... J6UG66 ...... MSETAR L VI G AKGHAALT V TEAE SA PR I ..... R9MCN0 ...... MI G KTKEISVT V DREKL A CT V ..... B0PBF1 ...... MADEINSISIQ V VPEHL A CA V ..... R7MJM6 ...... MKE I II G TKGNAEII A EKSTL A VN V ..... R7H2Y6 ...... MEP I TI G ARGTAQTV A DASKL A NA V ..... R5VLB0 ...... MQP I TI G TKASASVQ V TEAQL A VT V ..... D1PP05 ...... M L EP G IQGHAALR V TTEN TA RA L ..... C3J9P1 ...... M L PIHQTCQLEVT L QDHH SA IH V ..... G9YHC1 ...... MDFT L KANAAAEITEV V TEDN TA IKY..... E2ZCX0 ...... MMDFT L KEQSSAELTER V TEKN TA IAY..... R7MZQ7 ...... MEFN I KANSTAEVSEKCTENN TA IK L ..... U7UR89 ...... MEFT L KPQSTAEVSEI V TEDN TA IKF..... F9MNK7 ...... MDFT I RPNAQAEVSEI V NDNN TA IA M ..... D3LTB7 ...... MDFE V QKNSRAEVSEK V TDAN TA VA M ..... T1CHV2 ...... M I QI G DTYNCSTF V NEKN TA AA M ..... F9N406 ...... MA I EI G QVGMATVV V EANN TA EY M ..... J5AQJ6 ...... M I QV G QSATATVT V TESNI A KT M ..... K9D8B9 ...... MS V EV G LKGEATVV V NEQNI A KT M ..... R6UAG6 ...... ME I EI G KKHTEKTR V TDDML A CS V ..... G4Q7C4 ...... MTEFRI G SAAEATVM V MHTN TA KA M ..... D2RNA4 ...... MKEFRI G AVGEATDT V LHTN TA AA M ..... E0NZB5 ...... S...QKERFAMEFNEK V HV G ATHEVHGA V TEEN TA AT V ..... F5RQF3 ....AIDM....GEDAVS...IQRRIYMDYTSK I AV G MTHEAVDT V TDAN TA VT V ..... E4LMB1 ...... ME...KELELFMEFTST I AP G MTHEVQDS V TEEK TA RV V ..... C9LW87 ...... MVETK I EV G MKREAAAK V TEEN TA IE M ..... I0GTL3 ...... MELTD V KV G LQNVVTEE V TQEK TA RA M ..... V2XUL5 ...... M DK P TQHSLTWT V DDTM TA KR I ..... F4X9G0 ...... M EK P TEYTLTWE V TDEM TA KR I ..... R5G526 AHAKKIKAEGARFCDCPA...CTAVAAILADKADLYAPTYSLTWT V TDDM TA KR I ..... F2NH42 ...... MESPFKV G MINELRQKTGPEH SA RRF..... A0LL45 ...... MDTPFHA G MSHELKIKSLPEH SA RRF..... C3X5Q5 ...... MNPKNFF L VP G MAEKTELI V TEEQ TA RYW..... C2MAU3 ...... MNMESK P TTLTKQYI I EPKH SA AD M ..... R2S8F8 ...... MDQTFAKEYT V TADL TA EA M ..... S1MVB3 ...... M L AMNQKAQQTMI V TQKE TA QQ V ..... H6LHK5 ...... MKNE L KT G TSYEKEFL V TKED TA LA M ..... E6MGL5 ...... MTQEQISNK L RS G MEMEKDNT V SREL TA RV V ..... E3GGD1 ...... MQE L KV G MELKKDYM V TRTE TA QA M ..... R5KLD7 ...... MKE V KS G AFAEKTLV V NGEN TA VA A ..... R5XE94 ...... MKE I QLNAEASVSLE V TDDN TA VA A ..... R6J0V9 ...... MKT A VGEVQGEALTL V NTTN TA IA A ..... R7BG85 ...... MKEFHGEYKGEALVT V NSTN TA AA V ..... W1U6V8 ...... MSMI G TKGSFTRV I AATD TA AA V ..... H1D2D9 ...... M I IK G ITAQITRT I TEKD TA EK V ..... R7CTJ9 ...... ME I KE G MTGKAVLT V DTPH TA RV M ..... R6A5L1 ...... MS I TI G MKGTATKI V TEND TA KV V ..... F4GJM3 ...... MTLGIGA L EI G ASRTLVVR V DGTNM A TIY..... R6PY32 ...... MEFNN I VP G MTYEREYI V EESD TA SHF..... E4KRB3 ...... MKEIEKEFQ V TPEL SA AK M ..... K8E3X9 ...... MITKLSKTFI V KEEQ TA IE M ..... R2VCM1 ...... MKEITKKYL V KQEQ SA LK M ..... R2PBH5 ...... MKKITKQYK V SEDQ TA LA M ..... R2R932 ...... MKKLTEYYQ V TKDQ TA LS M ..... S0RW90 ...... MKKLIQLYQ V KEEQ TA LK M ..... R6GEG5 ...... M G IKGELNYT V QEED TA KA M ..... R5ZKL6 ...... MELKSVTIERK V ERANL A SV M ..... U2R6S0 ...... MIPFKMCLEDF I MEQKTIKIERV V EKQHL A SI M ..... B7CCX0 ...... MELKTIRIQRV V EQQNL A SV M ..... U2KTG5 ...... M I QV G MKHKESVE V TWEM TA EK L ..... R5AKF1 ...... MMEA L KT G ATAEKLYS V TEQN TA LA L ..... K6U6J6 ...... MEFN L KE G SSIVKKII V SDNDL A IK I ..... W6N7S3 ...... MEFN L KE G TVFTRKVK V TEND TA LK M ..... G7M699 ...... MELN L KE G TSIIKNLK V TENE TA IK M ..... V8G1J7 ...... MEFN L NE G TSYIKEFK V TENE TA IK M ..... D6BCU5 ...... M L EV G MKLEVEKV V TEND TA AK A ..... E3H9Z3 ...... M KE G MKLILEKT V KAEE TA AK V ..... C3WAL1 ...... M L KE G MNFTQSKV V KGSE TA AK V ..... H1PQ22 ...... M L KE G MSLTLEKI V NENE TA AK I ..... N0B8B7 ...... MERKMADISK L QI G LRGSASAL V VEQRL A PA V ..... F8J518 ...... MADLSQ L RA G LSGTASML V TDERL A TR V ..... R5RY61 ...... M QI G DTYEYKNV V TKEI TA GA L ..... J1HAQ4 ...... MNE L KI G QTISQDYT V TSADL A SK L ..... B2V410 ...... MNEPKI G QISKRELM V TKDTL A VN V ..... R7NT78 ...... M ...ENFVIEKV V TEDML A VN V ..... R6NZJ4 ...... MEE...KTLTKTIK V EEEQL A VS M ..... R5DLE4 ...... MSE I ...NTHSVTIK V EEENL A VS M ..... B8J205 ...... MKKK L EP G LSGTLEIT V SEAML A CN V ..... E2SKL5 ...... MNVKGRYVYERNQTAYKAHKKIT V EESML A SH V ..... C0EF68 ...... MED L KV G LTAAKTVT V GRDDL A VH V ..... B5EG61 ...... MKE L QV G LKHTFSYL V PKER TV PF L ..... W4LKY3 ...... MDIPD L QP G LSAEIT..TEVDD SL VVK..... Q01Y99 ...... MANIPIGTR G EQKRL... V TTENA I SF L .....

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! 157! α2 α3 η2 β2 flK . . T.T . 30 40 50 60 70 flK ....YPESPE.FAEFPE V F ATG F MV GL ME W A CVRA. M APY.. L E...P. G EG SL G TA I .C E3BLC4 ....FPEVEE.L Q VMPE V FT SG F MI GMF E F V CVKA. L KPF.. L N...WPSQQ SV G VGF.N W5WVP2 ....FPEAGE.FADMPD V FT TG C LV AL ME WSCIEQ. L SPH.. L D....S G QI SL G IA M .N Q8FQC6 ....YPESEE.FAAMPR V F ATG FF V GL IE W A CMDH. L KAS.. L P...E. G AI SL G VG V .D M1UKN7 ....YPESEL.F S AMPR V F ATG FF V GL IE W A CMEH. L KDS.. I S...DDNTI SL G VG I .D F4GGF8 .....PHGRGYAETL I ECI ATG Y LV AV VE S I CIKE. M QRH.. V D...HEREVV VG RS I .H C5CWG3 .....PHGREYA Q TL I ECL ATG Y LV AV VE S I CVRE. M QMH.. I D...AMMEVV VG RT V .H Q2JHG3 ...... G NP G V K V IG SP A LL TL IE RCSHTT. L QPF..YE...P.EEAA VG IRF.A W9CWY1 ...... G N SG L D V LS TP A IL GE IE R M CDAV. M KPR.. L A...D. G EM TV G VS V .T D2SED2 ...... E.LG P VHP V Y AT YS MA KHF E E A GRKL. L LSH.. L E...P. G EAG IG SA L .S I4EWF8 ...... E.LG P VHP V Y AT YR MA EHF E E A GRKL. L LRS.. L E...P. G EAA IG RS L .S F0RKI4 ...... E.LG P LHP V YS T YT LA KHF E E A GRKL. L LPF.. L E...P. G ED SI G IQ V .E H8GSG4 ...... E.LG P VHP V Y AT YW LA RHF E E A GRKL. I LPF.. L E...E. G EEG IG TQ L .E Q9RR87 ...... E.LGRVHP V Y AT YW LA KHF E E A GRKI. I LPF.. L D...E. G EGG IG SQ V .E K9ZXJ5 ...... E.LG P LHP V Y AT YW MA KHF E E A GRKI. I LPF.. L E...E. G EGG IG SE V .S E8U6U0 ...... E.LGRLHP V Y AT YW MA KHF E E A GRKI. I LPY.. L E...D. G EQG IG HA V .N Q1IWD4 ...... E.LGAVHP V Y AT YW LA KHF E E A GRKI. I LPF.. L E...E. G EGG IG TD V .Q C1CY65 ...... E.LGRLHP V Y AT YW MA RHF E E A GRKI. I LPF.. L E...D. G EGG IG TQ V .D D1C846 ...... E.LGRVHP V Y AT YW MA KH ME L V GRKI. L IPF.. L E...P. G EEG IG AA V .S D7CQM9 ....QP.EPE.LGKLHP V Y AT YW L TKH VE L V SRKI. I LPF.. L E...P. G DAG IG FE V .A D3PRJ9 ....HPDDPQ.LGKLHP V Y AT YW MA KH ME L A GRKI. I LPF.. L E...E. G EEG IG SK V .S D7BIK7 ....HPHDPR.LGKLHP V Y AT YW MA KH ME L A GRKI. I LPF.. L E...E. G EEG IG SK V .S F2NK44 ...... E.LGKVHP V Y AT YW M TKH ME L A GRKI. I LPF.. L E...E. G EDG IG HE V .E B7A757 ...... E.LG P VHP V Y AT YW MA KH ME L A GRKI. I LPF.. L E...E. G EEG IG SY V .E H5SBP7 ...... GRLIHP L YS T FW LA YH AE V A ARKA. I EPY..FD...E. G ENA LG AG I .E E6SLA6 ...... GRVIHP L YS T WS LV HH LE H A ARRV. L EPY.. L E...P.HEEG VG YA V .E F8I237 ...... G Q KVHP V L AT AQ MI EW ME W A GRKL. I LPY.. L E...D.DEDA VG YA V .D Q8EMG8 ...... DEVVHP A YS T AS MV YH ME W V SRDL. L LPF.. L K...E.QEE SV G AA V .K T0JGA7 ...... GEVVHP V YS T AS MV YH ME W V SRQC. I LPY.. L E...E.NEEA MG AA V .A U5L8R5 ...... G Q IVHQ L FS T AS MV YY ME W A SRKV. I LPF.. L E...E.SEEG MG NA V .S U6BAI1 ...... G Q LVHP A YS T VS MV YH ME W A ARQL. I LPY.. L E...A. G EEG VG GA V .S K0A8N8 ...... GVIVHP A YS T VS MV YH ME W A ARKL. I LPY.. L E...E.QEEG VG GA V .S W7LB64 ...... GEIIHP V YS T VT MI YH ME W A SRLL. I TPY.. L D...K.DEEG MG GA V .S S2XPF3 ...... GEVVHP V YS T VA M TYH ME W V SRKI. I LPF.. L E...E.HEEG MG AS V .Q W4RK43 ...... GKVVHP V YS T VS MV YH ME W V SRQI. I VPF.. L E...D.HEEG MG GA A .T I8UG48 ...... G S LVHP A YS T VS MV YH ME W A ARQI. I LPF.. L E...E. G EEG IG GG V .S A6CP21 ...... GEVVHP V YS T VS MV YH ME W A SRKI. I LPF.. L E...E.NEEG MG GA V .S E3IAI3 ...... G S IVHR A YS T VS MV YH ME W A SRKI. I LPY.. L E...E.HEEG MG AA V .F C0Z567 ...... G Q VVHP V MS T VS MI YY ME W V GRRV. I LPY.. L E...A.DEEGS G FA V .D F5SL03 ...... GKEVHPTLS T VA MI YY ME W V GRQV. I LPF.. L E...E. G EEG VG SE I .S D5WXP8 ...... GVQVHPTLS T VS MI YY ME W V GRRV. I LPF.. L E...E. G EEG VG AA I .S C7QDW7 ....G...... SG D L P V LG TP R LV AL AE Q A TVAA. A QPL.. L T...E. G QT TV G TR I .R M4ZY06 ....G...... SG D I D V LG TP R LI ALC E E A TMAA. L VGS.. L E...A.NSS SV G MR I .R D2AZ27 ....G...... SG D V P V L ATP R LL AF AE Q A TVQA. V AGA.. L E...P. G QT SV G TK V .A W2ELW3 ....G...... SG D V P V LG TP R LL AF AE ATTVKA. V QHH.. L E...P. G QT SV G TK I .R D6Y6X3 ....G...... SG D V P V LG TP R LL AL AE G A TVQA. V RPH.. L E...E. G KT TV G TK V .V Q47SH7 ....G...... SG D V P V LG TP R VL AL AE A A TVAA. L DGR.. L E...A. G HT SV G AE V .H R4L8I1 ....G...... SG D V P V LG TP R VL AL AE T A TVVA.TART.. L P...G. G LT TV G TR V .E U5VXT8 ....G...... SG D V P V LG TP R VL AL AE A A TVAA.TARQ.. I P...G. G VT TV G VR A .E G8SD04 ....G...... SG D V P V L ATP R VL AL AE A A TVAA.TARH.. L P...G. G IT TV G VR A .A A4X9A9 ....G...... SG D V P V LG TP R IL AL AE A A TVAA. I ARQ.. L P...S. G AT TV G VR V .E C4RK94 ....G...... SG D V P V LG TP R VV AL AE A A TVAA.TAGA.. L P...P. G RT TV G TR V .E W7W6W1 ....G...... SG D V P V LG TP R VL AL AE A A TVAA.TATR.. M P...S. G ST TV G VR V .E I0L862 ....G...... SG D V P V LG TP R VL AL AE A A TVAA.TAAT.. L P...P. G ST TV G TR V .E F4FFD5 ....G...... SG D V P V LG TP R VL AL AE A A TVAA.TARH.. L P...A. G TT TV G TR V .E E6VPY1 ....DDTWPG.FRDMPA V L AT AM MI GF VE QTCIEA. L RPY.. L P...P.QQR TV G TH V .N I4JL97 ....EPTWSG.FADMPP V L AT AM MI GF ME QTCIEG. L RPF.. L T...P.EQR TV G TH V .D N6ZAE8 ....DVSWPG.F Q DMPP V L AT AM MV AF IE QTCIMG. L RPF.. L S...S. G QH TV G IH V .D V2TPW7 ....SELFTG.F S DMPP V L AT AY LI GF VE WTCIEA. L HDY.. L M...P.SDK TV G TH V .N Q8RS32 ....SAAFTG.F S DMPP V F AT AY MV GF VE W A CIEA. L RPY.. L A...P.SQR TV G TH V .N D3PYM5 ....NPSFTG.FADMPR V L AT AF LV AF VE STCVEA. L SPL.. L P...S. G TQ TV G TL V .N V5PXQ4 ....YDDVEL.CRDMPA V L ATG Y LA GI LE C A CLQA. I RPY.. L D...WPREQ SL G TL V .S Q476Z6 ....YDDIAM.C T EMPD V L ATG Y MV GI ME C A CLQA. L RDY.. L D...WPREQ TL G TL V .S V2GN25 ....YDDVPG.CPEMPD V L ATG Y MV GI ME C A CLQM. L REY.. L D...WPREQ SL G TL V .S H5XC58 ....LPESEE.FKALPP V L ATG F LV GI VE WTCIRV. L AGH.. L D...D. G EQ TL G IH V .D A7HFD1 ....FPESPR.FVEMPQ V F AT AF MV GL LE W A CIEA. M QPH.. L D...G. G EQ SV G TG I .W M1SXE6 ....YPGSKE.AAVRPK V F ATG F LV GF LE L A CVKA. I ASH.. L D...WPEEQA VG TF I .S D8FEX3 ....YPESDE.F Q AMPN V F ATG Y LV GF IE WTCIQA. L KPH.. L D...WPQEQ TV G TH V .D D9SHK4 ....YPESPE.FLAMPE V F ATG F MV GF LE W A CIMA. I KPH.. L D...WPEEQ SV G TH I .N D5CM24 ....YPEAEE.FLAMPE V F ATG F LV GF LE W A CIKC. I NPH.. I D...WPAEQ TV G TH I .N S7VEV0 ....FPEFPE.G Q IMPN V F ASG Y MI GLF E F A CIKA. V KPH.. L D...WPDEQ TV G IGF.Q W6M343 ....YPEALE.F Q AMPE V F ATG F MV GL IE WTCIQA. L KPH.. L D...WPHEQ SV G TH V .D G7W6G7 ....YPEAEE.F Q AMPK V FG TG F MV GLF E W A CIKA. I NPH.. L D...WPNEQ TV G TD V .K F2NC79 ....LPESEE.F Q QMPK V L ASG F MV GL IE W A CIQA. I KPF.. L D...WPREQ TV G IGF.Q I4CEW2 ....FPESSE.F Q VMPK V L ATG F MV GL VE W A CIRA. I NPH.. I D...WPEEQ TV G TG I .T W5XXT0 ....LPENPD.F T AMPE V F ATG Y MV GV IE A A CMEA. I RPH.. L D...E.DEI SL G TH V .N E8WN56 ....YPESAL.FREMPE V F ATG Y LV GF IE W A CMEA. L APY.. L E...E.DER SV G TM I .N Q2RWF7 ....YPESAE.F Q AIPE V F ATG Y MI GL ME WCCVRS. L APA.. L E...D. G EG SL G IA I .N V5SDR4 ....YPESAD.FRAMPG V F ATG F MV GL ME W A CLEV. L KPH.. L D...A. G EG SL G IH V .D N0B387 ....YPEARE.F Q LMPT V F ATG F MV GL ME WTCLHI. I DPH.. L D...P. G EG SL G VH I .N D6V9R7 ....FPEARD.F Q IMPH V F ATG Y MV GL ME W A CMDM. I RPH.. L E...E. G EG TL G TL I .N J5PI50 ....YPEAPE.F S AMPK V F ATG F MV GL ME WTCVRL. M APH.. L D...A. G EG SL G VH V .D D3IB68 ....G...... SG D M Q V L ATP A LI AF ME N V AMLT. V RDV.. L S...D.EVT TV G GM I .E F3ZNZ3 ....G...... SG D L Q V L ATP A LV AA ME N A AMLA. V ADA.. L D...P.TDT TV G GH I .D F3QUN7 ....G...... SG D L E V L ATP V MM AL ME N A AMLA. V KEA.. L D...E. G MT TV G GH I .S D1W8U0 ....G...... SG D L P V L ATP A ML AL ME N A AMLA. V ANE.. L E...E. G QT TV G GH I .S U2MMW3 ....G...... SG D M P V LG TP A ML AL ME N A AMLA. V AEE.. L D...E. G TT TV G GH I .E E0NSD9 ....G...... SG D L P V L ATP A ML AL ME N A AMLA. V ADS.. L P...E. G TT TV G GQ I .A S8FGJ3 ....G...... SG D M D V L ATP A MM AL ME N A AMWA. V VHA.. L P...E. G ST TV G GS M .T D5ET49 ....G...... SG D M A V L ATP A MM AL ME N A AMLA. V ADA.. L P...E. G ST TV G GH I .A R5GHX3 ....G...... SG D M E V L ATP A LV AL ME N A AMEA. V IEE.. L D...D.EQT TV G AH I .D R6ZRJ4 ....G...... SG D L E V F ATP A LV AL ME N A AKQA. V ENY.. L P...E. G ST TV G AQ I .Q R6C452 ....G...... SG D M E V F ATP A LV AL ME N A AMNA. V KPY.. L P...K.DST TV G AM I .Q F0QZ92 ....G...... SG D M E V F ATP A LV AL ME N A AMKA. V QPE.. L P...E. G ST TV G AF I .E R6CGH3 ....G...... SG D M E V F ATP A LV AL ME N A AMNA. V KSA.. L P...E. G ST TV G AM I .Q B7BGY9 ....R...... SG D L P V L ATP A LV AL ME N A AMLA. V AAE.. L P...E. G AT TV G VK M .D

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! 158! R5W6M0 ....G...... SG D M E V F ATP A MV AL ME H A AMYA. V AAD.. L P...E. G ST TV G AS M .E R6WTD5 ....G...... SG D L E V F ATP S MV AL ME H A AMTA. V APA.. L P...E. G ST TV G AE M .N F2BXC3 ....K...... SG S L P V L ATP V L CAY IE ETCVKA.SENH.. L P...K. G FT TV G FH I .E R7L727 ....G...... SG D I P V F ATP A MI AL ME N A AMLA. A RNI.. A A...D. G ET TV G GF I .S E6S7M9 ....G...... TG D L D V V ATP R LV EWC E S A AFQV.CQHS.. V D...A.NHT TV G TM V .K W9GKR0 ....G...... SG D L E V L ATP R LI TWC E S A AFEV.CRPQ..TD...E.DRT TV G TF V .K W9GAD5 ....G...... SG D L E V L ATP V LI TWC E N A AFTM.CKSS.. I A...A.DRT TV G TM V .K D3QAD8 ....G...... SG D V P V LG TP R VL AL AE A A IVMS. L TRR.. I P...T.TMT TV G TR V .R W5THV3 ....G...... SG D V D V L ATP R IV AL AE E A TVRA. I ADA.. L S...G. G RT SV G TE V .S X0PXW9 ....G...... SG D V P V L ATP R LI AW ME A A TIAC. A APF.. I G...A. G QT TV G TA V .R D6KEI0 ....G...... SG D V P V L ATP R LI AW LE A A TVRA. A GAF.. L E...A. G RT TV G TA I .R V6KXP8 ....G...... SG D V P V L ATP R LI AW LE A A TVRA. A ARF..TE...S. G QT TV G TE V .R R4Z4Z8 ....G...... SG S V E V LG TP R VL AWC E A A SISA. L ADT.. L P...A. G MT TV G MR V .Q E2SCL7 ....G...... SG D L D V LG TP V L TAWC E E A TCAA. L D.... L P...A.EQT SV G VR V .L A3THU5 ....G...... SG S L P V L ATP R LL AW LE A A TCTS. L EPL.. I G...V. G RT SV G TR V .D R7XZ68 ....G...... SG S L P V LG TP R LL TWC E A A TCAA. L EGS.. L P...A. G GT SV G TR I .S A1SHD6 ....G...... SG D L P V LG TP R LL AW LE A A TCAA. I DPS.. L P...T. G ST SV G TR V .E B9DSI3 ....G...... SG G L S V LS TP S LV AF ME N A AYHL.GETL.LQD...P.KQS TV G SH I .S G5KAY0 ....G...... SG E L E V LS TP S LV GF LE N A ACLL. A KEK.IQN...D.LLT TV G AK M .T F5ZJ64 ....G...... SG S L E V LS TP S IV AF ME HTAYLY. A QET.IKD...S.DYT TV G TE I .V I7MYH5 ....G...... SG T L D V L ATP A LV SF ME N A AYLF. A QES.LE....T. G LT TV G SE M .A V6QBM2 ....G...... SG E L E V V ATP A LI AF AE N A CHQL. V D.... L A...A. G ET SV G TF I .E H3NJZ3 ....G...... SG D L P V L ATP A LI AL ME N A CYTY.SQEVLIED...P.QQT SV G AK I .E R9LQS4 ....K...... SG T V N V L ATP Q MI AW ME E A ASQC. L N.... L N...D.DLT SV G TK I .N F2I7N4 ....G...... SG S L E V LS SP W LL GYF E N A AVRF. L KDH.. L E...E.EET TV G TY A .Q U2QSW7 ....K...... SG D L E V L ATP S LV AF ME Q V SKEY. L NTF.. L E...D. G CG SV G IN I .N E5V1L6 ....G...... SG D L E V LS TP S LV AF ME NSAKNY. L NNF.. L N...E.ELG SV G SN I .N F3A7K0 ....G...... SG D L A V LS TP S LV AF ME N A AKNY. L NKF.. L P...E.EMG SV G SN I .N E4T2L8 ....G...... SG T L E V FG TP A LV AF ME NTAMKM. L S.D.. L P...T.EST SV G IS M .N T2NPG1 ....G...... SG G L A V L ATP R LV AM VE NTCWEA. L EPT.. L D...K. G FT TV G TKF.S C8ZYY8 ....G...... SG G L P V L ATP R LA AM VE A V CFEA. L ESR.. I A...S. G KT TV G TQF.T R2SKA9 ....G...... SG D L S V LS TP A LL AM ME NCAKEL. L SQQ.. L S...E.QET SV G FA V .E R3WMJ9 ....G...... SG E L K V L ATP A LA AM IE NTCMNS. V KNE.. L S...D.SET TV G TL L .S R2TEP8 ....G...... SG D L D V L ATP V MI AM VE NTAKEY. L HKE.. L A...S.EET SV G TV I .N S0KCS8 ....G...... SG G L A V L ATP A VV QM VE NTCYEY. L QQK.. L N...E.QQT TV G TK I .E I3TU50 ...... QG E V S A L AS AP VI WL AE L A CMRA. V AAA.. L P...P. G HT TV G VGF.D U2HCP0 ...... G G E A H A L ASP I MI IF IE QTCMQA.TDHL.. L G...A.DLM TV G YNF.E L8F1C4 ...... GNE L D V LS TP V LL WLS E I A AMKV. I EAA.. V T...A. P AM TV G LAH.D G2NQF4 ...... GND L P V L ATP V LL WLS E I A AMKV. L GTA.. V P...E. G DM TV G LAH.D C3GXB1 ...... END V E V L ATP V LL WL AE VSCMDA. I KNQ.. I E...E.NYM SL G LGHYE J8I2Q3 ...... RND V P V L ATP V LL WLT E LSCMKT. I EQS.E L S...T.NQM TV G YAH.E C8PLZ4 ...... DDT L E V FS TP T MV LK IE RTAALS. V LPF.. M E...E. G YTM VG VK I .D D4J4P3 .QLPA...... IELPA V FS TP S MI NL ME LTCAKL. M DNC.. L E...E. G KG SV G MS V .E U2L2J7 ...... N KG V P V FS TP E ML AF LE RTCRHC. V EPE.. L E...Q. G LG TV G IS V .E U5RWS7 ....G...... N NG V V M LS TP A MI KY ME YTTLHI. V DNV.. I P...K.NYRP VG TK I .D U3QHT6 ...... GES L R I Y ATP E LV RD IE QTCLDF. L QGY.. L E...D. G ES SV G AE V .S J3H1U4 ...... GED L R I Y ATP R LV DD IE QTCLDY. L LTF.. L D...E. G EN TV G AA V .D R4X4Z9 ...... GDD L R I Y ATP K LI DD IE RTCLDY. L LDF.. V D...D.EEN SV G TA V .D Q13AZ6 ...... GET L R V Y ATP E LV RDF E T V CREL. I LAH..TD...P. G ED SV G TG I .A H6SJ72 ...... GDS L R V Y AT YQ II SD IE Y A CRDL. L LAH.. V D...S. G WD SV G TA V .G M2Y638 ...... G P ESR V Y ATP Y LV YD LE TTCREF. L LEH.. L A...P. G ED TV G TR V .E M2ZCB4 ...... GDE A R V YN TP A LI YD ME VTARDL. L LEH..SD...A. G ED SV G TR V .E W0SEE6 ...... GDDCR V YS TP N LL YD IE M A CRDL. L LEH.. I D...P. G KD SV G TR V .E N6YXR0 ...... GDECR V YS TP A LL YD IE VCCRDL. L LQH.. I G...E. G KD SV G TR V .E W8U886 ....G...... N EG I K V LS SP S MI LL ME L A SSKV. L EPL.. L E...E.DER TV G ISF.D F8I584 ....G...... N AG V M V L ATP M VL DL ME I A SVQA. I LPA.. L P...D.DWI SL G IH A .D S6H1G1 ....G...... N AG V Q V VS TP A LI AL LE ETAGSG. V QAF..CV...P. G WG TV G TK V .D X7FEJ4 ....G...... N TG V K V VS TP A LI AL LE ETSADA. V AKD.. L P...S. G WV SV G TH V .D Q13BL0 ...... PYMPA V F ATP L MI LE ME M A SGEA. V HPK.. L P...D. G WV TV G TG V .D Q6NAK8 ...... PSMPA V F ATP M MI LV ME MTSGDA. I APK.. L P...P. G WV TV G SE V .D U1HEJ6 ...... P G MPM V Y ATP M MI LD ME M A SGDA. I RGA.. L Q...P. G WV TV G TE V .D H0TP42 ...... AHMPM V Y ATP M MI LE ME MTSGDA. I SAA.. L P...E. G WV TV G TE V .E K8P616 ...... ATMPP V Y ATP V MI LH ME E A CTAS. I AGL.. L P...E. G YV SV G ME V .N I0G1C1 ...... P G MPE V YG TP M MI LH ME M A AGSA. V QPS.. L P...A. G HV SV G MM V .N A6CFP8 ...... AAE I S V LS TP S LI WF LE Q A ALQF. L LPW.. L D...E.KSI SV G TH V .D L0DK57 ...... ARMPS I L ATP W LV AH LE Y A ARAA. I APC.. L E...D.HER SV G TF V .E M2WUQ8 ....G...... PDFPK A AS TP F VL GL AE V A CHNI. V AGE.. L Q...D. G EL TV G TA A .T G7D213 ...... ALLPQ V L ATP V MI LV ME N A ALNA. M RPY..FD...A. G ESA VG TA V .D B1M8N7 ...... N ILPP V F ATP M MV LI ME N A ALNA. V RQY.. L D...P. G ESA VG TK V .D K8NJU9 ...... PMLPP V F ATP M MI LA ME N A ALNA. I RAY.. L E...P. G ESA VG TV V .D F7ZKG2 ..AQG...... EALPE V F ATP F MI AD LE R A CAAL. L GPL.. L S...D. G EV SV G AR I .E K0DQR3 ..DAG...... ERFPN V LS TP W LI AH LE R A AAKA. L HPL.. L Q...D. G QL SV G SR V .V I5CHG7 ..AAG...... ERYPD V LS TP A LL TL IE R A CAGL. M HDA.. L R...D. G QL SV G VTT.H G8MI72 ..GAG...... ERYPD V LS TP A LL AL IE R A CADA. L RDA.. V G...D. G QL SV G VTT.H J7J7X3 ..DSD...... ETYPD V MS TP A ML GL ME R A CAEI. M RGE.. L E...D. G QL SV G VTT.Q G2LGZ5 DERYQ...... DAY I D V F AT IR MI AL ME L V CGRM. L ERI..QA...A. G EL SV G VE V .N D8NFT6 ..TAE...... DAFPP V Y AT SR MV GL ME L A AARV. M RQA.. L A...A. G EL SV G VT V .E A8ET68 ..SKD...... DAFPE V F AT AR MV AL ME CSAAKM. M LPL.. L K...D. G EL SV G VE V .N D5V0F7 ..SPE...... DNFPE V F AT AR MV AL ME CSAAKI. L IPF.. L E...E. G QL SV G VE V .N R5P9P0 ....G...... SG S L E V F ATP A MV AL ME NTAVHC. L KGH.. L E...E. G QD TV G IE I .N R8VSU0 ....G...... SG L V A V Y ATP M MI AG IE GTAAES. V APY.. L E...E. G RT TV G TQ M .N R7BPJ2 ....G...... SG T L R V F ATP A MI AL ME GTAAQS. V EPL.. L E...K. G QT TV G TE I .S R6KH82 ....R...... SG T L N V Y ATP A MV AL ME E A AYKS. I ESE.. L E...E. G QG SV G TS M .N R6GT38 ....S...... SG L L P V F ATP C MI QF ME ETARLS. V EPY.. L A...E. G QS TV G TL V .N J4TCP2 ....G...... SG S L E V FS TP I MI LL ME ECCFMS. V DDK.. L E...E. G FT TV G IS V .N H1LX05 ....G...... SG L L E V F ATP V MI AL ME ESAFMS. V NEI.. L D...E. G FT TV G IA V .D R6P4I1 ....G...... SG G L S V Y ATP A MI SL ME KSAYES. V QSL.. L E...E. G SG TV G TL M .N U2PAA7 ....G...... SG V L E V FG TP C ML AL ME KTASES. V APY.. L E...E. G CG SV G TQ V .T F7V6W4 ....G...... SG V L E V YG TP V MI AL IE KTACDS. V APY.. L E...E. G SG TV G TN L .T R5BBK6 ....G...... SG S V R V F ATP M MI AL ME RTSRMS. V KPY.. L E...D. G QE TV G TK V .D R6E7S6 ....G...... SG S L A V F ATP A MI AL ME KTARLS. V APY.. L E...E. G QS TV G TL V .N R5SHR8 ....G...... SG E L A V Y ATP A MI AL ME ETAYKS. V TAE.. L E...D. G MG TV G TL M .N R6DLF5 ....G...... SG E L M V Y ATP A MI AL ME KTAYTS. V ASE.. L E...E. G MG TV G TL M .N W7UZ64 ....G...... SG S L E V F ATP V MI ML ME K A ACSC. I TEY.. L E...G.DET TV G TE M .N E8RIW0 ....G...... SG L V E V Y ATP A MV AL ME KTCLKS. V LPY.. L P...E. G FG TV G IK V .D R5RJN6 ....G...... SG V M D V F ATP A ML AL ME NTAYKS. V LPY.. L N...E. G CG TV G TK V .D

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! 159! R7JYS9 ....G...... SG V M D V F ATP A ML AL ME KTAFMS. V AEH.. L N...E. G CG TV G TR V .E R5HR96 ....G...... SG T L D V L ATP V MI AR ME Q A AWTA. V APS.. L S...P.EEG TV G TW M .N E0E4V9 ....G...... SG G L E V FS TP S MI SL ME CTCKLC. A QEH.. L E...E. G LG TV G IS I .S B1C982 ....G...... SG A A E V FS TP D ML LL ME GTCFKL. A EEY.. L D...E. G ES TV G TA A .N F4X8V5 ....C...... SG A L P V FG TP F M TAL ME K A AWTS. I APY.. L A...P. G ES TV G TK L .D R7B458 ....G...... SG T V E V F ATP A MI NL IE YTCARS. V EDE.. L D...P. G QT TV G TN L .N U2BAI1 ....G...... SG G L R V L ATP V M CAL ME K A CWKS. A AGL.. L G...E. G ED TV G IS L .S U2SW53 ....G...... SG E L S V L ATP A MI AL IE ETSWRS. V AAG.. L A...E. G QG TV G TK I .D R9KEX9 ....K...... SG T L D V L ATP R MA AL ME ETAWRS. V AGE.. L E...E. G MG TV G IK L .D R9IHN6 ....K...... SG T L D V L ATP R MA AL ME ETAWKS. V AEH.. L E...Q. G MG TV G IQ L .N D4J9J7 ....G...... SG L L P V Y ATP S MI AL ME GTCAES. V QSE.. L A...E. G EG TV G VS V .D D4J2H1 ....A...... SG A L E V F ATP A MI AL ME ETCWKM. I QPE.. L E...E. G LG SV G TK V .N N2BI76 ....G...... SG L L D V Y ATP A MI AL ME ETCMKS. V QEE.. L D...E. G CG TV G TG L .T F7K912 ....G...... SG R L C V L ATP A LA GL VE RTCWES. V EPL.. L E...E. G ET TV G TA L .A R5KDY4 ....G...... SG T L S V F ATP A MI AL IE ETAWRS. V VPY.. L E...P. G QA TV G TR L .D R5CE45 ....G...... SG T L S V F ATP A MI AL IE QTAWKS. V EPY.. L D...E. G QG TV G TK L .D E2ZHD1 ....G...... SG T L E V F ATP A LV AL AE KTCWMS. V ADA.. L G...E. G NG SV G TK L .E R6WHR1 ....A...... SG T L D V F ATP S MI AL IE ETAWKS. V AQE.. L E...E. G MG TV G IS L .N R7C7Z2 ....R...... SG T L D V F ATP A MI AL ME KTAWTS. V APY.. L E...P. G EG TV G IH L .D C0CNB6 ....G...... SG T L D V F ATP A MI AL ME LTAWKS. V APY.. L E...E. G NG TV G SY L .D R6IH13 ....G...... SG S L D V F ATP C MV GL ME Q A AQMS. V APY.. L G...E. G EG TV G TK L .E R5CSL0 ....G...... SG E L E V F ATP Q MI AL ME Q A AASS. V AGE.. L E...E. G QG TV G TL L .N I5AU99 ....G...... SG E L M V L ATP C MI AM IE E A AWKS. I SAE.. L E...E. G QG SV G TL M .D R6BVQ8 ....K...... SG T L A V F ATP A MI AL IE ETAWKS. V ADE.. L E...E. G CV TV G TS L .N G2T231 ....G...... SG T L P V F ATP A MI AL ME NTAYKS. V ADE.. L E...E. G AG TV G TL M .N R5Q2H0 ....G...... SG L L P V Y ATP A MV AL ME G A ASES. V APY.. L E...E. G TT TV G TA L .N D7GQ38 ....G...... SG T L D V F ATP A M TAL ME KTAWMS. V ADQ.. L D...E. G CG TV G TL L .N R6ZN81 ....G...... SG L L P V F ATP A MV AL ME QTSWKS. V APY.. L N...E. G EG TV G TS L .N U2D4S0 ....G...... SG L L E V F ATP A MV AL ME KTAWMS. V APY.. M N...D. G EG TV G TM L .N R7B681 ....G...... SG L L P V F ATP A MV AL ME KTAWMS. V APY.. L N...D. G EG TV G TK L .E C9R9F2 ....G...... SG T V P V L ATP R LL AL ME A A AVAA. V AGA.. L P...P.EAT TI G VK A .E R5ICW6 ....G...... SG A L D V F ATP A VV AY ME M A ACYA. V NDL.. L E...E. G LS TV G TK I .D C0GGI3 ....G...... SG S V P V F ATP M LV AL ME N A AINA. L KGQ.. L E...D. G QT TV G MK V .D A6NV90 ....G...... SG A L P V F ATP M MA AL ME A A AMNA. V SAC.. L E...E. G QG TV G TK L .D R5B634 ....G...... SG A L P V F ATP Y MI AL ME N A ACNA. I ADG.. L E...E. G QS SV G TK L .D U2BG97 ....G...... SG L L P V F ATP Q MI AL ME N A AVDA.TQAS.. L E...E. G QG TV G TR L .D R5EG72 ....G...... SG D L L V Y ATP C MV AL ME G A ACEA. V AEG.. L E...E. G QT TV G TA L .N D4LA09 ....G...... SG S L P V Y ATP M MV AL ME Q A ACVA. V RDA.. L E...E. G ET SV G TA L .N U2M1B8 ....G...... SG S L P V F ATP M MA AL ME E A ACNA. V APF.. L S...E. G ET TV G TK L .D A0LP78 ....G...... SG E I E V L ATP A MA AL ME A A AVNC. V KPY.. L P...S.NET SV G VH I .E U2D856 ....G...... SG S L E V F ATP A MI AI ME N A ALKA. V QSE.. L P...E.DFT TV G IE V .S Q3A9K1 ....G...... SG S L E V F ATP M MV AL ME K A AVNA. V AVA.. L S...P. G ET TV G TK I .E E5UZI6 ....G...... SG D L N V F ATP A MV AL ME N A AMSA. V ADE.. L P...E. G ST TV G AM M .N D4IPX1 ....G...... SG D L A V F ATP A MV AL ME N A AMTA. V AGE.. L P...E. G AT TV G SE M .N R7D0I0 ....G...... SG D M N V F ATP A LV AL ME H A AMLC. V APH.. L P...E. G ST TV G SL I .H R5LPE1 ....G...... SG D L P V L ATP A MM AL ME N A AMTA. V APE.. L P...E. G ST TV G GH I .E V4RI11 ....G...... SG H V H V L ATP V MI GL ME E A ALAA. V EHL.. L P...E. G KQ SL G TR M .D B2A5Z4 ....G...... SG S V D V L ATP A MI AF ME E A ALKL. V DSQ.. L D...E.DRA TV G SL V .N V5SDQ6 ....G...... SG T A D V Y ATP A MI AL ME A A AVAA. I EKH.. L A...P. G ET SV G TH L .D U5Q519 ....G...... SG L L K V F ATP A MI AL ME KSAHLL. A KFS.. L P...S.NQD TV G IE V .N F5YR67 ....G...... SG E L P V Y ATP A MI AL ME G A AVAA. V QDK.. L P...P. G IS TV G TE L .Q B0TBP0 ....G...... SG G V E V Y ATP A MI GL ME G A ALAA. V DPL.. L P...E. G MA TV G TY I .A D5X9M4 ....G...... SG G V E V F ATP A MI AL ME N A ALNS. V EKF.. L P...P. G HT TV G IN I .Q Q67JY1 ....G...... SG A V D V F ATP M MI AL ME E A AARA. V EPF.. L G...E. G ET TV G TR V .D D6TKN4 ....G...... A G G V E V F ATP M MI AL ME N A SWQA. V APG.. L D...K. G HV TV G TL V .N F6DMU1 ....G...... SG G V N V F ATP A MI GL ME K A ALEL. A DSY.. L P...E. G QT TV G TL V .N K8DZX3 ....G...... SG G V R V F ATP A MI GL ME K A ALEL. A DQC.. L P...A. G QT TV G TL V .N A4J0S5 ....G...... SG G V R V F ATP A MI GL ME K A ALEL. A DQY.. L P...E. G ET TV G TL V .N A5D1P6 ....G...... SG SSR V F ATP A MI GL ME K A ALSS. V DPL.. L E...A. G LT TV G IR V .D F6CP13 ....G...... SG G A R V F ATP A MI GL ME K A ALSS. V DPL.. L E...P. G QI TV G IR V .D R6M9E2 ....K...... SG T L S V F ATP A MV AL ME E A SCMA. L KDY.. L E...E. G EG TV G IK L .D D6KHB2 ....K...... SG S L D V F ATP A M CAL ME E A AQAA. V QPY.. L E...E. G EG TV G IA L .S F0T2R2 ....G...... SG T L D V L ATP I MV AI ME N A ATRA. L ..D.. L P...E. G QT SV G TY L .E W0EBY5 ....R...... SG S L D V F ATP A LV AL ME A A AVNA. L ..Q..FE...A. G ES SV G TA L .E L0FAB3 ....G...... SG S L D V F ATP S LV AM ME E A AVGA. L ..A.. L E...D. G QS SV G VS I .E I4D8E6 ....G...... SG N L E V F ATP A MV AL ME Q A AVNA. L ..E.. L P...E. G QS SV G TS L .T G2G1T3 ....G...... SG H L E V F ATP A MV AL ME Q A AVNA. L ..N.. L P...A. G QS SV G TA L .N J7IWS0 ....G...... SG Q L E V F ATP A MI AL ME Q A AVNA. L ..E.. L P...E. G QS SV G TS L .S G7WFQ0 ....G...... SG Q L E V F ATP A MV AL ME E A AVNS. L ..Q.. L P...E. G QS SV G TS L .S H5XVU6 ....G...... SG Q L E V F ATP A MV AL ME Q A AVNA. L ..E.. L P...Q. G QS SV G TS I .T R6J6I5 ....G...... SG D L D V F ATP A MI AL ME Q A ACNA. I LPC.. L D...S.STS SV G IK M .D R6I9I9 ....G...... SG S L H V F ATP S MI AL ME Q A ACNA. V AAC.. L D...E.EST SV G TF V .N E4QCS6 ....Q...... SG L L D V F ATP S MI AL ME K A ALLC. V ENY.. L E...E. G YT TV G SK V .D F4A004 ....G...... SG A I S V F ATP S MI AL ME K A ALSA. V DLH.. L P...K. G YA TV G TR I .E G8M073 ....G...... SG D L D V Y ATP A MI CL ME RSASSA. V SLH.. L P...K. G YT TV G TA I .N B8I4C0 ....G...... SG N L D V F ATP A MI AL ME K A ASMA. V QPY.. I G...E.SSS TV G TM I .D G1UVA4 ....G...... SG L V T V YS T AM MI AG ME GTAVAS. V QDA.. L E...D. G QT TV G TR V .N B6WPN7 ....G...... SG E V R V L ATP M MI MG ME R A AMEA. I RPC.. L P...E. G MT SV G TR V .D H1HSX5 ....G...... SG T V H V L ATP M MI AN ME YTSAAS. V EEL.. L G...E. G KT TV G VK V .D S3MB87 ....G...... SG T V E V F ATP T MI AR ME DTATRS. V MSY.. L D...E. G KA TV G IH V .D R7K521 ....G...... SG L L P V LS TP A MI AF ME G V CQHS. V QPY.. L S...E. G YG TV G AE L .N U2F0D7 ....G...... SG E L E V Y ATP A ML AL ME Q A AAAC. L APL.. L K...E. G ET SV G AQ I .G G4KR18 ....G...... SG A L A V FG TP F MI AM ME N A ALTC. L QPG.. L E...E. G KG SV G VH M .D U2RDD7 ....G...... SG A L P V FG TP Y M SAL ME N A AMTC. L QSF.. L E...E. G QG SV G TH L .D R9LX73 ....G...... SG A L A V FG TP F MA AM ME N A ALTA. L QNF.. L E...E. G QG SV G TR L .D R6H0F9 ....G...... SG G L E V FG TP F MM AM ME H A AMDC. V QPD.. L P...E. G KG TV G VD I .Q R6D0Y4 ....G...... SG G L E V FG TP F MM GL ME Y A AMQC. V QPE.. L P...E. G KG TV G VE I .S R6BIU7 ....L...... SG L L P V Y ATP S LI AF ME YTCSET. V RPF.. L D...A. G MS TV G TL V .N R6G283 ....G...... SG S L L V YG TP A ML LL VE KTAVAL. L DGH.. L D...E. G MT TV G TN L .N B0MBZ2 ....G...... SG S L P V Y ATP A M TLL VE R A AVKL. L EGK.. L D...E. G MT TV G TN L .N R5YWL8 ....G...... SG S L P V Y ATP A MI SL IE QSAVNL. L AGK.. L D...E. G QT TV G TN L .N H1D405 ....A...... SG L L E V FS TP M MI AL ME K A AYTL. A EEH.. L E...E. G ES TV G VE I .G R6F6N6 ....G...... SG H L E V F ATP A MV GL ME NTAIHC. L EGM.. L E...P.DTD TV G IE I .H R5UUV1 ....G...... SG H L E V F ATP A MV AL ME NTAVKC. L EGE.. L E...K. G SD TV G IE I .D R7G5H6 ....G...... SG V V D V F ATP M MI AF ME NTAVKC. L EPF.. M E...E. G ET TV G IC M .N E4LXV6 ....G...... SG I V D V Y ATP M MI AL ME NTAAAC. L NQF.. L E...E. G ET SV G VM M .R

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! 160! H1BNL1 ....G...... SG I V S V Y ATP M MI AA ME H A AVQC. L QPF.. L E...E. G ET SV G VL M .H A1HRR7 ....G...... N AG A A V F ATP M LV AL ME Q A AIAA. V AGC.. L A...P. G EG TV G TR V .D F7NH57 ....G...... N AG A P V Y ATP M LA AL ME Q A AIAA. V SQA.. L Q...P. G QG TV G TR L .D M1E923 ....G...... SG L V N V LS TP R IV SL LE G A AVEA. I KNN.. L A...S. G ET SV G TL I .N C6Q0E0 ....G...... SG N I D V FS TP S MI AL ME NTSKSC. V DLH.. L P...F. G YT TV G IE V .N T0N2A7 ....G...... ATG I E V Y ATP A MI AL ME NTAKSA. V DLH.. L S...Y. G YT TV G TE V .C I7K9K8 ....L...... NG F I D V LS TP S LI GF LE C A AKNA. V DLH.. L P...T. G YS TV G IK V .D R7RUE8 ....A...... SG G V D V YS TP A MI GL ME C A AKEC. L DEF.. L D...E. G YS TV G IS L .N N1ZJT1 ....G...... SG T V T V L ATP K MI AW ME G V SLNA. V LPF.. L P...K. G YD TV G TA V .E A6TLR1 ....G...... SG G V E V L ATP M MI GL ME R A ALTA. V DPH.. L P...D. G FA TV G TH L .N R4KGW9 ....G...... SG D I S V F ATP A MI GL ME K A ALSS. V DPL.. L P...E. G YT TV G IK V .D J6UG66 ....G...... SG T I A V L ATP V MI RL ME A A ALDA. V EDS.. L P...E. G HH SL G VK L .D R9MCN0 ....G...... SG S L E V F ATP M MI AA ME Q A ACTL. L QEF.. L E...E. G QT SV G TM M .H B0PBF1 ....G...... SG D L D V L ATP M MI AA ME Q A ACGL. L ARF.. L E...P. G QT SV G TA I .D R7MJM6 ....G...... SG S L E V F ATP M MA ML ME KSACNA. L ADF.. M E...N.DET SV G TE L .N R7H2Y6 ....G...... SG S L P V F ATP M M TAL ME A A ACDA. V AAY.. L E...P.TET TV G TA L .H R5VLB0 ....G...... SG S L R V F ATP M LA AL ME KTACAA. V AEF.. L E...D. G ET TV G TA L .D D1PP05 ....G...... SG E L D V L ATP A VA AL IE K A CWKS. V AGE.. L D...M. G TG TV G TS L .S C3J9P1 ....G...... SG D L P V FG TP A MI AL ME K A SVQL. V APH.. L E...E. G AS TV G IH L .D G9YHC1 ....A...... SG T A P V Y ATP A LV GL ME H A AVKA. I GDQ.. L P...Q. G YS TV G IS M .N E2ZCX0 ....A...... SG T A P V Y ATP A LV GL ME H A AVKA. V DSQ.. L P...E. G FA TV G IS M .N R7MZQ7 ....G...... SG KSP V Y ATP A LV AL ME N A AINA.CDPQ.. L P...E. G YN TV G IA M .N U7UR89 ....A...... SG T A P V Y ATP A LV GL ME N A AVHA. V GSQ.. L P...E. G YG TV G IS M .N F9MNK7 ....G...... SG KSP V Y ATP A MV SL ME T A AIHA. I DPL.. L P...E. G YN TV G IG I .K D3LTB7 ....S...... C G KSP V Y ATP A MV AL ME M A AIHA. V DPQ.. L P...E. G TN TV G IA I .D T1CHV2 ....S...... SG D L A V FS TP S MA AL ME Q A ALTL. L SKH.. L S...A.EDT SV G IA L .N F9N406 ....G...... SG T L P V F ATP A MV AL ME K A AYTG. L QGD.. L G...D. G EG SV G IR I .E J5AQJ6 ....K...... SG S L E V F ATP A M CAL ME E A AQAA. V QPE.. L N...D. G EG TV G IS L .S K9D8B9 ....K...... SG A L E V F ATP A M CAL ME E A AVAA. L TGK.. L P...T. G DG TV G IS L .S R6UAG6 ....G...... SG S V Q V Y ATP M VV AL ME K A ASAL. A QQY.. V P...E. G CT TV G TN I .S G4Q7C4 ....K...... SG S L D V F ATP A LV AL ME Q A ACNL. V EPL.. L D...E.DTT SV G IS L .N D2RNA4 ....G...... SG S L D V Y ATP A ML AL ME K A ACSI. V NPC.. L D...E.ETT SV G IG V .N E0NZB5 ....G...... SG S L A V Y ATP A MA AL ME R A AAEL.CAAG..CP...E. G WT TV G TE I .A F5RQF3 ....G...... SG S L A V Y ATP A MA AL ME R A AAEL.CQMN..CP...E. G WT TV G TE L .H E4LMB1 ....G...... SG S L N V Y ATP C MA AL ME R A AAEL.CQRE..CP...V. G WT TV G TA L .S C9LW87 ....K...... SG S L P V F ATP A M TAL ME Q A AAEL. V DEL.. V P...S.DWT SV G IS L .N I0GTL3 ....G...... SG S L P V Y ATP A M TCL ME K A ATET. L ESL.. V P...E. G WT TV G IS L .H V2XUL5 ....G...... T AG A K I LS TP N ML AL ME ACALEL. A QEY.. L D...E. G MT TV G AE A .H F4X9G0 ....G...... S AGSK I LS TP N MV AL ME G A ALEL. A KAY.. L D...E. G MT TV G AE I .H R5G526 ....G...... S AGSK I LS TP N MV AL ME D A ALEL. A KSY.. L E...E. G QT TV G AE I .H F2NH42 ....F...... PDLPD A F ATP F LV GL ME G V SADL. M AKH.. L Q...S. G EQ SV G IG M .N A0LL45 ....Y...... PNLPD V LG TP I L GGL ME R V SAEL. I NAH.. L S...P. G EQ SV G IS M .N C3X5Q5 ....G...... SG KSD I L ATP A LV AV ME ATTQKI.TDGG.. L P...E.SWQ SV G TY I .G C2MAU3 ....G...... SG D M P V L ATP A LI AF ME N A AMLL. A RNY..TE...E. G ST TV G TM I .T R2S8F8 ....G...... SG G I P V L ATP A MV AM VE N A CYGE. L QKE.. L K...A.EET SV G GF L .S S1MVB3 ....E...... SG E L A V F ATP S LL AL VE KTAWQS. I ASC.. L S...A.EQT TV G TN I .V H6LHK5 ....G...... SG G A E V L ATP R LV AW ME N V AFEG. V EAA..QQ...P. G NT TV G TF I .E E6MGL5 ....G...... SG G L D V L ATP A LA AW IE N A AYEM. A DLW.. L P...E.EET TV G AN I .N E3GGD1 ....G...... SG G L E V L ATP I LV AW AE N A AYEM. A ELC.. L P...D.EQT TV G VN I .N R5KLD7 ....G...... SG S L P V LG TP Y MI AL ME N A TCAA. V EPY.. L E...E. G ET TV G TR I .D R5XE94 ....G...... SG S L R V LG TP F MI AL ME K A TCNA. V HEF.. L E...F. G ET TV G TC I .S R6J0V9 ....E...... SG S L P V LG TP F ML AL ME K A TCEA. V ADL.. L E...D.DET TV G TA M .N R7BG85 ....G...... SG S L P V Y ATP M ML SL ME E A TCQA. V SPL.. L D...D. G ET TV G TK I .D W1U6V8 ....G...... SG T I D V C ATP V IA AA ME A A AVAA. L APH.. L N...D.DET TV G TR I .S H1D2D9 ....R...... SG S L P V L ATP V L SAA ME E A ACKA. L APH.. L T...E. G ET TV G GS I .S R7CTJ9 ....K...... SG S L D V L ATP I L SAL ME E A ACAA. I AEA.. L D...E. G MT TV G GS I .S R6A5L1 ....K...... SG S L P V L ATP V L SAL ME E A ACDA. V KEG.. L A...E. G ET TV G GF I .G F4GJM3 ....D...... DRL L E V F ATP Q MI AF ME LSASSL. L EDY.. M E...E. G QA SV G AG V .D R6PY32 ....G...... N DK V P V F ASP K LL SW IE GTAIGT. V APF.. L P...D. G WE TV G TTF.D E4KRB3 ....G...... SG Q L E V L ATP A LI AM ME NTAQDL. L NGV.. L E...A. G QS SV G TQ I .Q K8E3X9 ....K...... SG D L A V L ATP A LV AM LE ETAKDS. I LEL.. L N...E. G ET TV G IE V .N R2VCM1 ....G...... SG D L E V L ATP A LV AM ME NCCKVL. L SSQ.. L S...S.EQT SV G FK M .N R2PBH5 ....G...... SG D L D V L ATP A LV AM ME NCAKIL. L APE.. L S...S.VQT SV G YK M .D R2R932 ....G...... SG D L E V L ATP A LV AM ME NCAKRL. L APE.. L S...S.NYT SV G FT M .N S0RW90 ....G...... SG D L E V L ATP A LV AM ME NCAKQL. L APE.. L S...S.EQT SV G FK M .T R6GEG5 ....G...... TG D L D I FS T SK MI AM LE LTVCKS. L EKH.. L N...K.EIT TV G KST.S R5ZKL6 ....K...... SG S L D V L ATP Q MI AW ME E A ACLC...LD.. L E...D. G QT SV G IS M .N U2R6S0 ....K...... SG S L D V L ATP Q MI AW ME E A ACLC...LE.. L E...E. G KT SV G TL M .N B7CCX0 ....K...... SG S L D V L ATP Q MI AW ME E A ACLC...LD.. L D...Q. G KT SV G IS M .N U2KTG5 ....G...... N EG V I V FS TP E ML SF LE ITCRHC. V EPH.. L K...E. G EG TV G IS V .D R5AKF1 ....G...... SG D L P V L ATP A LI AFF E N A AKDS. V ASM.. L P...G.SAT TV G TA I .S K6U6J6 ....G...... SG D L E V Y ATP A MI AL ME N A AKSL. V VNE.. I P...N.EFT TV G IE I .N W6N7S3 ....G...... SG D L E V Y ATP S MI AL ME N A AKSS. V AKD.. I P...E.DCT TV G IE M .N G7M699 ....G...... SG D L E V Y ATP A MI AL ME N A AKDI. A SPE.. L P...A. G FT TV G IE M .N V8G1J7 ....G...... SG D L E V Y ATP A MI AL ME N V SKSL. V IEE.. L P...S. G YT TV G ID M .S D6BCU5 ....A...... SG S V E V L ATP I MI AW ME G A SLNL. V QKE.. L E...N. G LT TV G TE V .N E3H9Z3 ....A...... SG A L D V FS TP M LI AF ME NTAFNL. A QKE.. L E...D. G DT TV G IS V .N C3WAL1 ....A...... SG A L E V FS TP M LI AF ME STAFTL. A QKE.. L G...E. G DT TV G IS V .N H1PQ22 ....A...... SG A L N V F ATP I LI AF ME N A SFEL. V QKE.. L P...A. G ET TV G IS V .D N0B8B7 ....G...... SG S A P V F ASP M LI AL ME A A AVDC. I EAQ.. L P...E. G HQ SL G VH L .D F8J518 ....G...... SG N V P V F ASP M LI AA IE A A AVDC. L DGF.. L P...Q. G HE SL G VH L .D R5RY61 ....G...... SG G L D I F ATP F LI CW ME A A AYYY. M KEQ.. L P...E. G KS SV G TM V .N J1HAQ4 ....G...... SG S V D V L ATP R LV AL ME G V CARM. L NTY.. L P...E. G IT SV G SE V .H B2V410 ....G...... SG S V E V F ATP M VA AI ME G A AADL. A QTF.. L E...D.IYT TV G TK I .T R7NT78 ....G...... SG S L R V L ATP T VV AL ME E A STKL. A DTF.. L D...E. G LT TV G TM V .E R6NZJ4 ....G...... SG G L A V L ATP A VV AL ME N A AFEL.CEEI.. L N..ND.ELT TV G TA I .S R5DLE4 ....G...... SG S L C V L ATP C VV AL ME N A AAAL. A DKV.. L D..ND.ELT TV G TM I .S B8J205 ....G...... SG L V D V FS T AM MI AW ME ATAVAT. V QEI.. L DHQGL. G MT TV G TK V .D E2SKL5 ....G...... SG I V N V Y ATP M MI AL ME NTAASC. L DQF.. L D...E. G ET SV G VM M .N C0EF68 ....G...... SG D A E V Y ATP K LV NL IE S V SASLV L EHL..SD...P.ELT SV G TL V .N B5EG61 ....YPESSY.F Q VMPE V F ATG Y MV GF ME W A CMDA. L APY.. L D...E. G ER TV G TM I .N W4LKY3 ....H...... V G GDG V LS TP S MI GL ME R A GIQA. V QPH.. L H...E. G QT TV G FH V .D Q01Y99 ....G...... VEG A R V LS TP H MI GL ME WTCRET. V QPL.. L D...A. G YD TV G TH V .N 1

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! 161! β3 β4 β5 flK TT . .. TT 80 90 100 110 120 flK V TH TAA T PP G LT V T V T A E L RS. V .. EGRRL S W R V S A H.. D GV.....D.E IG S G T H E R A V E3BLC4 L TH ISP T PPDFT I K V D I E L VE. M ..KKN RL R F K I I A H.. D GV.....D.V I SE G V H D R H I W5WVP2 M TH DGPCTT G TE L D I HCE V TA. A ..SA RRA S W N V E V RTSRGG.....V.L MG TAT H T R A I Q8FQC6 I TH DAPATE G AE L V V R V T A TG. V ..GK R S A E W S V E V T..AGE.....T.VQ G R G T H K R V V M1UKN7 I SH DAPATE G AT L E V K V E V VG. V ..GK R S V E W K V I V T..AGD.....T.VQ G A G T H K R A L F4GGF8 L EH RGPIPP G AE I R L SGW V ER. L ..G PR S V T F N V R A H.. D SH.....E.L V CE G HVTFV A C5CWG3 I EH LGPIPPTTR L R L RGW V EG. L ..GE R S A T F R V Q A Y.. D DH.....E.L V LD G TVTLV A Q2JHG3 L EH RSAAV AG DP L Q M R V E V VA. V .. EGRRI Q F Q V E I T..QQE.....R.L V MQ G SYTCL V W9CWY1 M RH RLPVR VG KH V EYR V T A GD.F..A. RKM D F SFE V R..NLD.....DEI V CD G T H R R A V D2SED2 V EH LAP S W VG DP V R V T A RCAE. V ..R G N RL TCECL A V.. D AG.....GRV LG R G TTVQV V I4EWF8 V EH LAPAW VG DM V Q V T A ECVE. L ..R G N RL TCACR A V..AAD.....GRE LG H G STVQV V F0RKI4 TF H TGSALP G ME V T V T A T L QE. V ..S GRRV VCA L R A V..SSL.....GDE IG H G TASQL V H8GSG4 V FH TASALP G MR V T V T A TFTR.Q..G GRRI HAD L F A V..NEL.....GDA VG H G TTTQV V Q9RR87 V RH TASALP G LA V R V T A S L DR. V .. EGRRV YAK L R A V..NEL.....GDE IG H G STTQV V K9ZXJ5 V TH TASALP G MQ V T V T A VFER.R.. EGRRI YAR M S A V..NEL.....GDL IG H G ATTQV V E8U6U0 V TH TASALP G MT V T V T A T L DR. V .. EGRRV YVN V R A V..NEL.....GDD IG H G TTTQV I Q1IWD4 V TH TASALP G MT V T V T A TFER. M .. EGRRI VCS L R A V..SEL.....GDE IG R G TTGQL V C1CY65 V TH TASALP G MT V T V T A TFER. M .. DGRRI LAS M R A V..NEL.....GDE IG T G TTTQM V D1C846 V RH LAPALP G ER L T V EGVHDR.T.. EG N RL YVRCR V V..NQW.....GEV IG E G ETTQV V D7CQM9 V TH VASALP G MT V R L E A TFVR.Q.. E KN RV YAR V R A W..NEL.....GDL IG E G TTTQV I D3PRJ9 V DH LASALP G MK V R I V A EHLR.T.. EG N RV HAQCT A W..NEL.....GDQ IG V G YTEQV I D7BIK7 V HH LASALP G MR V K I V A EHVR.T.. EG N RI YTACQ A W..NEL.....GDL IG E G ETEQV I F2NK44 V RH LASALP G MR V K I V A EHLR.T.. EG N RV YAACR A Y..NEL.....GDL IG E G RTTQV I B7A757 V RH LASALP G MR V R I I A RHER.T.. EG N RV HAV M E A Y..NEL.....GDL LG R G RTEQV I H5SBP7 L EH RNMCP LG VR V D I T A T V TQ.C.. DG N RI ICS L S A R..AGA.....I.E I AR G TQTQV V E6SLA6 V RH LAP T P VG AR V R A V A V L EA. V .. EG N RV VCR V E A Y..NDL.....E.K I AE G RQVQV V F8I237 I VH LAP T L VG ETFS A T A E L LA. I .. D KN RI IAR V T A E..NGR.....G.L IG R G QFTQV L Q8EMG8 L KH IAP S A LG TK V K L E A V V TS. I ..TE K Q V VTK V I A R..NES.....V.I IG E G EVKQS I T0JGA7 L NH IAP S A LG TN V K L T A A V SE. I ..TK R M V TTE L I V E..NDN.....G.R IG E G EVRQA I U5L8R5 V KH LAPAAE G SL L K V T A E V VS. L .. E RNL I FTS V K A F..HGD.....T.V IG K G QVTQA I U6BAI1 L KH LGMAAE G TR L I V T A T V TA. M ..TS RRV DAS I E V R.. D GQ.....T.I IG T G EVTQF I K0A8N8 L KH VGMAT LG SN I R I E A T V SE. L ..T P D RV VAK V E V R..HGD.....R.L IG L G EVKQI I W7LB64 A RH LSP S PE G SS I L I T A T V SK. L .. DG NS V YST V T V H..NGE.....M.L VG E G EVKQV I S2XPF3 L KH IAA S P LG SV V S L T A N V IE. L ..RDNF V KTK V T A R..NHL.....G.V IG K G EVMQV I W4RK43 V KH IAPCTE G AE V T V T A T V TG. L ..RANT I LTN V R A E..SNG.....R.L IG V G EVKQV V I8UG48 A KH LAP T PE G MN L T I T A T V TE. L ..T GK T V VCK V K V A..NEK.....M.V VG E G DVTQY I A6CP21 M KH IAP S SE G TT V T I T A T L TE. L ..QSNI V ITK A E A R..NEN.....G.L IG I G EVKQV I E3IAI3 V KH IAP T AE G ST V T V T A T V TE. L ..RDNV V VTK V E A K..NEV.....Q.V IG I G EVKQV I C0Z567 I KH VGPAV IG QE V TFR A SCVQ. V ..TE KRV VCE V T A D..TTR.....N.R VG L G TFVQA I F5SL03 I QH RAPAP VG KE V TFT A E V IE. V ..N P P KV VCR V R A E..HDK.....S.L VG A G TFVQA I D5WXP8 V EH RAPAP VG KE V TFT A E A VE. V ..T PRRV VCR V W A E..HDK.....G.I VG E G MFHQA I C7QDW7 FD H VYP T P VG GA V T A S A E L AH.R.. D D R L L R F T V A A H.. D AD.....GRL VG E G EIT R V I M4ZY06 V DH LQP T P VG AQ V S A E A T L EK. I .. DGRRL T F T V S V S.. D SG...... GL V AA G KIT R V L D2AZ27 L EH LVA S P VG TH V E I T A E L TE. V ..AE RRL V F TFT A R.. D RQ.....T.T V AT G TVE R V V W2ELW3 L DH LRP S P VG MH V E I VTE L TE. V .. DGRRL V F S V T A V.. D KT.....GAV V AT G TIE R V V D6Y6X3 L EH RAP S P IG MD V E V T A E L TA. V .. EGRRL V F SFT A V.. D RA.....GTL LG T G TIE R V V Q47SH7 L KH TAP T P VG AQ V V A T A R L VR. A .. EGRRL V F E V S V T..QEG...... RQ V AV G TVE R V V R4L8I1 V DH RAP T S VG RK V V A Q A T L TG.T.. DGRKL Q F A V T V K.. D GD...... TV V AEVHVE R V L U5VXT8 V EH RAP T P VG RM V T A L A T L AK. V .. DGRKL I F D V V V R.. D GE...... NL V AEVRVE R M V G8SD04 I DH HAPAA VG RT V T A Q A A L TR. V .. DGR T L A F D V T V R.. D GA...... TV I AEVRID R A I A4X9A9 L DH QAA T P VG RT V V A R A R L AE. V .. DGRRL L F A V S V T.. E DG...... ST V AE G RVE R L L C4RK94 L DH LAP T P VG RT V V A R A R L TA. V .. DGRRL G F E V T V T.. D AG...... EI V AR G RVE R T L W7W6W1 L EH RAA T P VG RT V A A H A E L VK. V .. DGKRL V F E V T V T.. D GP...... TV A AQ G RVE R A L I0L862 L EH LAP T V VG RT V R A Q A L L AT. V .. DGRRL S F E V T V A.. D GD...... QT V AR G RVD R I L F4FFD5 L EH LAA T P VG RT V T A Q A R L TA. V .. D K RRL T F E V T V V.. E GE...... QT V AQ G RVE R V V E6VPY1 V SH LAP T P VG MSFE A D V K L VE. I ..NSNS L L F T V SCR.. D EV.....G.L IG E G T H T R A I I4JL97 M SH TAA T P VG MT V T A T V E L VG. I .. EGK A L L F N V QCR.. D EH.....G.V IG E G T H R R A I N6ZAE8 V GH VAA T P VG MK V T A E V E L IE. I .. EGK A L L F K V ACR.. D EG.....G.L IG E G T H R R A I V2TPW7 L SH SAA T P IG LR I K A E V I L IN. I ..NK KKL T F S V YCT.. D GL.....D.I I CE G T H E R Y I Q8RS32 L SH SAA T P VG MQ V T A E V E L IE. V .. EGKRL T F K V LCR.. D EV.....D.V I CE G R H E R F I D3PYM5 I SH TAA T P VG LT V T A T V E L IE. V .. D K RRL R F A V ECR.. D DR.....D.V IG K G H H E R F I V5PXQ4 FT H LAA T P AG DT L R I RGT L VD. V ..A GR T L R F E V E A W.. D GL.....D.K V SA G T H E R V I Q476Z6 FS H LAA T PP G MT I T V KGQ L VE. V .. DGRRL R F E V S A W.. D GE.....D.K I TE G V H E R H A V2GN25 FT H LAP T P AG MT V T V KGV L TA. I .. EGRKL R F E L S A W.. D GE.....D.K I SE G V H E R H L H5XC58 L SH EAP T P VG GL L T V Q A E L SN. V .. D R R Q L T F T V H A F.. D DA.....A.T V CR G S H R R A I A7HFD1 V TH GAA T PP G FT V T V D V A V TK. V .. EGRRL T F S V R A H.. D GV.....D.A I CE G T H E R F V M1SXE6 V TH EAA T PP G ME V T A K V E L TE. V ..R GKKL I F S V E A Y.. D DA.....E.L V SK G S H E R I I D8FEX3 V SH MAA T PP G LE V T A N V R L VE. V .. DGRRL L F E V E V Y.. D DV.....D.L V SR G K H E R F V D9SHK4 V SH EAA T PP G LE V T A S V E L TI. V .. DGRRL T F A V S A H.. D GV.....D.T I AR G T H E R Y V D5CM24 V SH QAA T PP G LE V T A L V E L VE. V .. DGRKL V F Q V E A H.. D GV.....E.V I SK G R H E R F I S7VEV0 L NH TAA T PP G FT V T V K V K L TA. V .. EGKKL T F A I E A F.. D GI.....D.R I SR G T H E R F V W6M343 L SH TAA T PP G FT V T V K V R L VE.K.. DGKKL K F A V E A H.. D GI.....D.S I CQ G T H E R F V G7W6G7 L SH IAA T PP G FT V T V K L R L EK. I .. DGKRL L F H V E A H.. D GV.....D.L I SE G T H E R F I F2NC79 L SH SAP T PP G HT V T I R V E L SQ. V .. EGRKL T F M V Q A A.. D ES.....E.L I SQ G V H E R F I I4CEW2 L TH SAA T PP G LL V T I HGK L EQ. V .. EGRKL V F SFS A E.. D GV.....D.S I SE G T H E R F V W5XXT0 V SH VAP T VP G ST V T I D V T L EA. I .. E R R A L T F S I T A R.. D EY.....A.V I ST G T H Q R G V E8WN56 V TH SAA T PP G ME V T A Q V RCVE. V ..T GKR TV W E I E V H.. D QA.....D.L I SK G T H E R FT Q2RWF7 V SH LAP T PP G AR V V V E A K I IA. I .. DGRKV S W H V V A R.. D EV.....D.L IG E G TIG R A V V5SDR4 V SH SGA T VP G QV V T V D A TCTS. V ..T GRRI A F D V V A H.. D GV.....E.V IG K G R H E R M V N0B387 V SH VAA T VP G QT V T V E A ECTK. V ..A GRRI F F H V K A H.. D GI.....D.L IG E G E H Q R M I D6V9R7 V NH TAA T PP G LT I H V D V ECTD. V ..K GK L L R F N V K A H.. D GV.....D.V IG E G R H E R Y A J5PI50 V DH TAA T PP G LT V T V D A ECVA. V .. DGRRL T F Q V T A H.. D GV.....D.T IG K G R H E R V V D3IB68 V KH LKA S P IG AE V V V K A R L EK. V .. DG I KL Q F H L E A F..QGE.....K.L I AE G S H L R F I F3ZNZ3 M KH LRP T P LG EKFT A Y A K V VR. V .. EGKKI E Y S L I A E.. D GK.....G.V IG E G T H T R F V F3QUN7 SS H LKP T A LG KT I T A T A V L TG. I .. EGRKL T F K V V A E.. D ET.....G.T IG E G E H L R F I D1W8U0 SS H LKP S K VG AM V ETT A T L TK. V .. D R RKL F F H V V A R..QDG.....E.V IG E G D H L R F V U2MMW3 SS H LKP S K IG AE V E A T A T V TR. I .. DGKKL Y F S V V A R.. E GT.....T.V IG E G T H L R F I E0NSD9 SS H LSP T A IG AI V E A E A V L EK. V .. DGKKL H F R I T V R..QGD.....K.L IG E G T H L R F I S8FGJ3 SS H LRP S K IG TL I A A T A L L EK. I .. EGRKL T F R V E A R..QHG.....T.V IG E G V H V R F I D5ET49 SS H LKP S K VG DT V T A T A E V VK. V .. DGKKI E F K V S A Y..QGD.....V.L IG E G S H L R F V R5GHX3 V AH LRP T P IG ET V H A T A V L TA. V .. EGRKL T F S V K A E.. D GH.....G.V IG E G T H V R Y I R6ZRJ4 TS H IKP S A LG EE V T A T A V L EE. V .. EGRKL T F R L K A T.. D SQ.....G.I VG E G T H I R Y I R6C452 TS H IKP S G LG EV I S A T A I L EE.E.. EGRKL T F K V S A S.. D SK.....G.I IG E G I H I R Y I F0QZ92 TT H VKP S A LG EE V S A T A V L EN. V .. EGRKL A F K V T A S.. D TK.....G.V VG EAT H I R Y I R6CGH3 TS H IKP S A VG ET V K A T A V L QE. V .. DGRKL T F S V S A V.. D SQ.....G.A VG ECT H V R Y V B7BGY9 M TH VRP S GG G EE V Y A E A T L VE. V .. EGRKL T F S L V A R.. D SV.....D.V IG E G T H I R Y I

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! 162! R5W6M0 TT H VRP S A VG AV I A A E A E L VE.T.. EGRRL T F R V A A F.. D GE.....A.L IG E G T H V R Y I R6WTD5 TT H IKP S G LG AS V T A T A I L TE. V .. DGRKL T F N V G A R.. D AE.....G.L IG E G V H V R Y V F2BXC3 L DH IAP S A V NSE I S V V A K L .IH V .. DGKKL E F E A I A K.. E K.....TK.T IG HAK H L R M I R7L727 A TH LRP S K IG ET V S A T A T L .VK A .. EGKKL F F E I E A K.. D SQ....GA.L IG KSE H V R F I E6S7M9 FE H IKG S Q VG SE V T V HCAE.PIN.. DGRRL VCF V S I K.. D AE....GD.E LG H G EVH R A I W9GKR0 I EH VKG T P VG AE V S V SCAE.PIS.. DGRRL VCF V T V T.. D PD....GE.T L AS G EVH R A V W9GAD5 FE H VKG S P V DAV V T V NCAE.PIN.. DGRRL VCH V T V I.. D SE....GE.E I AR G EVH R A V D3QAD8 L RH LAAVP LG RM V A A R A R L .RGR.. DG N KL T F D I S L V.. D GD....NN.L V AD G EIE R A L W5THV3 V RH RRP S LP G TT L T V C A R L .VE V .. D EA RL T F R V V A R..AG.....EV.L V AD G TVR R T V X0PXW9 I EH LGP T P VG AG V E I A A EPPAA A ..V GHRL T F T V R A I.. D SS....GK.V V AS G EID R A I D6KEI0 V EH LRA T A VG GR V E V A A EPPAAP.. DG T RL T F R V K A V.. D DS....GR.L V AT G EID R A I V6KXP8 V RH VRA T P VG GR V E V S A EPAAS A ..A G G RV T F V V R A V.. D GS....GR.L V AT G EID R A I R4Z4Z8 L EH IAP S A IG AV I R V DSS L .DK V .. EGRRL T F T V S A H.. E .....DGR.L V AA G KVT R V V E2SCL7 V NH LAA S P VG ME I K I D A H V .TSR.. DGR A V T F E V T A D.. D AH....GT.R V ADARIE R A V A3THU5 L QH SAA S P VG AT V I V T A DP.AY A .. DGR LNRLR V S A R..HTD....GK.V V AT G EIT R V I R7XZ68 L EH LAA S P VG AV L E V S A TT.AY V .. DGR LHR F T V S A R..HVD...GGR.V VG A G EVT R V V A1SHD6 V EH VAA S P VG QQ V E V T A S A .SY V .. DGR LHR F T V A A R..NVGAAGAGK.L A AT G EIT R V V B9DSI3 I QH LKA S K IG DP V T I N I TDLK. V ..N GRR FD F R I E A F..V.K....DT.F V AEAS H T R I A G5KAY0 I DH LKA S K IG NS I T V I I TEVT.N..Q GRK YD F Q L E A F..V.G....KE.L I AKAS H T R VC F5ZJ64 I SH LAA S K IG QA I T I I I T A LK.E.. EGRR YD F R I E A Y..A.G....EK.L V AKAC H T R VR I7MYH5 I QH LAA S A IG KA V T I V I T A LK.E.. EGRK YD F R I E A F..A.G....EQ.L IG KAC H T R VR V6QBM2 L NH LKA S R IP EE I K V D V KDVT.R.. EG N RL S F IFE I R.. D .S....SH.L I AT G K H Q R A I H3NJZ3 V NH LLA S P I KGT V Q V V I DQVH.Q.. EGRHI H F E M T A Y..V.Q....DK.K V AT G L H E R V I R9LQS4 V SH IKA S P LG AV I R I V A T I TNV V ..ND R T I E F W V Q A F..Q.G....DD.L IG E G S H E R V I F2I7N4 L EH LAP S L L NEE I R I HCE L VD.H.. D D RH YH F Q M Q A Y..C.Q....DQ.L IG RLD H R R VK U2QSW7 I NH IAP T L IG KT V L V KGC V KEI I ..KE K I I I F S L E A F.. E .N....NK.K IG DAN H T R A I E5V1L6 I NH LAP T L IG KS I I V KGT I KEI I ..KE K I I I F S L E A Y.. E .G....DK.Q I ATAD H T R V I F3A7K0 I NH IAP T L IG NS I T V RGK I TEV I ..KE K I I K F S I E A Y.. E .K....DK.K IG DAE H T R V I E4T2L8 M QH LKA S P VG AT V ECT A T I TA. I .. EGRK YS F A I K A I.. D AS....GD.L IG EAM H E R V I T2NPG1 I DH LKP S P VG AT I T V F I Q.WE....KEQGFQ F DYQ V T.. D GA....QQ.L I AK G T H Q R A A C8ZYY8 I EH RAA S G IG AT V Q V CCEEIS....NE K TYE F TFR V Y.. E .N....DQ.L L AT G T H H R A A R2SKA9 L KH LAP S P VG AV I E V Q A V I TE.Q..N GKKA T F E I E A Y.. D .N....QR.M IG QAH H T R V I R3WMJ9 L TH QRP S K VG QE I T V ETE I SK.K..T GK L I D F SFQ V F..M.K....DV.L V SE G T H Q R A V R2TEP8 A KH LRP S K VG AN I L V K V K V DS.Q.. E TS KI N F SFD V F.. D .N....EE.L V AT G I H Q R A V S0KCS8 L RH VKP S K MG AV I T V E V S L LK.Q.. D F KKA T F AFSCR.. D .N....EN.L I AT G T H T R VQ I3TU50 L RH LAP T P AG VT I E I T A R L NR. A ..I GR S L L F E V RGT.. D GA.....G.I I LD G RMT R H I U2HCP0 I KH LAP T PPDWE V T V D A E L IS. V .. EGR M M T Y K V S V R.. D AA.....G.V VG E G I H T R C A L8F1C4 SV H LAP T PT G EV V T L T A T L RQ. V .. DGKKL T F A V E A A.. D RH.....A.T V LR G V H T R A V G2NQF4 SA H LAP T P VG DD V T V S A T L SR. A .. D D RKL T F D V E A R.. D SH.....G.S V LR G R H T R A L C3GXB1 M KH LAP S LYNEK V T I HSK L IK. V .. D KN KL L F E V E A F.. D ES.....N.K I LS G F H E R A V J8I2Q3 M KH LAA T P MG HT I N I ESE L VE. V ..S GKKL I F K V VGY.. D FH.....D.K V VE G Y H T R F I C8PLZ4 V QH TAA T LE G MK V F V HTE L ID. I SEN GK F L T F K A E V S..NEK.....G.I IG A G T H V R A I D4J4P3 V RH LAS T PC G KK V RCE V E I TE. V .VN GKKV T F A V N A Y.. D EN.....G.L IG T G T H K R A V U2L2J7 L NH LAS T P I RMQ V RCECE L TE. I .HN. KRL V F Q V K L F.. D EK.....E.L VG KAK H E R Y I U5RWS7 V EH IKPIP A NMK V V V K V T L IS. I .. EGKKL R Y N V E A F..NEK....NC.K VG F G IYEQQ I U3QHT6 V QH SGA T P LG GE V R I S A E V TA. I .. DGR S V T F S V T A E.. D AA.....E.T V CS G T H R R F V J3H1U4 I RH VGA T L LG MS V N I V A T V TR. I .. EGR S V T F N V E V R.. D DV.....E.L V ATAA H T R V V R4X4Z9 I AH TGA T L LG MT V R I E V S V AS. I .. EGR A V K F D V T V F.. D DI.....E.Q V CT G S H S R F V Q13AZ6 I SH GGA T LC G MH V D I T V T V SK. I .. EGR L V T F E L R A N.. D AI.....E.E I SR G S H S R F V H6SJ72 L SH VAA T P LG AR V N I T V R V AE. I .. EG S RV R F E A E V R.. D TA.....E.V V AR G T H E R FC M2Y638 M DH AAP T P LG MW V D I T V T V AA. V .. EGR A V TLAFT A A.. D AL.....D.R V AS G R H S R F V M2ZCB4 V DH LAP T L LG QW V E I T V T V EE. V ..K G PG V T F SFT A K.. D SL.....D.N IG K G V H K R F V W0SEE6 L DH VGA T L LG MW V E I T V T L TA. V ..N G GA V S F DFT A R.. D SV.....E.E V AR G K H N R F I N6YXR0 L DH VGA T L LG MW I E I T V K L VQ. V .. DG PA V V F EFS A R.. D AL.....E.E V AR G R H N R F V W8U886 I KH MAP T P AG MS V V A NSE L VE. A ..K G I K FK F N V E V L.. D DL.....E.K IG E G TISMA V F8I584 L RH LQA T P LG SR V H A TSR V VQ. V .. DG Q KV T F Q V A V F.. D EW.....E.K V AE G T H T R YC S6H1G1 I AH LSPAWP G IE V QCE A V L ES.T..K GR T L S F A V S A S..QQG.....R.L V MR G H H E R VR X7FEJ4 V HH HKPAWPYQE V RCT A T L IS. A .. D E KRL Q F F V Q A H..QAG.....A.L I MS G K H E R A L Q13BL0 I RH LTPAL VG HV V RTVSR V TA. V .. E Q R T V S F E V ACY..VGT.....R.R VG D G R H I R G L Q6NAK8 I RH LAPAF VG QT V RTT A V V SA. V .. E R R V I R F D V ASF..LGD.....R.K VG D G R H A R G L U1HEJ6 I RH LAAAL VG AT V RTTSK V VV. V .. E R R V I R F E V E A F.. E GT.....R.K LG E G R H A R G L H0TP42 V KH LAAAP VG AT I RTT A E V IE. V .. E R R V I R F A V T A F..HGD.....L.K IG E G R H G R G L K8P616 V RH LAA T P VG RT V R V SSR V RQ. I .. DPK S V L F D V E A W.. D GS.....R.K IG D G T H R R G V I0G1C1 I RH LAA T P VG RT V R A I A Q V VA. V .. E A R S I L F E V E A W.. D GN.....R.K IG D G T H R R G V A6CFP8 V EH LAP T P VG AT V NCT A Q L IF.H.. DG PVYR F K V E A Y..AGD.....E.K I AK G L H S R R I L0DK57 V EH LAP S PE G SA V VCR A R V IH. V .. DG AV V T F Q V E A H.. D DV.....E.L I AR G L H R R R V M2WUQ8 I EH LLP S A VG AT L T A R A T L VE.R.. DGRRL H F T V E V F.. D GD.....N.VCATLT H S R A V G7D213 V KH FAA T P VG HE V R A T A E V IN. V .. EGRRV D F K V S A S.. D DK.....E.E IG S G T H Q R I V B1M8N7 V TH MAA T P VG HR V R A E A E V VG. V ..A GR Q I Q F R V A A W.. D ET.....E.Q IG A G T H E R M I K8NJU9 V RH LAA T P AG QR V T A Y A E V TH. V .. EGRRI V F A V S A R.. D ET.....E.E IG K G M H E R M V F7ZKG2 V SH TAP T P IG GQ I S A T A TFTE.Q..V G ALYW F D V V A A.. D AA.....G.Q IG K G RIA R A I K0DQR3 FD H FAP T P LG ATFT A Y A KFTG. M .. EG PLYL F D L W A E.. D AG.....G.A IG K G T H A R A V I5CHG7 L SH LAP T PP G ID V S A T A TFRG. V .. EG ALYW F D I V A A.. D AG.....G.P VG TAS H A R A I G8MI72 L HH LAP T PP G VE V S A T A TFRG. I .. EG SLYW F D V A A S.. D PG.....G.P VG TAS H A R A I J7J7X3 L TH SAP T P V DAQ V S A T A SFRG.R.. EG SLFV F D V V V H.. D PK.....G.Q VG S G T H A R A I G2LGZ5 V RH LAP T P VG AT V T A T A TYLG.N.. DGK FHR F R V E A F.. D PA.....G.K IG E G E H T R A I D8NFT6 V SH MAA T P IG VE V T A E A RYVG.Q.. DGK LFV F E V S A R.. D RG.....G.E IG R G M H K R A I A8ET68 I KH LAP T LE G DT A IST A TFVG. M .. EGK LFK F E I E V V.. D SG.....G.V IG N G T H T R A I D5V0F7 I KH LAP T LS G DV A IST A TFEG. M .. EGK LYK F S L E V V.. D SG.....G.V IG T G T H T R A I R5P9P0 V KH IKA T P VG DK V NCK A K V TE. I .. EGRRI R F E I E A E.. D EK.....G.Q IG YAI H D R F I R8VSU0 V SH VAA T P AG MK V HFETE L VE. I APN GK I L T F K V A A Y.. D EA.....G.L IG E G T H Q R A I R7BPJ2 I KH VSA S P VG AH I RCEST L TE. V ..ND R A L T F E V K A Y.. D NA.....G.L IG E G T H H R F I R6KH82 I KH IAS T P IG ME V I A KTE L IE. V .. D R RRL V F K V E A F.. D ER.....G.K IG E G I H E R F I R6GT38 V KH LAS T F VG SK V TCETE L IE. V .. D R RKL V F S V K V Y.. D ET.....E.L VG E G I H E R F I J4TCP2 V KH VSA T P LG MK V E I KSD L IK. V .. DGR A L T F K V E A Y.. D EK.....G.L IG E G I H E R F I H1LX05 I KH IAA T P VG MK V V A KSE L LK. I .. DGR E L T F K V E A Y.. D EK.....G.L IG E G I H K R F I R6P4I1 I QH ISA T P VG MK V T A ESE L IA. I .. DGRKL T F K V T A F.. D EV.....G.K IG E G T H E R F I U2PAA7 I SH VAA T P IG MT V RCETE L TE. V .. DGRRL V F K V A A Y.. D EA.....G.L IG E G T H E R F I F7V6W4 I NH VAA T P VG MQ V TCETE L VE. V .. D R RRL V F Q V K V T.. D EA.....G.L IG E G T H E R F I R5BBK6 V TH VSS T P VG LK V WCESE V VD. I .. D R RRI V F K V A A Y.. D EK.....G.L IG E G T H E R F V R6E7S6 V KH LSA S P VG MT V TCKTE L VD. I .. D R RRL V F H V ECY.. D TA.....G.L IG E G D H E R F I R5SHR8 V KH VSA S P VG ME I TCETE L TE. V .. DGRRL V F T V K A Y.. D TK.....G.L IG E G V H E R F V R6DLF5 V SH IAA S P IG MK I RCESE L VE. V .. EGRKL T F K V E A Y.. D ET.....G.K IG E G K H E R F I W7UZ64 V KH TSA T PKNMK V C A E A E L LE. V ..N GR E L I F S V K A Y.. D EA.....G.D IG S G I H K R F L E8RIW0 I SH GKA T P VG MK V TCEST L VE. V .. D R RRL V F E L L A Y.. D EK.....G.E IG R G R H E R F I R5RJN6 I EH MAA S P VG MK V ECDTE L IE. V .. DGRRL V F K V E A S.. D EK.....G.L IG K G T H E R F I

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! 163! R7JYS9 I EH VAS S P IG MK I TCDSE L TA. I .. EGRKL V F K V E A Y.. D SK.....G.L IG K G T H E R F I R5HR96 V KH LSA T P IG VE V TCR A E L TE. V .. DGRRL V F R V T A Q.. D SQ.....G.L VG E G I H E R V I E0E4V9 TN H KAA T P IG ME V TCECE L VE. I .. D RS RL V F K V RCY.. D EL.....E.E IG VCT H E R F I B1C982 FK H LAA T P LG MK V RCECE L IE. V .. D R RRL L F K V D V Y.. D EQ.....E.K VG E G T H E R F I F4X8V5 V SH DSA S P IG IK V W A ESE V TE. V .. DGKRI ELK V A A Y.. D EK.....G.M IG H G T H Q R F I R7B458 I SH DAP T P IG ME V SCKSE L VK. V .. DGR A L S F T V E V V.. D EQ.....G.I VG R G T H D R F I U2BAI1 V RH VAA T P VG MK V WCKSC L TQ. V .. EGRKL T F H V E A Y.. D ET.....G.L IG T G E H E R V L U2SW53 V AH LAA T PC G MK V RCETE L VE. V .. DGRRL V F S A T V F.. D EM.....G.K IG E G R H E R F I R9KEX9 L GH LAP T P VG MK V WCESL L TG. V .. DGRKL T F S I T V F.. D GA.....G.K IG E G L H E R F I R9IHN6 L EH LAP T P VG MK I WCETV L KE. V .. DGRKL T F D L T V Y.. D ET.....G.K IG A G V H E R F I D4J9J7 I KH IAA T P IG MK V RCESL L KE. V ..N GKKL V F E V N V Y.. D EK.....G.L IG T G T H K R A I D4J2H1 I SH MAP S A IG KT V MCE A T L IE. M .. DGRRL T F E V VCS.. D EN.....G.V VG M G T H E R F I N2BI76 I HH VSA T P VG MN V RCISK L VE. V .. DGRKL V F D V Q A F.. D DA.....G.L IG Q G T H E R F I F7K912 I KH LAP T P VG MK V WCESR L VQ. A .. DGRRM V F Q A E V F.. D ES.....G.K IG E G E H E R F L R5KDY4 I SH VSP T P LG MT V KCETE L IE. A .. DGRKL K F K A E V Y.. D ES.....G.L IG I G T H E R F I R5CE45 I SH LSP T P VG MT V KCETE L TG. I .. DGR C L R F E A N V Y.. D ET.....G.L IG K G V H E R F I E2ZHD1 L EH TAP T P VG MT V TCESE L VA. V .. EGRKL T F K V A L H.. D EK.....G.P VG G G T H E R F V R6WHR1 V EH VAP T P VG MK V RCETE L VK. V .. DGRKL D F K V E V Y.. D EC.....D.L IG K G T H S R F I R7C7Z2 I SH DAP T P LG MT I TCESE L VE. I .. DGRKL V F S V S A K.. D EK.....GTV IG R G T H E R F V C0CNB6 I HH NAP T P VG MT V TCESE L IQ. V .. DGRKL T F R L T A R.. D AA.....G.V VG E G T H E R F I R6IH13 I SH DAA T P LG MK V W A ESE L IE. I .. D R RRL V F E V K A F.. D EC.....G.Q IG A G R H E R F I R5CSL0 V SH DAA T P LG MK V W A ETE L VE. V .. D R RRL V F D V K A Y.. D EC.....G.L IG Q G K H E R F I I5AU99 V KH TAP T P MG MK V TCETV L KE. V .. D R RRL V F E V K A Y.. D ET.....G.I IG E G I H E R F L R6BVQ8 V KH LAA S P VG MK V TCNTT L VE. V .. D K RRL V F S V E V E.. D ET.....G.K VG E G T H E R F I G2T231 V KH VAA T P VG ME V TCETK L VE. V .. D R KRL V F E V K A Y.. D AA.....G.V IG E G T H E R F I R5Q2H0 I KH TSA T PT G MR V YCEST L TE. V .. DGRRL V F S V S A Y.. D EC.....G.E IG S G T H E R F I D7GQ38 V TH DAP T P LG MT V WCETE L VE. V .. DGRRL V F D V A A Y.. D AK.....G.K IG G G R H E R F I R6ZN81 I SH SAA T P LG MK V WCESE L TE. V .. D R RRL V F H V TCY.. D EA.....G.V IG E G T H E R F I U2D4S0 I KH LAA T P AG MI V WCESE L KE. V .. DGRRL V F H V E A F.. D ES.....G.K IG E G E H E R F I R7B681 I AH NAA T P VG MQ V WCESE L VE. V .. DGRRL V F K V E A F.. D AK.....G.S IG G G T H E R F I C9R9F2 I EH LAP T P VG ME V T A V A E L VE. V .. EGRRL V F R V E A R.. D QV.....E.V IG R G R H E R V L R5ICW6 I AH IKA S P IG ET I T A K A T L KE. I .. DGRRL V F D V T A S.. D SK.....G.E I AN G T H E R F I C0GGI3 V AH TAA T P I DMT V T A H A E L VE. V .. EGRRL R F K V T A V.. D DA.....G.P VG E G T H D R F V A6NV90 I TH DAA T P VG MK V W A E A T V TA. V .. DGRRI S F T V K A Q.. D AC.....G.P IG S G T H E R F V R5B634 V SH DAA T P VG MH V T A K A E L VE. V .. D R RRL V F R V T A E.. D DA.....G.P IG Q G T H E R F L U2BG97 V SH DAA T P VG MQ V W A E A E V TA. V .. DGRKL T F A V T A Y.. D ET.....G.P IG K G T H E R F I R5EG72 I EH ISA T P VG LE V R A E A E V TA. V ..A GK V I T F A V R A F.. D EA.....G.E IG H G T H Q R V V D4LA09 I LH TAA T PP G ME A H A V A E V TE. V ..S GR E I T F H V V A Y.. D RC.....G.K IG E G M H K R V V U2M1B8 I SH DAA T P VG LT V T A T A E I TG. V ..N GR E I S Y H V T A E.. D GV.....G.V IG S G V H K R F L A0LP78 TS H IAA T PP G MG V R A E A T L EG. V .. EGRRL F F R V E A Y.. D DR.....E.K IG E G R H E R V I U2D856 V KH IKS T PS G MK V K A E A Y L EK. V .. D R KRL F F K V E A Y.. D ES.....G.K IG E G T H I R Y I Q3A9K1 V SH IKA S P VG AL V E A K A K L IS. V .. EGRKL T F L V E A F.. D EQ.....G.K IG E G R H E R VR E5UZI6 TT H IKP S A VG DT V S A T A V L KE. V .. EGRKL T F E V R A Q.. D SK.....G.V IG E G T H V R Y I D4IPX1 V TH IKP S G LG AE I T A T A V L AE. V .. EGRKL T F N V G A R.. D AE.....G.L IG E G I H V R Y I R7D0I0 TS H LKP S A LG AS V E A T A E L TE. V .. DGRKL T F R V S A H.. E GD.....K.L IG E G T H V R F I R5LPE1 SS H LKP T P VG AE V K A E A T L TK. V .. DGRKL Y F T V K A M..QGD.....T.I IG E G T H L R F I V4RI11 A SH IAA T P VG MK V E A V A E L VE. V .. DGRKL V F E V T A R.. D AM.....E.I IG E G R H E R V V B2A5Z4 V KH LAP T P VG DK V K V N A V L KE. V .. DGKRL V F E V T A H.. D SN.....N.K IG E G E H E R F I V5SDQ6 V SH KAA T PP G LA V S A T A T L LA. V .. DGRKL T F A I E A R.. D AT.....E.Q IG DAR H T R V I U5Q519 I KH TRA T P LG AK V Y A E A E L IE. I .. D N KKL T F S V V A F.. D EM.....G.E IG R G T H M R Y I F5YR67 I KH LAA S P LG IE V R A W A E L IE. V .. DGRRL R F K V E A A.. D AA.....G.K IG E G V H E R F V B0TBP0 V SH LAA T P VG MT V RCE A E L TE. V .. DGKKL V F K V V A Y.. D EA.....E.K IG E G V H H R F V D5X9M4 SS H LAA T P VG ME V I A RSE L IR. I .. EGKKL V F K V E A Y.. D EK.....D.K IG E G I H E R F I Q67JY1 V EH LAA T PP G MK V R A T A V L ER. V .. DGRRL L F R V E A F.. D DR.....E.R VG S G T H E R V I D6TKN4 V KH LAA T P VG QN V R A V A T L HE. I .. DGRRL V F Q V E A Y.. D ER.....Q.K IG E G T H E R F I F6DMU1 I KH TAA T P LG LG V K A K A E L ME. I .. DGKRL V F R V E A F.. D ES.....G.P IG A G V H E R F V K8DZX3 V KH TAA T P VG MR V V A R A T L TE. I .. DGKRL V F R V E A T.. D ET.....G.P VG E G T H E R F I A4J0S5 I KH TAA T P VG MK V V A R A E L IQ. I .. EGKKL S F V V E A R.. D ET.....G.P VG A G K H E R F I A5D1P6 V KH KAA T P VG ME V V A KSR L VE. V .. DGKRL V F E V S A W.. D EA.....G.L IG T G I H E R F I F6CP13 V EH LAA T P IG ME V V A RSE L VE. I .. DGKRL V F R V E A R.. D EQ.....E.L IG R G T H E R F I R6M9E2 I SH IAP T A L DDV V I A I A T L DK. I .. EGRKL T F I V E A K.. D TH.....K.V IG K G I H E R F I D6KHB2 I TH EAP T P LG AT V T A K A T V SA. V .. EGRKI T F D I E A S.. D GV.....G.I IG R G T H E R F V F0T2R2 I KH LAA T P VG MK V W A V A E I TG. I .. EGRKL S F K I E A Y.. D EK.....E.K IG E G E H E R F I W0EBY5 I IH SAA T P IG MK V W A E A V L VE. V .. D R RRL V F E I D A Y.. D EV.....E.K IG S G R H E R F I L0FAB3 I KH TAA T P LG MK V W A V A E L LE. I .. D R RRL V F K L E A F.. D EK.....E.L IG T G V H E R F L I4D8E6 I NH IAA T T IG VK I T A T A E L IE. I .. D R RRL V F T V E A S.. D EA.....G.Q IG A G K H E R F I G2G1T3 I SH TAA T P LG AK V S A T A E L IE. I .. D R RRM V F S V E V Y.. D EV.....G.Q IG V G K H E R F I J7IWS0 I KH NAA T P LG AN V F A I A E L VE. V .. D R RKL V F T V E A F.. D DA.....G.Q IG V G N H E R F V G7WFQ0 I KH NAA T P IG AL V K A T A E L ME. I .. D R RRL V F N V E A F.. D EA.....G.Q IG V G K H E R F I H5XVU6 I KH TAA T P LG AK V F A T A E L LE. I .. D R RRL V F Q V E A C.. D EA.....G.Q IG I G N H E R F I R6J6I5 TT H EAA T L LG EK I I A T A K L IA. V .. EGRKL T F K V G A C.. D DH.....G.P IG N G T H E R F I R6I9I9 I TH DAA T G MG KK V T A K A T L TA. V .. EGRKL V F E I T A A.. D ED.....K.Q IG K G T H E R F I E4QCS6 I LH LAP T PK G MK V K A V A Q L IA. I .. E D RKL T F K V E A Y.. D SF.....E.K IG E G V H E R F I F4A004 V EH IAA T P VG MK V R A D A E L IE. I ..N GR A L T F K V R A F.. D EN.....E.M IG Q G V H Q R Y I G8M073 V KH MAA T P IG MK V K A V A E L IS. I .. EGRKL I F K V E A F.. D AK.....E.K IG E G Q H E R Y I B8I4C0 V KH IAA T P IG MN V A A R A E L IE. I .. DGKKL I F S V E A F.. D GK.....D.K IG E G R H E R F I G1UVA4 V AH LAA T PC G MK V RFVTE L TA. V SPN GK G L T F N V A A Y.. D EA.....G.L IG E G V H E R V V B6WPN7 I SH MAP T PC G MK V RFE A K L TA. V SAN GR G L T F T V A A Y.. D EA.....G.P IG E G S H E R V V H1HSX5 V SH VAA T PK G MK V TFD A E L LE. I SAN GK I L T F H V E A H.. D EC.....G.L IG E G T H Q R A V S3MB87 V RH LDA T PE G MK V F I HTE L VE. I NAN GK F L T F T V E A R.. D EH.....G.V IG S G I H E R A I R7K521 V RH VSA T P VG MR V K A ECE L ... V SV DG NMYT F E V R A F.. D ET.....G.L IG E G T H K R C V U2F0D7 ST H LSP S P LG AT V R A T A T L ... L SF DGR T A R F A V T A E.. D DF.....G.K IG E G E H T R A V G4KR18 V SH DAP T P VG MK V W A E A E I TG. V SEN GK M V D F K V R A W.. D EK.....G.P IG H G T H T R A I U2RDD7 I SH DAP T P IG MK V W A E A E I TA. V SEN GK M V D F A V K A W.. D ES.....S.P IG S G T H T R A I R9LX73 I HH DAP T P IG MK V W A E A E I TG. V SEN GK M V D F K V S A W.. D ER.....G.P VG G G T H T R A I R6H0F9 SS H LAP T P VG MT V R A T A T V TG. V SEN GK L I T F R V E A S.. D DW.....G.P IG E G T H T R A V R6D0Y4 SS H LAP T P VG MK V T A T A E V TG. I SAN GK M I T F K V T A H.. D AD.....G.L IG E G I H T R A V R6BIU7 I KH TSA S P VG ST I TCECE L A...QI DGRKL I F N V I A H.. D DF.....G.P VG E G V H E R F V R6G283 V DH VSA S P IG CE V SCE V T L T...EI D R KKL V F A V E V K.. D PA.....G.V IG K G T H E R F I B0MBZ2 I SH VSA T P VG CQ V TCQCH L T...EINR KKL E F Q A E I M.. D NK.....G.R IG I G T H E R Y I R5YWL8 I AH VSA T P VG GT I KCHCS L T...EI D R KRL V F H V E V T.. D NK.....G.R VG I G T H E R F I H1D405 SK H MKA T P IG VT V K A V A T I T...KI EGR F I T F Q V Q A F.. E ED.....GTL IG E G T H L R C I R6F6N6 V QH TKA T A VG KK V MCK A T I V...EI DGRRI R F E I E A T.. D EK.....G.T IG HAI H D R F I R5UUV1 V QH LKA T A IG GQ I SCQ A T I T...QV EGRKI S F E I E A W.. D EK.....G.K IG SAR H D R F I R7G5H6 ST H DAA T P VG MK V TCT A T I T...AV E R KKI S F S I V A K.. D EK.....D.V IG KAT H D R F L E4LXV6 TT H DAA T P AG MT V T A E V E I T...AV D R KKV S F H I I A R.. D ER.....D.L IG TAD H D R F I

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! 164! H1BNL1 TT H DAA T P AG MK V R I S A E I I...AV D R KKV T F S I I A Q.. D EK.....D.I IG KAS H E R F V A1HRR7 V QH LAA T P VG MT V R A T A E L V...EVA GKRL V F A V S A Y.. D DR.....E.K VG E G W H E R Y I F7NH57 I SH LAA T P VG MS V R A V A T L T...EIT GKKL V F A V E A Y.. D DL.....E.K VG Q G Q H E R Y I M1E923 I EH IAP T P VG MK V W A S A K L I...RV DGRRL I F E V L A R.. D EV.....E.Q V AR G T H E R F I C6Q0E0 V KH IKA S P IG MK I RCE A S L V...KV D K KKL V F N V E A W.. D EK.....G.K IG E G N H I R Y I T0N2A7 I KH LKA T P VG SK V RCE A V V D...NI DGKKI L F S V Q V W.. D GK.....G.L IG E G T H I R Y I I7K9K8 V KH LAA T P IG MK V K A I A E L V...EV DGKRL I F K V E A Y.. D EM.....E.K I ME G T H E R F I R7RUE8 I KH LAA T P IG MK V R A E A E I V...AV EGKKV V F K V E A F.. D EV.....E.K IG E G I H E R Y I N1ZJT1 I KH SAA T P VG MK V R V VSE L I...EI DGK A L T F K V K A Y.. D EI.....D.K IG S G I H K R A I A6TLR1 V KH MGA T P VG MK A Y A E A K L I...KV EGKKL T F E I T A R.. D EE.....E.K VG E G T H E R Y I R4KGW9 V EH VAA T P VG GV V R A QSR L L...KI DGRRL V F E V Q A S.. D DT.....R.V VG R G T H Q R F I J6UG66 V AH TAA T P VG MR V E A E A E L T...AI DGR T L T F R V E A R.. D AV.....E.T IG A G T H V R V V R9MCN0 V AH SAA T P LG MQ V T A A A T I T...AQ EGRKV E F E V S A R.. D AC.....G.E IG R G T H T R F V B0PBF1 I KH TAA T PC G MAYT A S A R L T...GR EGR C A S F E V W A E.. D EI.....G.E IG R G S H T R V V R7MJM6 I KH ISA T PESMK V T A S A E I T...EVN GR E I T F K V E A F.. D ES.....G.K IG E G L H K R F I R7H2Y6 I TH DAA T PQ G MT V R A E A E V T...GVN GR E I S F A V T A Y.. D EA.....G.Q IG T G T H K R F L R5VLB0 L AH TAA T PE G MT V T A T A E I T...AVS GR E I T F A I T A S.. D EA.....G.E VG HCT H K R F L D1PP05 L VH TAP T P IG MV V RCDCE L T...AVAD RRL T F T A R V Y.. D DV.....S.E V AHAT H E R Y I C3J9P1 V HH TRA T A IG QT V R V M A T L T...EV DGRKL I F D I R A E.. D SQ.....G.E IG H G TLE R F I G9YHC1 I KH TSA T P VG MT V K A K A V L I...DQ D R RRL T F K I EGY.. D DA.....G.S VG EAV H E R F I E2ZCX0 I KH TSA T P VG MD V T A K A V L T...EQ D R RKL T F K I T A C.. D AA.....G.P VG E G I H E R F I R7MZQ7 V KH VAA T P IG LT V T A K A T L V...EQ D R KKL T F K I E A H.. D EA.....G.L VG EAT H D R F I U7UR89 V KH LSA T P IG LT V T A K A T L T...AQ D R RKL T F K V E A F.. D TA.....G.A VG EAV H E R F I F9MNK7 V KH MSA T P LG MK V T A K A T V V...AQ D R KH FDVK I E A F.. D EA.....G.L IG TAE H E R F I D3LTB7 V KH LSA S P VG MT V R A E A T L T...AQ D R RHL T F T I E A F.. D DA.....G.L IG TAT H E R F I T1CHV2 I TH SKA S K LG AE I T A T A T V T...AI DGRKI S F D V E A W.. D PD.....G.L IG K G T H E R F V F9N406 TS H EAA S P LG AT I T A M A T V V...AV DGRKV T F E V V A K.. E GE.....K.V IG R G T H E R F I J5AQJ6 I SH DAP S P MG AT I T A T A T V T...AV EGRKI S F A I E A S.. D GV.....G.V IG K G S H E R F V K9D8B9 I SH DKA S A VG AQ I R A E A E V T...AV DGRKI S Y K V ATY.. E GD.....I.C IG K G T H E R F V R6UAG6 I DH LAA T AS G AE V Y A T A E L T...ES DGRR FE F A V E A Y.. D NA.....G.L I AK G T H Q R FS G4Q7C4 I SH DAA T A VG RQ V T A K A V L V...GI DGRKL S F K L T A S.. D EH.....G.I IG Q G S H E R F L D2RNA4 L SH DAA T A VG KT V T A K A V L V...GV DGRKL T F K I T V S.. D NY.....G.T IG Q G T H E R F L E0NZB5 I RH KAA T P VG LD V R A V A E V T...AV DGR A V TLK V T A Y.. D TR.....E.E IG A G T H T R F A F5RQF3 I KH RAA T PT G LT V R A I A E V T...TV DGR A I SLK V T A Y.. D ER.....E.E IG S G T H T R F A E4LMB1 I SH RAA T P VG LS V R A V A E V T...AV D E RKI NLK V T A Y.. D AR.....E.E IG S G T H E R F A C9LW87 I RH KAA T A LG RS V H A E A E V K...NF DGR Q V T F A V K A F.. D EA.....G.E IG E G L H E R V L I0GTL3 V AH KAA T P VG MK V R A E A E V T...AV EGRKV T F T V R A F.. D DA.....E.E IG V G T H E R F A V2XUL5 V RH LAP T P VG MQ V T A T A T L RE. V .. E R RKM W F D I E V H.. D EK.....G.KC G E G S H L R I M F4X9G0 CR H LAP T P VG MK V S A T A R L RS. I .. E R RKM W F D L E V F.. D EK.....G.KC G E G S H L R I M R5G526 CR H LAP T P VG MK V T A T A K L RS. I .. E R RKL W F D I E V N.. D EK.....G.KC G E G S H L R V I F2NH42 L KH TAP T P LG ME V R V VTE L IG. I .. EGKKL T F K L E A F.. D EK.....E.K IG EAT H E R F I A0LL45 L KH TAA T P LG ME V R V RTE V IA. V .. EGRKL T F R L E A F.. D EA.....E.K IG EAT H E R F I C3X5Q5 L TH SSV T P VG AK V S I H A V L TH. I .. DGK E L T F E I S A F.. D ES.....G.I I AE G S H K R I I C2MAU3 TD H QHA T P IG ET I T V T A Q L SS. I .. EGRKL T F A I E A T.. D QT.....G.V V AQAT H E R F I R2S8F8 I EH LAP S P VG AT V T V R A A L YE.H..A G T KV V F SFT V Y.. E GE.....K.L VG K G N H E R F I S1MVB3 L DH LAP T P VG LQ V TCHTN L TT. I .. D R KRL V F SFE V Y.. D EV.....D.L I AK G T H E R F I H6LHK5 L KH LAA S S VG MK I R I K A T L VA.T.. E T R V L N F D I D A W.. D GL.....E.K IG EAI H Q R F I E6MGL5 L DH MAP T P VG MK V R V K V H L DA. I .. EGRKL V F S I E A Y.. D TA.....Q.Q I AG G T H T R F I E3GGD1 L NH IAA T P VG MK V R I K V V L SN. I .. E S RRL D F T V E A W.. D TV.....Q.K IG E G T H Q R F V R5KLD7 V TH DRA S KT G ET I T A R A E I RE. V ..R GRKI V F S V E A T..NEK....GE.R IG G G TIE R F A R5XE94 V SH DKA S G V SEI V T A F A K I TA. A .. EGRKI T F S V Y A E..NES....GD.V IG K G VIE R F V R6J0V9 I TH IKA S A IG EV I T A K A I L TE. V .. DGRKL T F H V T A R..NDK....NE.L VG S G TVE R F V R7BG85 I TH DKA S S MG TT V S A V A K L EE. V .. DGRRL V F S V S A K.. D ND....GN.V IG K G TIE R F V W1U6V8 L TH TAP S L VG MT L T A C A E I TA.H..T GR QYE F T V T V R.. D EI.....G.V V AE G T H T R V L H1D2D9 L IH KAP T LP G HV I T V T A T V TE. V ..K GKKI S F H I T A A.. D EN.....G.D IG EAD H D R F L R7CTJ9 L VH KAP T LP G DT V T A T A V V TG. V ..K GKKI T F T I R A E.. D SA.....G.E VG TAD H T R F I R6A5L1 L AH KQP T L VG ST V R A E A T V TV. V ..K GKKI TLH L I A Y.. D EA.....G.E IG EAE H I R F V F4GJM3 I RH TSA T PE G MD V T I T A T V TG. I .. D R RRV D F A V E A R.. D AC.....G.Q VG S G V H T R F I R6PY32 FK H LAA T P VG MK V R V VTE L TE. V ..N GK L L T F A V K A Y.. D EV.....D.K I CE G T H G R A I E4KRB3 CN H LIA S A IG SS I I V K A R L IK.E.. D Q R FYS F E L E A F.. D GE.....N.M I AK G S H Q R A I K8E3X9 L KH VRP S R I SEE I QCE A E L IE.Q..TKNI L R F T L K A L..AGE.....S.I I AE G T H Q R A I R2VCM1 V KH LYP T S VG EE I R I E A T L ME. V .. D N KK HS F S I K A Y.. D NK.....Q.L IG EAT H Q R V V R2PBH5 L KH LVP T A VG ST V K V D A E I LE. V .. DGKKI S Y S I K A F.. D DD.....Q.V IG EAV H Q R V I R2R932 L KH IAP T A IG AD V K V E A ETER. I .. D TN KV S F L I K A F..VDE.....Q.L VG EAT H Q R V I S0RW90 L KH LVP S T VG AE V K V E A E M LE.N.. E RN RV S F S I K A F.. D GK.....Q.L IG EAD H Q R V I R6GEG5 I QH FCP T P VG TV I KFESE L VK. I .. DGK N L T F E I K A L.. D PF.....G.V MG A G I H E R T I R5ZKL6 TS H DAP S A LG AT I S I T A K M TE. V ..Q GRKV L Y D V E A Y.. D GK.....T.C IG K G K H A R F I U2R6S0 V SH DMA S P LG AT I E I H A T L LK.Q.. E N RKL V F E V Q A F..QNG.....K.S IG K G Q H E R F I B7CCX0 V SH DMA S P LG QT I T I E A K M VH. V .. EGRKI D Y E V Q A F..QDG.....K.S IG K G V H S R F V U2KTG5 L KH MAS T P IG MN V TCECE L IE. I .. DGKRL V F N V E L W.. D EG.....E.K VG KAV H E R F I R5AKF1 V SH RAP T V VG KT A R V ITT L YE. V ..A GRKL R F R L E A Y.. D AA.....G.L I AE G E H E R C L K6U6J6 V KH IKS S R IG AN I ECK A T L VK. V .. DG M KL F F E V E A Y.. D DN.....G.I IG K G T H T R Y I W6N7S3 V QH VKS S P VG AN I ECK A V L TK. I .. D R KKL F F E V E A R.. D DK.....G.V IG K G S H I R F I G7M699 V KH IKS S P VG AN I ECK A I L TK. V .. E N KKL F F D V E A S.. D DH.....G.T IG K G F H I R Y I V8G1J7 V KH IKS S P IG AN I KCKST L TK. V .. DGKKL F F D V E A S.. D DQ.....G.T IG K G S H I R F I D6BCU5 I KH LKG T L VG KT V K I ISV L KE. I .. D R KKL I F D V E V L.. E DG.....I.V VG T G T H T R F I E3H9Z3 I KH LKA N L VG DL L KCE A L L TK. I .. DGKKL F F N V K V T..YND.....E.I VG D G E H I R Y I C3WAL1 I KH LKA N L IG DE L TCT A T L DK. I .. DGKKL D F S V K V F..HGE.....T.L VG E G E H S R F I H1PQ22 I KH LKA N L VG DK I KCVSV L EK. I .. DGKKL D F S V K V Y..HGD.....T.L VG E G K H G R F I N0B8B7 V TH SSP T P LG FN V T A T A T L K...SI DGRKL T F D V I A N.. D GI.....E.Q IG S G S H T R V V F8J518 V TH TSP T P IG YT V T A T A T V K...SV DGRKI T F D V S A T.. D GI.....D.R IG S G T H T R I I R5RY61 V RH VSP S P IG AE I T A K A E L IA. I SEN GK MYT F K V E A Y.. D NK.....G.L IG E G T H E R A I J1HAQ4 I KH LAP T VE G VT I H L T A K L I...ES DGRK FT F E V E A S.. D NV.....G.L I STCT H E R AS B2V410 V NH LAA T AE G VQ V F A E A E L T...AV DGRK YS F S L K A F.. D NK.....G.L I AT G E H E R VC R7NT78 I QH ISP S P IG AK I K V ESK L I...SN DGR SFK F E V T A Y.. D NA.....G.M I AN G T H N R VS R6NZJ4 I EH TSP T P LG AE I S A T A V L K...EN DGR FFN F E V S A S.. D AK.....G.I I AK G T H T R VS R5DLE4 I EH TSP T P LG AE V T A T A V L K...SV DGR MFN F E V F A E.. D KK.....G.E I AR G T H T R VS B8J205 I SH MAA T PC G MK V IFT A E L VG. M SSN GK G L T F K V S A R.. D EV.....G.L IG E G L H E R V V E2SKL5 TT H DAA T P AG ME V E A E V E I TA. V .. D R KKV S F H I I A R.. D AK.....D.T IG TAQ H D R F I C0EF68 I SH LAA T PE G MK V T A E V E I TE. V .. DGRRI V F S V T A R.. D EK.....D.L I CK G T H E R F V B5EG61 V TH EAA T P AG ME V T A T V T L VE. V .. DGKR TV W E I E A R.. D EV.....E.V IG R G R H E R F V W4LKY3 V MH IAA T PK G KN V T V R A E L LD. V .. DGRKL R F K V E A H.. D ED.....K.K VG E G T H R R A I Q01Y99 V AH LAGAP IG MT V TFT A E V VA. I ..TE RRV Q F R V E A R.. D EK.....E.K IG E G T H E R A I

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! 165! α4 flK 130 flK I HLE KF NA KV RQKTPAG...... E3BLC4 I NCAN F NK KI LKKLSMMKCLEK...... W5WVP2 V DRAT F SST V DRQVATIGGQYLEHP...... Q8FQC6 V DREQ F TGT V NNLADSFGAIRI...... M1UKN7 I SRQQ F VS HA NNLASQFGANELKESSK...... F4GGF8 A DRSALES KI AHKVNVSAR...... C5CWG3 A HRASIES RI ATKVDALEAARGGGQQ.G...... Q2JHG3 V DLP RF LA R YGLTAEDPSRCSSCSR...... W9CWY1 I ISAD F MR RL AGSTP...... D2SED2 L SEEAVQA RI GERARLQQR....TGS.G.D...V..LPDAR...... I4EWF8 L PQQALDD RL RRP...... F0RKI4 L PEE K IRAGFEALRERFAASRQPS...... H8GSG4 TAGT R LQDGFAELRRRWEAARD...... Q9RR87 L PQATIDAGFNELGRRWAESQEGR...... K9ZXJ5 L PQS R IDENFAQLAQRFEEYRAGR...... E8U6U0 A PQA R LDAGFADLAARWHAHQTTHQR...... Q1IWD4 L SQA K MDANFERLRARWQASRD...... C1CY65 L PQE K IDANFDRLRERWAAHQR...... D1C846 L PAAEIEA R FARQRGRLDATREEMRA...... D7CQM9 V PQA K LEAGFAALRERWRARKEATA...... D3PRJ9 L PRA K LLRIFEKLQARWQEQHK...... D7BIK7 L PKE K LERIFHKLQERWASFQQAKS...... F2NK44 L PKA K IQAAFDKLRARWESQKTEVNR.G...... B7A757 L PKA K VEALFARLRARWEAFRQGAVS.G.D...EKRKPEES...... H5SBP7 L PSE R IQAM I ARAYEQHGVQLPQKS...... E6SLA6 L DRAA F RR RI AEVQQRLGTAAAGTPGQGRT...SREAPGV...... F8I237 L PKKALED KI RDLSHHVSSDKEP...... T...ANE...... Q8EMG8 L PKNTMKS KL DIQIAAK...... T0JGA7 L PKHIIQE KV RAASIGGNVSISS...... Q...LERKPITFL...... U5L8R5 L PRS K IKRFTEKIRLD...... U6BAI1 L EKS R IQE KL QNNVPTK...... K0A8N8 L KKQLIQE KL TPRVF...... W7LB64 L KKS R IAKL L KID...... S2XPF3 L SKETIAN KL KEASV...... W4RK43 L PKE K ITEL L TGS...... I8UG48 L SKE K ITQ KL QSMRMENTQK...... A6CP21 L PKE R IRMMGK...... E3IAI3 L PKQ K IDQM L QKQQLHS...... C0Z567 FKKDEIKQ R FEALQAEINQEASR...... E...L...... F5SL03 L PKQGILE RI RQME...... D5WXP8 L PRQ K ILD RI ASMK...... C7QDW7 V NAE RF MA KL ...... M4ZY06 I DRD KF MG K CCSTN...... D2AZ27 V DRE RF LA KL SR...... W2ELW3 V DRE RF LS RL ...... D6Y6X3 V DRE RF LA RL ...... Q47SH7 V PRE RF LSG L RPGE...... R4L8I1 I DRQ RF IA KA IGS...... U5VXT8 L DRQ RF IA KA LGDA...... G8SD04 V DRQ RF IA KA LGD...... A4X9A9 V DRQ RF IE RA GRSS...... C4RK94 V DRR RF VE RA SGAPARAATA...... W7W6W1 V DRQ RF VE RA LGPS...... I0L862 V DRQ RF VD RA GRPS...... F4FFD5 L DRQ RF IE RA GRSS...... E6VPY1 I DLQ RF AQ RA NEKRERWKCAKATHPS...... I4JL97 I DLG RF TQ RL QEKAVRAGS...... N6ZAE8 I DVA RF MQ RL RDKSAKAA...... V2TPW7 I NED KF LQ KV KDKSLSAIKNISII...... Q8RS32 V EAQQ F IR RV TSKGERG...... D3PYM5 I DRRA F DAS A EAKRANPS...... V5PXQ4 V DEA RF QG KL REKAARLGLSV...... Q476Z6 I DAA RF NE KV AAKAARAAG...... V2GN25 I DAA RF ND KV AAKRERAAAAPR...... H5XC58 I DTE RF LA RM SKRTAS...... A7HFD1 I DRA RF DR KI QEKLAASTSC...... M1SXE6 I NKRE F EE R TRAKRS...... D8FEX3 I NKE KF DA KM KKKSKIKNTQS...... D9SHK4 I NKE KF DN KL RDKREKINQR...... D5CM24 I NRE KF EA KI GEKMRRSDT...... S7VEV0 I DPV KF NAA V AEKRNKHIS...... W6M343 I NAA KF NA KA VEKAGQRS...... G7W6G7 I DAA KF NE KV TRKSEIERITE...... F2NC79 I DAD RF RI KA ASKAR...... I4CEW2 I DAI RF NS RA SEKQAKYGDTT...... W5XXT0 V NRE RF VG RL PQ...... E8WN56 I RLEQ F KS RL KTKAEAAGIIIA...... Q2RWF7 V RWSS F TQ RL AEKAATIAARRATKPSGTD...... V5SDR4 V PWA RF VS HV NDKAQGAGADPIAAVTASGG...... N0B387 V DWE KF EQ RV NDKAKRARLSPITRGTG...... D6V9R7 V MWD KF TA RV SKKAAKAGVAA...... J5PI50 V SWD KF NA R TGEKAKAAGLASAEGE...... D3IB68 V NRQE F MS RV LKAE...... F3ZNZ3 V NRE KF MS RL ...... F3QUN7 V DRK KF MA KL QG...... D1W8U0 V DKQ RF MQG...... U2MMW3 V DRE RF LS KV IGH...... E0NSD9 V DRE RF LS RL ...... S8FGJ3 V DRET F LA KL G...... D5ET49 V DRE RF MS KL G...... R5GHX3 V DRH RF LG KL KKQD...... R6ZRJ4 V DIC RF MS KL K...... R6C452 V DRK RF LS KL DTESSK...... F0QZ92 V DRE RF MS KL ...... R6CGH3 V DRE RF LA KL KK...... B7BGY9 V DRT KF LS KL KG......

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! 166! R5W6M0 V DRA KF LS KL ...... R6WTD5 V DRA RF MA KL SK...... F2BXC3 V NEE KF MS KL SNK...... R7L727 V GRE KF LAS I Q...... E6S7M9 V DPD RF MT R CRKLV...... W9GKR0 V DRE RF MS K CHPVA...... W9GAD5 V DPE RF MS K CQRLV...... D3QAD8 V DRAD F LT RL D...... W5THV3 V DRKD F LA RA RDS...... X0PXW9 V DRD RF LDASSPGDDNR...... D6KEI0 V DRQ RF LA K TAGA...... V6KXP8 V DRQ RF LA R TPDPAADR...... R4Z4Z8 V NTDE F LS KL D...... E2SCL7 V DRE RF LA RL PGVTP...... A3THU5 V DTD RF LG RI Q...... R7XZ68 V DAE RF MS RV ...... A1SHD6 V DAE RF LS RL ...... B9DSI3 I DCE KF LKN L ...... G5KAY0 V NKET F LE KL ...... F5ZJ64 V DIQ KF LG KL KKS...... I7MYH5 V DSQT F MA KL VD...... V6QBM2 V DVD RF LSS I K...... H3NJZ3 I DPD R LLA KV DANFIDEV...... R9LQS4 V NSE KF VN K TYNFK...... F2I7N4 V NKES F MK KA QDNSSLQ...... U2QSW7 I DNK KF MK KL LDNN...... E5V1L6 I NNK KF MK KL ISK...... F3A7K0 I NNT KF LE KL NN...... E4T2L8 V NID KF MS KV LS...... T2NPG1 V EIS RF MK KV NENQS...... C8ZYY8 V DIEG F LK KV SEKES...... R2SKA9 V DNEQ F MR KM KENHNM...... R3WMJ9 V QIEQ F LK RI ...... R2TEP8 I LTDV F LD KL ALRK...... S0KCS8 V NCE KF LSQ L D...... I3TU50 A EPAVVLQQ M TRRHGTTRPSV...... U2HCP0 V GRDS F HE RL AERRDRAPVAQ...... L8F1C4 V DRD RF AA RL TARNA...... G2NQF4 I DRK RF TD KL SRRTA...... C3GXB1 V KKE KF SK KL EEKREAIKVNG...... J8I2Q3 I DKE KF LKN L AQKELVK...... C8PLZ4 V NKK RF ME KL RAAVSEG...... D4J4P3 I NKAA F NM...... U2L2J7 V SPDALKA KI ADKIRRHQKEENESEKNGNSGTCC...... U5RWS7 V NLEQ F LN R ...... U3QHT6 V DIE R LRA RV QKKTASMQGAA...... J3H1U4 V NVA K LRS RV QAKAEAAAAGDVEDG..VLSPSQFSASR...... R4X4Z9 V NVD K LKA RV REKANRVGALSA...... Q13AZ6 V EIE K LRK KV AEKAAKSREAQG...... H6SJ72 L PVASLDK RL KAKVARLAS...... M2Y638 V DVE K TRE RL MAKARKAGL...... M2ZCB4 V GVEQTKA RL LAKAEKAKSAS...... W0SEE6 V GVE K TAQ RL KAKLAKAS...... N6YXR0 V GVD K TRQ RL LGKLAKAQAA...... W8U886 I KMD RF LEG A IAKKL...... F8I584 L PRLE F DR RV AAKKVRPKPI...... S6H1G1 V DLS RF SLP.QPISAGEPIEFFFDF...HSPWCYLAMPRLREIAAAHGRAIHWRPMHLAN X7FEJ4 A PLS RF ERADGKLPEGAPLEFFFDF...HSPWCYLAFPRLREIAAAYGRMIVLRPFHLAN Q13BL0 I DVDA F TK R FKDWSGS...... Q6NAK8 V DVAL F TK R FAEG...... U1HEJ6 I NVE RF NK RL AGTSAQ...... H0TP42 V NVAQ F KK R FGVP...... K8P616 I NVAA F EK R FGVN...... I0G1C1 V DVAE F ER R FGVTRAAAEMV...... A6CFP8 I QASQLAR RL QALETKSRK...... L0DK57 I DVD RF AR RV ARKQAGR...... M2WUQ8 A QAQ K ITD RL AQHGG...... G7D213 I DLGS F NE RL AEKGTSLRNGGPDSF...RH....KGLPA...... DRGDG...... B1M8N7 V DIE R LGK RL AGKQAAPSRRV...... K8NJU9 V DLA R IEK RL EAKRAQSRANRKSES...... F7ZKG2 V DRA R LMTT A AGRA...... K0DQR3 V PGVAVEK RA AKRLGQTA...... I5CHG7 V DRAAIEA RA SKRGAR...... G8MI72 V ERSAIEA RA AKRRRAE...... J7J7X3 V DRAAIEK KA ASRR...... G2LGZ5 V TVE R LLAG A QKRLMSR...... D8NFT6 V SKE R LMRG A VQRTGGK...... A8ET68 V SNE R LMNG A KKRVEK...... D5V0F7 V TKD R LMSG A NKRVGK...... R5P9P0 I DPV KF MA KL ...... R8VSU0 V AKE RF EA KA QSKLG...... R7BPJ2 V DAA KF YK KA TGKLA...... R6KH82 I NNED F QN K TNNK...... R6GT38 I DNR KF ID KL EAKKQSLNK...... J4TCP2 V NNE KF QA KA DSKSDK...... H1LX05 V NND KF QA K TDSKLEK...... R6P4I1 I NNE KF QA K TNSKK...... U2PAA7 V QNE KF QT KA NNKLAK...... F7V6W4 V NNE KF QA KA NSKKA...... R5BBK6 I DVA KF QA K TEEKIKDR...... R6E7S6 I DEA KF MA K TDGKLQ...... R5SHR8 I VNE KF QA K TDSKKEA...... R6DLF5 V ANE KF QS KA DAKLEK...... W7UZ64 V YGD KF IQ K TYSKI...... E8RIW0 I DPK KF ME KL ...... R5RJN6 I ESE KF RE K TYNKLKKDAE......

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! 167! R7JYS9 I ESE KF QE K TNRKLEQ...... R5HR96 V QND RF LT KA QKKRNPK...... E0E4V9 I DNA KF LAN V DAKIERAKNK...... B1C982 I DQA KF EA KA EAKKK...... F4X8V5 V TDE RF LA K TAKKLEG...... R7B458 V DKE RF QS KA NAKLNK...... U2BAI1 I QAQ RF QE KA DAKRKPEKGKE...... U2SW53 V DNA KF QA KA DAKKHA...... R9KEX9 I EET KF QA KA DKKKQA...... R9IHN6 I EEE KF QS KA DRKKEA...... D4J9J7 I NNEA F MA RL A...... D4J2H1 I NNE KF MA KA NGLK...... N2BI76 I END KF FK KA EKKLEA...... F7K912 V RAE RF QQ KA DAKGNLQQGKEQEING...... R5KDY4 I TAD KF QA KA ESKKS...... R5CE45 I SSD KF QI KA DSKLISRD...... E2ZHD1 V NDA KF AA KA EAKKG...... R6WHR1 V KND KF QI KA NGKLEKA...... R7C7Z2 I LEE KF QE KA NRALLG...... C0CNB6 V DNA RF SQ KA EKKLG...... R6IH13 I NNE KF IA KA NAKGAK...... R5CSL0 I QND KF LA KV NAKGVK...... I5AU99 I DNE KF QA KA EGKKAGE...... R6BVQ8 V QAE KF QN KA DAKLNR...... G2T231 I DNE RF LA KA EAKKN...... R5Q2H0 I KSE KF MA K TNAKLEK...... D7GQ38 I QNE KF QA KA NKKALAE...... R6ZN81 I GNE KF LA KA EQKKNASKGE...... U2D4S0 V KNE KF MD KA LAKKEK...... R7B681 I KNE KF LA KA QSKKEN...... C9R9F2 V DVA KF LA KA QAKRSG...... R5ICW6 I KVD KF MA KL Q...... C0GGI3 V DQA KF MA KV EQKSG...... A6NV90 I SND RF LA R TNQKKEAPHVN...... R5B634 I MAD KF LA KA EAKKG...... U2BG97 I QND KF LA KA QGKKKGN...... R5EG72 V NSQ RF LD KA YSKL...... D4LA09 V RAE RF LE K TNGKMAK...... U2M1B8 V DAE RF LS KA QKRGQ...... A0LP78 V DRE KF LG KV NKKCAGPS...... U2D856 V NSEE F MG K KK...... Q3A9K1 V NIE KF LE KV SSREK...... E5UZI6 V DKE KF MS KL SQ...... D4IPX1 V DRE KF MA KV K...... R7D0I0 V DRE RF LA KL ...... R5LPE1 V DRE KF MS KL ...... V4RI11 V TGAS F QK RI DEKMPD...... B2A5Z4 I NLS KF MN KL S...... V5SDQ6 I DRT RF EA KA RAKGGPA...... U5Q519 I NPA KF MD KI S...... F5YR67 I ENE RF LK K TSEKKQ...... B0TBP0 I QTE RF LA K QAAKKKG...... D5X9M4 I DRE KF MA K CEGKKQSG...... Q67JY1 V RME RF LQ RA GEKRLP...... D6TKN4 V KLDQ F LKN L ED...... F6DMU1 I VKD KF IQ KA ESKAKAKE...... K8DZX3 I NLE KF LQ KV ESKLSR...... A4J0S5 V NQE KF LQ RA ESKLD...... A5D1P6 V KHES F IK KA EARLKE...... F6CP13 V NQE RF LS R TAAKLKTTS...... R6M9E2 I NNE KF MN KL QKN...... D6KHB2 I NNE KF MA KV HSRANAN...... F0T2R2 I NAE KF LE KA YAKLS...... W0EBY5 I QED RF LS KA NQKSGNLKNQ...... L0FAB3 I DAE KF MK K TLSKRSE...... I4D8E6 I DIEP F LN KA QKRNREI...... G2G1T3 I DIDS F LV KA QTRI...... J7IWS0 I DVDS F IA KA QARNQGM...... G7WFQ0 I DVDP F LA K TQTRKQGM...... H5XVU6 I DIEP F LA K TQNRKQES...... R6J6I5 I DKA KF IA KL QSKR...... R6I9I9 V NKE KF MA KL G...... E4QCS6 V NRE RF LN K TYQKVR...... F4A004 V NIE RF IN KA NSKFKRER...... G8M073 V ESA RF RD KV YGKLNS...... B8I4C0 I NTQ KF IT KV DSKLEG...... G1UVA4 V NKE KF ES R TRDKARQA...... B6WPN7 V DRE KF QS RA QAKGGQE...... H1HSX5 V DRV RF DE K TQAKLENK...... S3MB87 I DVG RF ME KV NKKKNA...... R7K521 I NDE KF MK K TLSKLENRKQSF...... U2F0D7 V GAQ RF LQ RM REKKPL...... G4KR18 I NNE RF MA K CAAKLDG...... U2RDD7 V KNE KF LA K CNAKLEKK...... R9LX73 I NCQ RF LD K CNGKLEP...... R6H0F9 I AND RF LQ K CNSKLTEQK...... R6D0Y4 I DNA RF LQ K CNDKLAHV...... R6BIU7 I NND KF MS KV AEKQAALQMHSAE...... R6G283 V DAE KF QN KA NGKFDK...... B0MBZ2 V AADS F LDN A KQKLN...... R5YWL8 V ASEP F ME KA AKKLED...... H1D405 I NRQ KF ID KL NKR...... R6F6N6 I YPE KF MS KL ...... R5UUV1 I DPE RF MA KL ...... R7G5H6 V IKE KF EA KA KQKAMN...... E4LXV6 V KKEA F EA KA LSKLENS......

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! 168! H1BNL1 V MKE KF EA KA QAKLC...... A1HRR7 I KTAS F LE KV AAKAGSGI...... F7NH57 I QTEP F FN KI AAKGKK...... M1E923 V NFD RF IK KA EKKKSF...... C6Q0E0 V NSEE F MK KV QE...... T0N2A7 V NIE KF MS KL ...... I7K9K8 I DVE RF MQ RV NNKVK...... R7RUE8 V DIN KF IS RV NNKNSKEE...... N1ZJT1 V ELD RF IK R TKEKGKKA...... A6TLR1 I PLE KF IA RA KEKGEK...... R4KGW9 V KVDE F LQ RV GSKK...... J6UG66 V EGA RF LQ RL ARKTQAR...... R9MCN0 V DAA KF LS K TNAKREAAE...... B0PBF1 V DAK RF QE KA DAKRRLNQ...... R7MJM6 I YGE RF TE KA KSKLK...... R7H2Y6 V EQQ RF LD KA AKRGQQ...... R5VLB0 V KSES F MK KA AAKKESL...... D1PP05 V DND RF TH KA SAKLNAD...... C3J9P1 V YRD KF MA KL A...... G9YHC1 I EAAP F IE KA NAKLKK...... E2ZCX0 I EAAP F TE KA NSKLK...... R7MZQ7 I ESAP F LA KA EAKKAAAK...... U7UR89 I ESAP F LA KA EGKKK...... F9MNK7 V ESAP F LA K TEAKLKK...... D3LTB7 I DSAP F LA KA EAKKKNNTQL...... T1CHV2 V NRE RF LS KL ...... F9N406 I NNE KF MA KI VTG...... J5AQJ6 I NNE KF MT KV GARKASN...... K9D8B9 I NNE KF MS KL K...... R6UAG6 V KIE KF LA KA EERLKK...... G4Q7C4 V KKE KF MN KL KTRYDR...... D2RNA4 V NKA KF LG KL AAKDQK...... E0NZB5 V AVQ KF MA KA EGKKV...... F5RQF3 V SVE KF MA K TEGKR...... E4LMB1 V ASE KF FA KA ESKR...... C9LW87 V NRE KF MS KV SAK...... I0GTL3 V QKE KF LS KA QAKH...... V2XUL5 V PNKDRSQG...... F4X9G0 V TPKGIQD KA GQ...... R5G526 V NSKAMSE KA EKKAE...... F2NH42 I NAD KF MK KL VSKATD...... A0LL45 I QAD KF NA KV AEKAAKMA...... C3X5Q5 SRTEVLKRL L RKKK...... C2MAU3 V SRQ RF LE RL GIKE...... R2S8F8 V KKE RF LS KL S...... S1MVB3 V NRMQ F SE KA QQKLHQK...... H6LHK5 V EAK RF NE KA AKKLESSRQEES...... E6MGL5 V NKA RF MQ KV QDKKNQ...... E3GGD1 V QKM KF MG KV LQKKNPK...... R5KLD7 V IRE KF MA KL G...... R5XE94 V KSE KF LS KV YGD...... R6J0V9 V LAE KF LR KV EGK...... R7BG85 V LAD KF MK KV NG...... W1U6V8 V NTS RF LAN A DARTRK...... H1D2D9 V DGV RF ME KA EQRNGKKQ...... R7CTJ9 V EADP F MDN A GKRKNKE...... R6A5L1 V EAKP F ME KA EKRI...... F4GJM3 V DREA F MK K TMEKTKEKTQDLMMEQKV...... R6PY32 I DLK KF LS RV NEKGKK...... E4KRB3 I NVE KF MA HL ...... K8E3X9 V NSE KF MA KL TK...... R2VCM1 V EIAN F LE KL K...... R2PBH5 V ETAA F LK KL K...... R2R932 V ETAA F LK KV K...... S0RW90 V ETAA F LK KV K...... R6GEG5 V DKQY F TM KA LVKKAK...... R5ZKL6 V DAK KF LS KV YPD...... U2R6S0 V DAN RF LE K TYAK...... B7CCX0 V DAQ KF MD K TYKK...... U2KTG5 V DMN K LLK KI DKKKEEYCK...... R5AKF1 V FSEA F MD RA NERAESEA...... K6U6J6 V NIND F ME K TKNK...... W6N7S3 V NSHD F ME K TKK...... G7M699 V NSKD F MA R TKDN...... V8G1J7 V NSED F MR K TKNC...... D6BCU5 I DPI KF YE KL KNTK...... E3H9Z3 V NEK KF IE KI RG...... C3WAL1 V NEE KF LA KL KK...... H1PQ22 V NEE KF LS KL KG...... N0B8B7 V DTA RF MA RL TAKSLQQT...... F8J518 V DTA RF MA RI AAKSASRP...... R5RY61 I DCE RF MK K CLTKLED...... J1HAQ4 I KREN Y LK RV AERAKQ...... B2V410 V KKDS F VN KA IERKNS...... R7NT78 V KSE KF QK KA DEKFE...... R6NZJ4 V KAE KF QK KA DEKFNG...... R5DLE4 V KSE KF QM KA DSKFDE...... B8J205 V HKE KF ES R TRDRKLCGC...... E2SKL5 V KKES F EA RA LSKLEK...... C0EF68 V ART KF IS KA KAKLN...... B5EG61 I DYE KF SK RV AAKGNK...... W4LKY3 I QIGNHG...... Q01Y99 I NVA KF AT RL AEKTK......

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! 169! A)1)2(

β1 η1 α1 α2 α3 η2 F8JPF9 T.T 10 20 30 40 50 60 F8JPF9 RFTHDFV V PPHK TV RH L YPESPEFAEFPE V FA T G F MV GL ME W A C V R AM APY L EP. G EG SL W5WVP2 TFTKHYT I HPGH TV RD I FPEAGEFADMPD V FT T G C LV AL ME WSC I EQ L SPH L DS. G QI SL Q8FQC6 THEFRYT V PGNKA V PD L YPESEEFAAMPR V FA T G FF V GL IE W A C M DH L KAS L PE. G AI SL M1UKN7 TFTFEYV V PDNKA V PD L YPESELFSAMPR V FA T G FF V GL IE W A C M EH L KDS I SDDNTI SL D3PYM5 RHS A RLT V DESL TV PA V NPSFTGFADMPR VL AT AF LV AF VE STC V E AL SPL L PS. G T QTV H5XC58 TAQ L EYV V PAER TV PY L LPESEEFKALPP VL AT G F LV GI VE WTC I R VL AGH L DD. G E QTL W5XXT0 THT M TYQ V TPER TV PN L LPENPDFTAMPE V FA T G Y MV GV IE A A C M E AI RPH L DE.DEI SL R0HVN0 THQ M TYA V PANR TV PN L LPEAAEFETMPD VL AT G Y MV GI IE W A C M E LL RSH L DD. G EI SL S5XAI0 THQ M TYI V PANR TV PH L LPEAAEFETMPD VL AT G Y MV GI IE W A C M E LL RPH L DD. G EI SL W9CWY1 AAT V TKE V SPA DSA ERW GN S...... G L D VL ST P A IL GE IE R M CDA VM KPR L AD. G EM TV D2SED2 SAV L DVV V TPEM TV RFDEL G .....PVHP V YA T YS MA KHF E E A GRK LL LSH L EP. G EAG I I4EWF8 TAE L VVV V TPEM TV RFGEL G .....PVHP V YA T YR MA EHF E E A GRK LL LRS L EP. G EAA I L8F1C4 SAT L DHT V APE DSA MNW G .N...... E L D VL ST P V LL WLS E I A A M K VI EAA V TA. P AM TV G2NQF4 SAQ L VHR V TER D A A TNW G .N...... D L P VL AT P V LL WLS E I A A M K VL GTA V PE. G DM TV R7BPJ2 TNTEEIM V SDSN TA LS MGSG ...... T L R V FA T P A MI AL ME GTA A QS V EPL L EK. G Q TTV R7B458 TNTETTM V TEQN TA LA LGSG ...... T V E V FA T P A MI NL IE YTC A RS V EDE L DP. G Q TTV E6S7M9 MRQFQHT V TPD DTA AK LGTG ...... D L D VV AT P R LV EWC E S A AFQ V CQHS V DA.NH TTV W9GKR0 MRTFQHT V TPA DTA TA VGSG ...... D L E VL AT P R LI TWC E S A AFE V CRPQTDE.DR TTV W9GAD5 MRQFQHT V TPDN TA AS VGSG ...... D L E VL AT P V LI TWC E N A AFT M CKSS I AA.DR TTV M2WUQ8 TGS L SQT V SDV DTA AMF G PD...... FPK AA ST P F VL GL AE V A CHN IV AGE L QD. G EL TV D3QAD8 SGR V ETI V TQA DTA QS LGSG ...... D V P VL GT P R VL AL AE A A I V MS L TRR I PT.TM TTV M4ZY06 RGE A TLV V TEA DTA CA LGSG ...... D I D VL GT P R LI ALC E E A T M A AL VGS L EA.NS SSV W5THV3 EAEFSIE V TAA DTA SA LGSG ...... D V D VL AT P R IV AL AE E A T V R AI ADA L SG. G R TSV D2AZ27 RAQ L LIM V EKE DTA TK VGSG ...... D V P VL AT P R LL AF AE Q A T V Q AV AGA L EP. G Q TSV W2ELW3 RAE V LIM V ERG DTA IR VGSG ...... D V P VL GT P R LL AF AE ATT V K AV QHH L EP. G Q TSV D6Y6X3 RAT V SVT V GQP DTA IQ VGSG ...... D V P VL GT P R LL AL AE G A T V Q AV RPH L EE. G K TTV R4Z4Z8 RGE V EFT V ADA DTA RS LGSG ...... S V E VL GT P R VL AWC E A A S I S AL ADT L PA. G M TTV Q47SH7 TGT A ELT V TTA DTA EA LGSG ...... D V P VL GT P R VL AL AE A A T V A AL DGR L EA. G H TSV R4L8I1 SAK V ELT V TDA DTA QS LGSG ...... D V P VL GT P R VL AL AE T A T V V A TART L PG. G L TTV G8SD04 SAR V ELT V TDA DTA QS LGSG ...... D V P VL AT P R VL AL AE A A T V A A TARH L PG. G I TTV I0H1J4 SAR V ELT V TDA DTA QA VGSG ...... D V P VL GT P R IL AL AE A A T V A A TARQ I PG. G V TTV U5VXT8 TAR V ELT V TDA DTA QA LGSG ...... D V P VL GT P R VL AL AE A A T V A A TARQ I PG. G V TTV C4RK94 SAR V ELT V TEA DTA LA AGSG ...... D V P VL GT P R VV AL AE A A T V A A TAGA L PP. G R TTV I0L862 TAQ V ELT V TDT DTA QA VGSG ...... D V P VL GT P R VL AL AE A A T V A A TAAT L PP. G S TTV F4FFD5 AAR V EMT V TDD DTA QA VGSG ...... D V P VL GT P R VL AL AE A A T V A A TARH L PA. G T TTV W7W6W1 TAR V ELT V TDA DTA QA VGSG ...... D V P VL GT P R VL AL AE A A T V A A TATR M PS. G S TTV D9T3T3 TAR V ELT V TDA DTA QA VGSG ...... D V P VL GT P R VL AL AE A A T V A A TATR M PS. G S TTV E8S960 TAR V ELT V TDA DTA QA VGSG ...... D V P VL GT P R VL AL AE A A T V A A TATR M PS. G S TTV A4X9A9 TAR V ELV V TVA DTA QA IGSG ...... D V P VL GT P R IL AL AE A A T V A AI ARQ L PS. G A TTV A8LVV3 TAQ V ELT V TDA DTA QA VGSG ...... D V P VL GT P R VV AL AE A A T V ATTAHQ V PG. G A TTV C7QDW7 QAQTTLN V TDA DTA RA LGSG ...... D L P VL GT P R LV AL AE Q A T V A AA QPL L TE. G Q TTV X0PXW9 RRA V RYE V GES DTA TA LGSG ...... D V P VL AT P R LI AW ME A A T I AC A APF I GA. G Q TTV J1RSR8 ... M RYE V VES DTA TA LGSG ...... D V P VL ST P R LI AW ME A A T V TC A ASF I GA. G Q TTV Q0SJ66 ... M RYE V VES DTA TA LGSG ...... D V P VL ST P R LI AW ME A A T V TC A ASF I GA. G Q TTV W8H6P1 ... M RYE V VES DTA TA LGSG ...... D V P VL ST P R LI AW ME A A T V VC A APF I GA. G Q TTV L2T6Q0 ... M RYE V VES DTA TA LGSG ...... D V P VL AT P R LI AW ME A A T V AC A ASF I GA. G Q TTV I0WLQ9 ... M RYE V VES DTA TA LGSG ...... D V P VL AT P R LI AW ME A A T V AC A ASF I GA. G Q TTV D6KEI0 ... M HYE V TEA DTA EA LGSG ...... D V P VL AT P R LI AW LE A A T V R AA GAF L EA. G R TTV V6KXP8 ... M RHE V TDA DTA VA VGSG ...... D V P VL AT P R LI AW LE A A T V R AA ARFTES. G Q TTV E2SCL7 MTT L TKT V RAD DTA RA VGSG ...... D L D VL GT P V L TAWC E E A TCA AL D.. L PA.EQ TSV A3THU5 SVE A TFT V TED DTA AA LGSG ...... S L P VL AT P R LL AW LE A A TCTS L EPL I GV. G R TSV R7XZ68 QAT L TFT V TES DTA IA VGSG ...... S L P VL GT P R LL TWC E A A TCA AL EGS L PA. G G TSV A1SHD6 TAT L TFT V TDD DTA AT LGSG ...... D L P VL GT P R LL AW LE A A TCA AI DPS L PT. G S TSV

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! 170! β2 β3 β4 β5 F8JPF9 TT . TT 70 80 90 100 110 120 F8JPF9 G TA I C V TH TAA T PP G LT V T V T A E L R.S V E GRRL S W R V S A H D G.....VDE I GS G THE RAV W5WVP2 G IA M N M TH DGPCTT G TE L D I HCE V T.A A SA RRA S W N V E V RTSR...GGVL M GTATHT RAI Q8FQC6 G VG V D I TH DAPATE G AE L V V R V T A T.G V GK R S A E W S V E V TAG.....ETVQGR G THK RVV M1UKN7 G VG I D I SH DAPATE G AT L E V K V E V V.G V GK R S V E W K V I V TAG.....DTVQGA G THK RAL D3PYM5 G TL V N I SH TAA T P VG LT V T A T V E L I.E V DK RRL R F A V ECR D D.....RDV I GK G HHE R F I H5XC58 G IH V D L SH EAP T P VG GL L T V Q A E L S.N V DR R Q L T F T V H A F D D.....AAT V CR G SHR RAI W5XXT0 G TH V N V SH VAP T VP G ST V T I D V T L E.A I ER R A L T F S I T A R D E.....YAV I ST G THQ R G V R0HVN0 G TH V NFS H SAP T VP G ST V T I D V E V A.E I NR R A V T F N I T A A D E.....FAT I ST G THQ R G V S5XAI0 G TH V NFS H AAP T VP G ST V T I D V E V T.E I NR R A V T F N I T A A D E.....FAT I ST G THQ R G V W9CWY1 G VS V T M RH RLPVR VG KH V EYR V T A G..DFA RKM D F SFE V RNLD....DEI V CD G THR RAV D2SED2 G SA L S V EH LAP S W VG DP V R V T A RCA.E V R G N RL TCECL A V D AG....GRV L GR G TTVQ VV I4EWF8 G RS L S V EH LAPAW VG DM V Q V T A ECV.E L R G N RL TCACR A VAAD....GRE L GH G STVQ VV L8F1C4 G LAHDSV H LAP T PT G EV V T L T A T L R.Q V D GKKL T F A V E A A D R.....HAT V LR G VHT RAV G2NQF4 G LAHDSA H LAP T P VG DD V T V S A T L S.R A DD RKL T F D V E A R D S.....HGS V LR G RHT RAL R7BPJ2 G TE I S I KH VSA S P VG AH I RCEST L T.E V ND R A L T F E V K A Y D N.....AGL I GE G THH R F I R7B458 G TN L N I SH DAP T P IG ME V SCKSE L V.K V D GR A L S F T V E V V D E.....QGI V GR G THD R F I E6S7M9 G TM V KFE H IKG S Q VG SE V T V HCAEP.IND GRRL VCF V S I K D AE....GDE L GH G EVH RAI W9GKR0 G TF V K I EH VKG T P VG AE V S V SCAEP.ISD GRRL VCF V T V T D PD....GET L AS G EVH RAV W9GAD5 G TM V KFE H VKG S P V DAV V T V NCAEP.IND GRRL VCH V T V I D SE....GEE I AR G EVH RAV M2WUQ8 G TA A T I EH LLP S A VG AT L T A R A T L V.ERD GRRL H F T V E V F D G.....DNVCATLTHS RAV D3QAD8 G TR V R L RH LAAVP LG RM V A A R A R L R.GRD G N KL T F D I S L V D GD....NNL V AD G EIE RAL M4ZY06 G MR I R V DH LQP T P VG AQ V S A E A T L E.K I D GRRL T F T V S V S D S.....GGL V AA G KIT RVL W5THV3 G TE V S V RH RRP S LP G TT L T V C A R L V.E V DEA RL T F R V V A RAG.....EVL V AD G TVR R T V D2AZ27 G TK V A L EH LVA S P VG TH V E I T A E L T.E V AE RRL V F TFT A R D RQ....T.T V AT G TVE RVV W2ELW3 G TK I R L DH LRP S P VG MH V E I VTE L T.E V D GRRL V F S V T A V D KT....GAV V AT G TIE RVV D6Y6X3 G TK V V L EH RAP S P IG MD V E V T A E L T.A V E GRRL V F SFT A V D RA....GTL L GT G TIE RVV R4Z4Z8 G MR V Q L EH IAP S A IG AV I R V DSS L D.K V E GRRL T F T V S A H E D.....GRL V AA G KVT RVV Q47SH7 G AE V H L KH TAP T P VG AQ V V A T A R L V.R A E GRRL V F E V S V TQE.....GRQ V AV G TVE RVV R4L8I1 G TR V E V DH RAP T S VG RK V V A Q A T L T.GTD GRKL Q F A V T V K D G.....DTV V AEVHVE RVL G8SD04 G VR A A I DH HAPAA VG RT V T A Q A A L T.R V D GR T L A F D V T V R D G.....ATV I AEVRID RAI I0H1J4 G SR A E I EH RAA T P LG RR V V A Q A T L T.K V D GRKL L F E V T V H D G.....DLL V AEVRVE RVL U5VXT8 G VR A E V EH RAP T P VG RM V T A L A T L A.K V D GRKL I F D V V V R D G.....ENL V AEVRVE RMV C4RK94 G TR V E L DH LAP T P VG RT V V A R A R L T.A V D GRRL G F E V T V T D A.....GEI V AR G RVE R T L I0L862 G TR V E L EH LAP T V VG RT V R A Q A L L A.T V D GRRL S F E V T V A D G.....DQT V AR G RVD RIL F4FFD5 G TR V E L EH LAA T P VG RT V T A Q A R L T.A V DK RRL T F E V T V V E G.....EQT V AQ G RVE RVV W7W6W1 G VR V E L EH RAA T P VG RT V A A H A E L V.K V D GKRL V F E V T V T D G.....PTV A AQ G RVE RAL D9T3T3 G VR V E L EH RAA T P VG RT V A A H A E L V.K V D GKRL V F D V T V T D G.....PTV A AQ G RVE RVL E8S960 G VR V E L EH RAA T P VG RT V A A H A E L V.K V D GKRL V F D V T V T D G.....PTV A AQ G RVE RVL A4X9A9 G VR V E L DH QAA T P VG RT V V A R A R L A.E V D GRRL L F A V S V T E D.....GST V AE G RVE RLL A8LVV3 G VR V E L DH RAA T P VG RT V V A R A R L A.K V D GRRL L F E V S V T E G.....GST V AE G RVE RLL C7QDW7 G TR I RFD H VYP T P VG GA V T A S A E L A.HRDD R L L R F T V A A H D AD....GRL V GE G EIT RVI X0PXW9 G TA V R I EH LGP T P VG AG V E I A A EPPAA A V GHRL T F T V R A I D SS....GKV V AS G EID RAI J1RSR8 G TA V R I EH RGP T P VG AT V E I A A EPQAA A V GHRL T F T V R A T D SS....GKV V AS G EID RAI Q0SJ66 G TA V R I EH RGP T P VG AT V E I A A GPPAA A V GHRL T F T V R A T D SS....GKV V AS G EID RAI W8H6P1 G TA V R I EH RSP T P VG AG V E I A A EPPAA A V GHRL T F T V R A T D SS....GKV V AS G EID RAI L2T6Q0 G TA V R I EH RGP T P VG AS I E I A V EPPAA A V GHRL T F T V R A T D SS....GKV V AS G EID RAI I0WLQ9 G TA V R I EH RGP T P VG AS I E I A A EPPAA A V GHRL T F T V R A T D SS....GKV V AS G EID RAI D6KEI0 G TA I R V EH LRA T A VG GR V E V A A EPPAAPD G T RL T F R V K A V D DS....GRL V AT G EID RAI V6KXP8 G TE V R V RH VRA T P VG GR V E V S A EPAAS A A G G RV T F V V R A V D GS....GRL V AT G EID RAI E2SCL7 G VR V L V NH LAA S P VG ME I K I D A H V T.SRD GR A V T F E V T A D D AH....GTR V ADARIE RAV A3THU5 G TR V D L QH SAA S P VG AT V I V T A DPA.Y A D GR LNRLR V S A RHTD....GKV V AT G EIT RVI R7XZ68 G TR I S L EH LAA S P VG AV L E V S A TTA.Y V D GR LHR F T V S A RHVD...GGRV V GA G EVT RVV A1SHD6 G TR V E V EH VAA S P VG QQ V E V T A S A S.Y V D GR LHR F T V A A RNVGAAGAGKL A AT G EIT RVV 1

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! 171! α4 F8JPF9 130 F8JPF9 I HLE KF NA KV RQKTPAG...... W5WVP2 VDR AT F SST V DRQVATIGG....QYLEHP.. Q8FQC6 VDR EQ F TGT V NNLADSFGA....IRI..... M1UKN7 I SR QQ FV SHA NNLASQFGA....NELKESSK D3PYM5 IDR RA F DAS A EAKRANPS...... H5XC58 ID TE RFL ARM SKRTAS...... W5XXT0 V NR ERFV GRL PQ...... R0HVN0 V NR DRFV SRL PEAPKEN...... S5XAI0 V NR EKFV SRL PEAPKEN...... W9CWY1 I ISAD FM RRL AGSTP...... D2SED2 L SEEAVQA RI GERARLQQRTGSGDVLPDAR. I4EWF8 L PQQALDD RL RRP...... L8F1C4 VDR DRFA ARL TARNA...... G2NQF4 IDR KRF TD KL SRRTA...... R7BPJ2 VD AA KF YK KA TGKLA...... R7B458 VDK ERF QS KA NAKLNK...... E6S7M9 VD PD RFM TR CRKLV...... W9GKR0 VDR ERFM SK CHPVA...... W9GAD5 VD PE RFM SK CQRLV...... M2WUQ8 A QAQ K ITD RL AQHGG...... D3QAD8 VDR AD FL TRL D...... M4ZY06 IDR DKFM GK CCSTN...... W5THV3 VDR KD FL ARA RDS...... D2AZ27 VDR ERFL AKL SR...... W2ELW3 VDR ERFL SRL ...... D6Y6X3 VDR ERFL ARL ...... R4Z4Z8 V NTDE FL SKL D...... Q47SH7 V PR ERFL SG L RPGE...... R4L8I1 IDR QRFI AKA IGS...... G8SD04 VDR QRFI AKA LGD...... I0H1J4 LDR QRFI ARA MER...... U5VXT8 LDR QRFI AKA LGDA...... C4RK94 VDR RRFV ERA SGAPARAATA...... I0L862 VDR QRFV DRA GRPS...... F4FFD5 LDR QRFI ERA GRSS...... W7W6W1 VDR QRFV ERA LGPS...... D9T3T3 VDR QRFV ERA VRQS...... E8S960 VDR QRFV ERA VRQS...... A4X9A9 VDR QRFI ERA GRSS...... A8LVV3 VDR RRFV ERA ARPS...... C7QDW7 V NAE RFM AKL ...... X0PXW9 VDR DRFL DASSPGDDNR...... J1RSR8 VDR DRFL AA A SPAKPDR...... Q0SJ66 VDR DRFL AA A SPAKPDR...... W8H6P1 VDR DRFL EA A SRA...... L2T6Q0 VDR DRFL EA A SWA...... I0WLQ9 VDR DRFL EA A SRA...... D6KEI0 VDR QRFL AK TAGA...... V6KXP8 VDR QRFL AR TPDPAADR...... E2SCL7 VDR ERFL ARL PGVTP...... A3THU5 VD TD RFL GRI Q...... R7XZ68 VD AE RFM SRV ...... A1SHD6 VD AE RFL SRL ......

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! 172! A)1)3(

β1 F8JPF9 TT 1 10 F8JPF9 ...... MKDG M RV G ERF T HD A6L1R5 ...... ME TGLT YT ST H5SBP7 ...... U5Q519 ...... ME TG KKLIIE R6XD43 ...... M IE LGI KGRQE R5BBK6 ...... M VE IGI KGRQE R6T5U3 ...... ME TGI IG Q EE R5RS32 ...... ME KGI IG Q GE R6E7S6 ...... M LE KGI KG Q EE R6WHR1 ...... ME SGI KF T LE R5KDY4 ...... ME IGV KNVIE R5CE45 ...... ME IGI KNVIE E4T2L8 ...... MIHS T FT QS R5UUV1 ...... M V QV G KS FT QK R5P9P0 ...... M LE TGIT YKQS R6F6N6 ...... M LE TG KS YT QE F9Z5K8 ...... M LE TG KS YT QE C2MAU3 ...... MNMESK P TT LT KQ F4KKS4 ...... MNMERKAT T LT KQ C3J9P1 ...... M L PIHQ T CQ LE S4NC03 ...... M I QI G DT YN CS T1CHV2 ...... M I QI G DT YN CS R5ICW6 ...... MATND I KV GIS GT ST D3IB68 ...... M I KE GL EH S AH F3ZNZ3 ...... M L KE GL DY S TE R5GHX3 ...... M KTK L YY T ST E5CHM3 ...... M LE KGLQ FIKE A7M138 ...... M LE KGLQ FIKE C3QYF9 ...... M LE KGLQ FIKE D7K9U9 ...... M LE KGLQ FIKE R5DIC1 ...... M LE KGL KH T SR B7BGY9 MKPKTGAKVRKKSIRTKSGLKNKFIIFGVGFVRLRERITEYGTINNEVTM LE KGL KH T SR A7AE80 ...... M LE KGL KHISR R5W6M0 ...... MP E KGL KYVAR R6CGH3 ...... ME KGLT YT SV R6ZRJ4 ...... ME KGLS YT SK F0QZ92 ...... ME KGLT YT SK R5TUD7 ...... ME KGL VY T SK R5MRP7 ...... ME KGLT YT SK R6C452 ...... ME KGLT YT SQ R7AZ58 ...... ME TGLT YS ST B5CUE7 ...... ME TGLT YS ST R7D0I0 ...... M LE KGLT FT AH R7NKX3 ...... ME TGLT YT ST U6RF88 ...... ME TGLT YT ST E5UZI6 ...... M ME TGLT YT ST C6Z502 ...... ME TGLT YT ST D1K928 ...... ME TGLT YT ST C3REJ4 ...... M ME TGLT YT ST C3Q5P4 ...... M ME TGLT YT ST R6WTD5 ...... MDMTMTQK LE KGLS AT AT R5WX43 ...... M LE KGLS ARSA D4IPX1 ...... M LE KGLS ARSA R7JGL8 ...... M ME KGLS AQ SK I3YPV8 ...... M LE KGLS AQ SR E4MCD9 ...... M LE KGLS AQ SR R6Y1D2 ...... MDII LE KGLS AQ SR F3XSV1 ...... ME IGLT HT ST R5NFJ0 ...... ME IGLT HT ST F3QUN7 ...... ME IGLT HT ST S8FGJ3 ...... M LE EGLT HT ST D1W0P3 ...... MM L KE GLS HT SV U7UUE8 ...... M L KE GLT HT SR D1W8U0 ...... MM L KE GLT HT SR E0NSD9 ...... M L KE GL KHVSR U2MMW3 ...... M L RE GLS HT SR D5ET49 ...... M IE VGL KH T ST R5CKQ7 ...... M LE TGLT HT SS R5LPE1 ...... M LE TGLT HT SS E7RLI7 ...... M L KE GLT HT SR

(

! 173! η1 α1 α2 α3 η2 β2 F8JPF9 TT 20 30 40 50 60 70 F8JPF9 FV V PPHKT V RH L YPESPEFAEFPE V F AT G F MV G LM E W A C V R AM APY L EP G EG SL G TA I CV A6L1R5 VV V SKENV AA T MGS ...... GDL N V F AT PAMVALM E NAAM S AV ADE L P EGSTTV G AM M NT H5SBP7 ...... MA A SLD G R...... LIHP L YS T FW LA YH AE V AA RK AI EPYFD EG E N A LG AG I EL U5Q519 KL V KETDT A EYY GS ...... G L L K V F AT PAMIALM E KS A HL LA KFS L PSN Q D TV G IE V NI R6XD43 EI V TEELT A SH IGS ...... G T V R V F AT PMMIALM E RTSRLS V KPY L E EGQ E TV G TK V NV R5BBK6 EI V TEDLT A SH IGS ...... G S V R V F AT PMMIALM E RTSRMS V KPY L E DGQ E TV G TK V DV R6T5U3 LI V NEMNT A EA LGS ...... G S L A V F AT PAMI T LM E KT A RLS V APF L E DGQSTV G TL V NV R5RS32 IT V TANDT A EA LGS ...... G S L A V L AT PAMIALM E KT A RLS V APH L E EGQSTV G TL V NI R6E7S6 IT V NENNT A EA LGS ...... G S L A V F AT PAMIALM E KT A RLS V APY L E EGQSTV G TL V NV R6WHR1 KV V TNEMT A RN M A S ...... G T L D V F AT P S MIALI E ET A WKS V AQE L E EG MG TV G IS L NV R5KDY4 TT V TENNT AV A MGS ...... G T L S V F AT PAMIALI E ET A WRS V VPY L EP GQ A TV G TR L DI R5CE45 IK V TKENT AA A LGS ...... G T L S V F AT PAMIALI E Q T A WKS V EPY L D EGQ G TV G TK L DI E4T2L8 TK V SDHQT AA Y LGS ...... G T L E V FG T PALVA F ME N T AM K ML S.D L PTE STSV G IS M NM R5UUV1 MT V QLKDT AA VY GS ...... G H L E V F AT PAMVALM E N T AV KC L EGE L EK GS D TV G IE I DV R5P9P0 IT V QPKDT AA VY GS ...... G S L E V F AT PAMVALM E N T AV HC L KGH L E EGQ D TV G IE I NV R6F6N6 II V GPKDT AL AH GS ...... G H L E V F AT PAMV G LM E N T AI HC L EGM L EPD T D TV G IE I HV F9Z5K8 II V GPKDT AL AH GS ...... G H L E V F AT PAMV G LM E N T AI HC L EGM L EPD T D TV G IE I HV C2MAU3 YI I EPKHS AA D MGS ...... GDM P V L AT PALIA F ME NAAM L LA RNYTE EGSTTV G TM I TT F4KKS4 YL I EPKHS AA E MGS ...... GDM P V L AT PALIA F ME NAAM L LA RNYTE EGSTTV G TM I TT C3J9P1 VT L QDHHS AI H VGS ...... GDL P V FG T PAMIALM E K A S V Q LV APH L E EG A STV G IH L DV S4NC03 TF V NEKNT AA A M F S ...... GDL A V FS T P S MAALM E QAAL T LL SKH L SAED TSV G IA L NI T1CHV2 TF V NEKNT AA A M S S ...... GDL A V FS T P S MAALM E QAAL T LL SKH L SAED TSV G IA L NI R5ICW6 TK V CKENC A SL MGS ...... G A L D V F AT PAVVA Y ME M AA CY AV NDL L E EG L STV G TK I DI D3IB68 II V EPEHS AL S VGS ...... GDM Q V L AT PALIA F ME NVAM LT V RDV L S D EV TTV G GM I EV F3ZNZ3 RI V EKSNL AV T MGS ...... GDL Q V L AT PALVAAM E NAAM L AV ADA L DPTD TTV G GH I DM R5GHX3 ST V TAENT AI A LGS ...... GDM E V L AT PALVALM E NAAM E AV IEE L D D E QTTV G AH I DV E5CHM3 VV V KPENL AV A MGS ...... GDL P V L AT PAMVA F ME NAAL S AV EDQ L P EGNTTV G AM I QS A7M138 VV V KPENL AV A MGS ...... GDL P V L AT PAMVA F ME NAAL S AV EDQ L P EGSTTV G AM I QT C3QYF9 VV V KPENL AV A MGS ...... GDL P V L AT PAMVA F ME NAAL S AV EDQ L P EGNTTV G AM I QT D7K9U9 VV V KPENL AV A MGS ...... GDL P V L AT PAMVA F ME NAAL S AV EDQ L P EGNTTV G AM I QT R5DIC1 TV V LPDNF AL A M R S ...... GDL P V L AT PALVALM E NAAM L AV AAE L P EG A TTV G VK M DM B7BGY9 TV V LPDNF AL A M R S ...... GDL P V L AT PALVALM E NAAM L AV AAE L P EG A TTV G VK M DM A7AE80 TV V QPDNF AL T MGS ...... GDL P V L AT PALVALM E NAAM L AV AAE L P EG A TTV G AK M DM R5W6M0 TT V GADNT AL A MGS ...... GDM E V F AT PAMVALM E H AAM Y AV AAD L P EGSTTV G AS M ET R6CGH3 AE V TAANT AI A LGS ...... GDM E V F AT PALVALM E NAAM N AV KSA L P EGSTTV G AM I QT R6ZRJ4 TI V NRNNT AI A MGS ...... GDL E V F AT PALVALM E NAA KQ AV ENY L P EGSTTV G AQ I QT F0QZ92 VK V TQANT AL A LGS ...... GDM E V F AT PALVALM E NAAM K AV QPE L P EGSTTV G AF I ET R5TUD7 VK V TQANT AL A LGS ...... GDM E V F AT PALVALM E NAAM K AV QPE L P EGSTTV G AF I ET R5MRP7 TT V CKGNT AL T IGS ...... GDM E V F AT PALVALM E NAAM N AV KPY L P EGSTTV G AM I QT R6C452 TT V CKGNT AL T LGS ...... GDM E V F AT PALVALM E NAAM N AV KPY L PKD STTV G AM I QT R7AZ58 VQ V TPTNT AL A LGS ...... GDM E V F AT P T LVALM E NAAM H AV QPA L P EGSTTV G AM I QT B5CUE7 VQ V NSTNT AL A LGS ...... GDI E V F AT PALVALM E NAAM N AV KPV L P EGSTTV G AM I QT R7D0I0 TT V TENNT AA T LGS ...... GDM N V F AT PALVALM E H AAM LC V APH L P EGSTTV G SL I HT R7NKX3 VT V SEENI AA T MGS ...... GDL E V F AT PAMVALM E NAAM N AV VGE L P EG A TTV G AM M NT U6RF88 VT V SEENI AA T MGS ...... GDL E V F AT PAMVALM E NAAM N AV VGE L P EG A TTV G AM M NT E5UZI6 VV V SKENV AA T MGS ...... GDL N V F AT PAMVALM E NAAM S AV ADE L P EGSTTV G AM M NT C6Z502 VV V SKENV AA T MGS ...... GDL N V F AT PAMVALM E NAAM S AV ADE L P EGSTTV G AM M NT D1K928 VV V SKENV AA T MGS ...... GDL N V F AT PAMVALM E NAAM N AV AGG L P EGS I TV G AM M NT C3REJ4 VV V SKENV AA T MGS ...... GDL N V F AT PAMVALM E NAAM N AV AGG L P EGSTTV G AM M NT C3Q5P4 VV V SKENV AA T MGS ...... GDL N V F AT PAMVALM E NAAM N AV AGG L P EGSTTV G AM M NT R6WTD5 TT V TAANT AL V MGS ...... GDL E V F AT P S MVALM E H AAM T AV APA L P EGSTTV G AE M NT R5WX43 AT V AAGNT AA A MGS ...... GDL A V F AT PAMVALM E NAAM T AV AGE L P EG A TTV G AE M NV D4IPX1 AT V AAGNT AA A MGS ...... GDL A V F AT PAMVALM E NAAM T AV AGE L P EG A TTV G SE M NV R7JGL8 VT V DGGNV AA T MGS ...... GDL Q V F AT PAMVALM E NAAM K AV ADH L P EGSTTV G AE M NV I3YPV8 TT V TNENT AA A MGS ...... GDL P V F AT P S MVALM E NAAM T AA AAA L PA GSTTV G AE M NV E4MCD9 TT V TNENT AA A MGS ...... GDL P V F AT P S MVALM E NAAM T AA AAA L PA GSTTV G AE M NV R6Y1D2 TT V TAANT AA T MGS ...... GDL G V F AT P S MVALM E H AAM T AV AAA L P EGSSTV G AE M NV F3XSV1 LT V ENKHL AI Q AGS ...... GDL E V L AT PVMMALM E NAAM L AV KEA L D EG M TTV G GH I SS R5NFJ0 LT V ENKHL AI Q AGS ...... GDL E V L AT PVMMALM E NAAM L AV KEA L D EG M TTV G GH I SS F3QUN7 LT V ENKHL AI Q AGS ...... GDL E V L AT PVMMALM E NAAM L AV KEA L D EG M TTV G GH I SS S8FGJ3 LV V AREHT AV A LGS ...... GDM D V L AT PAMMALM E NAAM W AV VHA L P EGSTTV G GS M TS D1W0P3 LT V TEAYT A QA LGS ...... GDL P V LG T PAMLALM E NAAM L AV ANE L D EGQTTV G GY L GA U7UUE8 LT V TDAQT A QV IGS ...... GDL P V L AT PAMLALM E NAAM L AV ANE L E EGQTTV G GH I SS D1W8U0 LT V TEAQT A QV IGS ...... GDL P V L AT PAMLALM E NAAM L AV ANE L E EGQTTV G GH I SS E0NSD9 LT V SNAQS AI AQ GS ...... GDL P V L AT PAMLALM E NAAM L AV ADS L P EGTTTV G GQ I AS U2MMW3 LM V CNDNS AI T LGS ...... GDM P V LG T PAMLALM E NAAM L AV AEE L D EGTTTV G GH I ES D5ET49 LV V TDDVT AV K IGS ...... GDM A V L AT PAMMALM E NAAM L AV ADA L P EGSTTV G GH I AS R5CKQ7 LT V TEALT A KA MGS ...... GDM P V L AT PAMMALM E NAAM L AV APE L P EGSTTV G GH I ES R5LPE1 LT V TEALT A KA MGS ...... GDL P V L AT PAMMALM E NAAM T AV APE L P EGSTTV G GH I ES E7RLI7 LI V NSKNT AL S MGS ...... GDL E V L AT PAMLALM E NAAM L AV ADE L P EGSSTV G GH I EC 1

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! 174! β3 β4 β5 α4 F8JPF9 TT TT 80 90 100 110 120 130 F8JPF9 TH TAA T PP G LT V T V T A E L RS VEGRRL S W R V S A H D GV.DE IG S G T H E R A VI HLE KF NA KV R A6L1R5 TH IKPS A VG DT V S A T A V L KE VEGRKL T F E V R A Q D SK. GVIGEG T H V RYIVDK E KFM S KL S H5SBP7 EH RNMCP LG VR V D I T A T V TQC DG N RI ICS L S A RAGA.IE I AR G TQTQV VL PSE R IQAM I A U5Q519 KH T R A T P LG AK V Y A E A E L IE ID N KKL T F S V V A F D EM. G E IG R G T H M RYII NPA KFM D KI S R6XD43 SH V SS T P VG LK V WCESE V VE ID R RRI V F K V A A Y D EK. GLIGEG T H E RFVID VA KF KA K TE R5BBK6 TH V SS T P VG LK V WCESE V VD ID R RRI V F K V A A Y D EK. GLIGEG T H E RFVID VA KF QA K TE R6T5U3 KH L SA S P VG MK I TCRTE L KE ID R RRL V F H V ECS D EA. GIIGEG E H E RFIID EA KFM A K TE R5RS32 KH L SA T P VG MK V TCRSE L VD ID R RRL V F H V ECT D EA. GLIGEG E H E RFIID EA KFM A K TE R6E7S6 KH L SA S P VG MT V TCKTE L VD ID R RRL V F H V ECY D TA. GLIGEG D H E RFIID EA KFM A K TD R6WHR1 EH V A PT P VG MK V RCETE L VK VDGRKL D F K V E V Y D EC.D LIG K G T H S RFIV KND KF QI KA N R5KDY4 SH V S PT P LG MT V KCETE L IE ADGRKL K F K A E V Y D ES. GLIG I G T H E RFII TAD KF QA KA E R5CE45 SH L S PT P VG MT V KCETE L TG IDGR C L R F E A N V Y D ET. GLIG K G V H E RFII SSD KF QI KA D E4T2L8 QH LK A S P VG AT V ECT A T I TA IEGRK YS F A I K A I D ASGD LIGE AM H E R V IV NID KFM S KV L R5UUV1 QH LK A T A IG GQ I SCQ A T I TQ VEGRKI S F E I E A W D EK. G K IG SAR H D RFIID PE RFM A KL . R5P9P0 KH IK A T P VG DK V NCK A K V TE IEGRRI R F E I E A E D EK. G Q IG YAI H D RFIID PV KFM A KL . R6F6N6 QH T K A T A VG KK V MCK A T I VE IDGRRI R F E I E A T D EK. G T IG HAI H D RFII YPE KFM S KL . F9Z5K8 QH T K A T A VG KK V MCK A T I VE IDGRRI RIE I E A T D EK. G T IG HAI H D RFII YPE KFM S KL . C2MAU3 DH Q H A T P IG ET I T V T A Q L SS IEGRKL T F A I E A T D QT. GVV AQAT H E RFIV S R Q RFL E RL G F4KKS4 DH Q H A T P IG ET I T V T A Q L SS IEGRKL T F A I E A T D QA. GVV AQAT H E RFIV S R Q RFL E RL G C3J9P1 HH T R A T A IG QT V R V M A T L TE VDGRKL I F D I R A E D SQ. G E IG H G TLE RFIV Y R D KFM A KL A S4NC03 TH S K A S K LG AE I T A T A T V TA IDGRKI S F D V E A W D PD. GLIG K G T H E RFVV N R E RFL S KL . T1CHV2 TH S K A S K LG AE I T A T A T V TA IDGRKI S F D V E A W D PD. GLIG K G T H E RFVV N R E RFL S KL . R5ICW6 AH IK A S P IG ET I T A K A T L KE IDGRRL V F D V T A S D SK. G E I AN G T H E RFII KVD KFM A KL Q D3IB68 KH LK A S P IG AE V V V K A R L EK VDG I KL Q F H L E A FQGE.K LI A EG S H L RFIV N R QE FM S RV L F3ZNZ3 KH LRPT P LG EKFT A Y A K V VR VEGKKI E Y S L I A E D GK. GVIGEG T H T RFVV N R E KFM S RL . R5GHX3 AH LRPT P IG ET V H A T A V L TA VEGRKL T F S V K A E D GH. GVIGEG T H V RYIVDR H RFL G KL K E5CHM3 TH LKPT A LG DT V L V T A T L LE VEGRKL T F S V V A S D SQ. G K IGEG T H I RYVVDR N KFM E KL A A7M138 TH LKPT A LG DT V L V T A T L LE VEGRKL T F S V V A S D SQ. G K IGEG T H I RYVVDR N KFM E KL T C3QYF9 TH LKPT A LG DT V L V T A T L LE VEGRKL T F S V V A S D SQ. G K IGEG T H I RYVVDR N KFM E KL T D7K9U9 TH LKPT A LG DT V L V T A T L LE VEGRKL T F S V V A S D SQ. G K IGEG T H I RYVVDR N KFM E KL T R5DIC1 TH VRPS GG G EE V Y A E A T L VE VEGRKL T F S L V A R D SV.D VIGEG T H I RYIVDR T KFL S KL K B7BGY9 TH VRPS GG G EE V Y A E A T L VE VEGRKL T F S L V A R D SV.D VIGEG T H I RYIVDR T KFL S KL K A7AE80 TH VRPS GE G EE V H A E A T L VE VEGRKL T F S L V A R D SA.D VIGEG T H I RYIVDR A KFL L KL K R5W6M0 TH VRPS A VG AV I A A E A E L VET EGRRL T F R V A A F D GE.A LIGEG T H V RYIVDR A KFL S KL . R6CGH3 SH IKPS A VG ET V K A T A V L QE VDGRKL T F S V S A V D SQ. GAVGE CT H V RYVVDR E RFL A KL K R6ZRJ4 SH IKPS A LG EE V T A T A V L EE VEGRKL T F R L K A T D SQ. GIVGEG T H I RYIVD IC RFM S KL K F0QZ92 TH VKPS A LG EE V S A T A V L EN VEGRKL A F K V T A S D TK. GVVGE AT H I RYIVDR E RFM S KL . R5TUD7 TH VKPS A LG EE V S A T A V L EN VEGRKL A F K V T A S D TK. GVVGE AT H I RYIVDR E RFM S KL . R5MRP7 SH IKPS G LG EE I S A T A L L EE AEGRKL T F K V S A S D PK. GIIGEG I H I RYIVDR E RFL S KL . R6C452 SH IKPS G LG EV I S A T A I L EEE EGRKL T F K V S A S D SK. GIIGEG I H I RYIVDR K RFL S KL D R7AZ58 TH LKPS G LG EE V T A T A E L TE VEGRKL T F K I S A S D RK. GLVGEG T H I RYIVDR E RFL S KL . B5CUE7 SH LKPS G L NEE V T A T A E L TE IEGRKL T F K V S A S D SK. GLVGEG I H I RYIVDR E RFL S KL . R7D0I0 SH LKPS A LG AS V E A T A E L TE VDGRKL T F R V S A H E GD.K LIGEG T H V RFIVDR E RFL A KL . R7NKX3 TH IKPS A MG DT V S A T A V L KE VEGRKL T F E V H A K D SK. GVIGEG M H V RYIVDR E KFM S KL S U6RF88 TH IKPS A MG DT V S A T A V L KE VEGRKL T F E V H A K D SK. GVIGEG M H V RYIVDR E KFM S KL S E5UZI6 TH IKPS A VG DT V S A T A V L KE VEGRKL T F E V R A Q D SK. GVIGEG T H V RYIVDK E KFM S KL S C6Z502 TH IKPS A VG DT V S A T A V L KE VEGRKL T F E V R A Q D SK. GVIGEG T H V RYIVDK E KFM S KL S D1K928 TH IKPS A VG DT V S A T A V L KE VEGRKL T F E V R A Q D SK. GVIGEG T H V RYIVDK E KFM S KL S C3REJ4 TH IKPS A VG DT V S A T A V L KE VEGRKL T F E V R A Q D SK. GMIGEG T H V RYIVDK E KFM S KL S C3Q5P4 TH IKPS A VG DT V S A T A V L KE VEGRKL T F E V R A Q D SK. GVIGEG T H V RYIVDK E KFM S KL S R6WTD5 TH IKPS G LG AS V T A T A I L TE VDGRKL T F N V G A R D AE. GLIGEG V H V RYVVDR A RFM A KL S R5WX43 TH IKPS G LG AE I T A T A V L AE VEGRKL T F N V G A R D AE. GLIGEG I H V RYIVDR E KFM A KV K D4IPX1 TH IKPS G LG AE I T A T A V L AE VEGRKL T F N V G A R D AE. GLIGEG I H V RYIVDR E KFM A KV K R7JGL8 TH IKPS G VG AE I V A S A V L TE VEGRKL T F N V G A R D AE. GMIGEG V H V RYVVDR E KFM S KV R I3YPV8 TH IKPS G LG AE I T A T A V L TE VEGRKL T F N V G A R D AG. GMIGEG V H I RYVVD CE KFM A KL G E4MCD9 TH IKPS G LG AE I T A T A V L TE VEGRKL T F N V G A R D AG. GMIGEG V H I RYVVD CE KFM A KL G R6Y1D2 TH IKPS G LG AE I T A T A V L TG VEGRKL T F N V G A R D AE. GMIGEG V H V RYVVDR E KFM A KV R F3XSV1 SH LKPT A LG KT I T A T A V L TG IEGRKL T F K V I A E D ET. G T IGEG E H L RFIVDR K KFM A KL Q R5NFJ0 SH LKPT A LG KT I T A T A V L TG IEGRKL T F K V I A E D ET. G T IGEG E H L RFIVDR K KFM A KL Q F3QUN7 SH LKPT A LG KT I T A T A V L TG IEGRKL T F K V V A E D ET. G T IGEG E H L RFIVDR K KFM A KL Q S8FGJ3 SH LRPS K IG TL I A A T A L L EK IEGRKL T F R V E A RQHG.T VIGEG V H V RFIVDR ET FL A KL G D1W0P3 SH VKPS K LG AT V E A T A T L TK VD R RKL S F H V V A K E NG.E VIGEG E H L RFIVDR Q RFM QG.. U7UUE8 SH LKPS K VG AM V E A T A T L TK ID R RKL F F H V V A KQDG.E VIGEG E H L RFVVDK Q RFM QG.. D1W8U0 SH LKPS K VG AM V ETT A T L TK VD R RKL F F H V V A RQDG.E VIGEG D H L RFVVDK Q RFM QG.. E0NSD9 SH L S PT A IG AI V E A E A V L EK VDGKKL H F R I T V RQGD.K LIGEG T H L RFIVDR E RFL S RL . U2MMW3 SH LKPS K IG AE V E A T A T V TR IDGKKL Y F S V V A R E GT.T VIGEG T H L RFIVDR E RFL S KV I D5ET49 SH LKPS K VG DT V T A T A E V VK VDGKKI E F K V S A YQGD.V LIGEG S H L RFVVDR E RFM S KL G R5CKQ7 SH LKPT P LG AE V K A E A T L TK VDGRKL Y F T V K A MQGD.V VIGEG T H L RFIVDR E KFM S KL . R5LPE1 SH LKPT P VG AE V K A E A T L TK VDGRKL Y F T V K A MQGD.T IIGEG T H L RFIVDR E KFM S KL . E7RLI7 SH LKPT G MG EE V E A T A I L NK VEGRKL Y F H V T A KQGD.T VIGEG E H L RFIIDR E RFL A KL .

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! 175! A)1)4(

F8JPF9 T 1 F8JPF9 ...... MKDG M RV E6VPY1 ...... MNEFKECLP L EV I4JL97 ...... MNNPTCTS L SI N6ZAE8 ...... MPSHPPI L TA V2TPW7 ...... MKE L TI Q8RS32 ...... MKDAMAINHP I PL E3BLC4 ...... MVFNNLIGHLKIMKK L EV R7WV65 ...... MPSPE L VP V5PXQ4 ...... MPSPE L VP V5UF45 ...... MPSPE L VP V2GN25 ...... MSPD L KP Q476Z6 ...... MSPE L RP B2AGA5 ...... MSPD L RP R7XDM1 ...... MSPD L RP Q0KF81 ...... MSPD L RP G0ET77 ...... MSPN L RP U3QKD1 ...... MSPE L RP E8WN56 ...... MKDT L AA B5EG61 ...... MKE L QV Q2RWF7 ...... MRDV L KV V5SDR4 ...... MKPT L EA D6V9R7 ...... MKPS L TA K8NP64 ...... MKPS L TV B6JGW2 ...... MKPT L VA J5PI50 ...... MP F8J907 ...... MKDT L HP D8JZE0 ...... MKPS L QA N0B387 ...... MRTAAVKPS L QA A7HFD1 ...... MKST L AP F2NC79 ...... MKDA L KP I4CEW2 ...... MKSS L QP M1SXE6 ...... MKTS L KP D8FEX3 ...... MKKT L KP D9SHK4 ...... MKDT L KP D5CM24 ...... MKDT L KP W6M343 ...... MRES L QP S7VEV0 ...... MKET L KP F4GGF8 ...... MNHESIS C5CWG3 ...... MNQQGVS D8NFT6 ...... M L TI A8ET68 ...... MS L EI D5V0F7 ...... ME L EI F7ZKG2 ...... MKQD L QI B2JRL7 ...... MTT L EK K0DQR3 ...... MNPNFLNPISTLIFSMTT L GK J7J7X3 ...... M I NS K8RJQ7 ...... M I ER G8MI72 ...... M I EK R4X3Z9 ...... M I EP I5CHG7 ...... M I EP W6X1M9 ...... M I EP U3QHT6 ...... MKNG L QP B3R275 ...... M QP F8GSV9 ...... MKNG L QP J3G8Y6 ...... MNFST M HA J2WAB7 ...... MNFST L EA U7DRC6 ...... MNFST L EA J3H1U4 ...... MNFST L HP J3GW14 ...... MNFST L HP R4X4Z9 ...... MINDS L RV G8MJ21 ...... M RA Q13AZ6 ...... MRPT L IA H6SJ72 ...... MKTT L HE M2Y638 ...... MEKNVMKES L VV Q2W7Y1 ...... MKST L VA M2ZCB4 ...... MKST L VA W0SEE6 ...... MSET L QA N6YXR0 ...... MSDT L QP S6H1G1 ...... MSTPII W9B1R2 ...... MKMSVT R4YBG9 ...... MKMSVT H3MVK1 ...... MKMSVT X7FEJ4 ...... MKMSVT K8P2Z0 ...... MSTLEK V TV F7QFC8 ...... MSTLEK V TV K8P616 ...... MNVLEK V AV U1GWE8 ...... MNPFDK V TT G7DNP5 ...... MSPLDK V TV Q89XN0 ...... MNPLER L TA I0G1C1 ...... MNPLEK V TT U1HEJ6 ...... MDARDF I KI W1K0M8 ...... MDARDF I TI I2QHF9 ...... MDARDF I KA Q89DI4 ...... MDARDF I TT G7D4M7 ...... MDARDF I KV H5YA88 ...... MDARDF I KV J2VKT2 ...... MDARDF I KV I0GET7 ...... MDARDF I KV H0TP42 ...... MDARDI I KI H0S5U0 ...... MDARDF I KP H0SDP2 ...... MDAREI I KP H0SXL7 ...... MDARDI I KP A4Z0L3 ...... MDARDI I KP M4ZEE9 ...... MDAREI I KP A5ECQ7 ...... MDAREI I KP Q13BL0 ...... MTTTHALIVEIAEALMDARDV L TV E6VGD3 ...... MDARDI L SV Q6NAK8 ...... MDARDF I TV I3TU50 ...... MSEIGDLIATLPG L TP U2HCP0 ...... MLDFSFLIPKESALT I QP C5AK92 ...... MS I QP F2NH42 ...... MESPFKV A0LL45 ...... MDTPFHA

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! 176! C3X5Q5 ...... MNPKNFF L VP G7D213 ...... MRQ I PL B1M8N7 ...... MQD I PL K8NJU9 ...... MKD I PI W4LKY3 ...... MDIPD L QP B6WPN7 MRDAALALLDDRLRGDVTAWYAWLAVHRGLPLDRKAVPVQEQEISVSATGRDIMETMPKA B8J205 ...... MKKK L EP G1UVA4 ...... MKTA L QV D9YAY2 ...... MKTA L QV E8RIW0 ...... MDIQ I PT D8JR26 ...... MADLSK L TI N0B8B7 ...... MERKMADISK L QI F8J518 ...... MADLSQ L RA A0LP78 ...... MNDRNK L QP V5SDQ6 ...... MTASERSTS L IP V4RI11 ...... MDQ L KP J6UG66 ...... MSETAR L VI

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! 177! β1 η1 α1 α2 α3 η2 F8JPF9 T T 10 20 30 40 50 60 F8JPF9 G ERFTHDFV V PPHK TV RHLYPESPEFA...E.FPE V F ATG F MV GL ME WAC V R AM APY L EP E6VPY1 GI GARESVT V APFH TV PELDDTWPGFR...D.MPA V L AT AM MI GF VE QTC I E AL RPY L PP I4JL97 GL RHSATLT V AARH SV PEVEPTWSGFA...D.MPP V L AT AM MI GF ME QTC I EG L RPF L TP N6ZAE8 GL RHSEQFT V EPRH TV PEVDVSWPGFQ...D.MPP V L AT AM MV AF IE QTC I MG L RPF L SS V2TPW7 GL KYTKEIF V DKSL TV PNVSELFTGFS...D.MPP V L AT AY LI GF VE WTC I E AL HDY L MP Q8RS32 GL RHSQTLR V DDSL TV PAVSAAFTGFS...D.MPP V F AT AY MV GF VE WAC I E AL RPY L AP E3BLC4 GM CYSYTYQ V PLNK TV PYQFPEVEELQ...V.MPE V FT SG F MI GMF E FVC V K AL KPF L NW R7WV65 GL TFEWTYR V PKKAV V PELYDDVELCR...D.MPA V L ATG Y LA GI LE CAC L Q AI RPY L DW V5PXQ4 GL TFEWTYR V PRKAV V PELYDDVELCR...D.MPA V L ATG Y LA GI LE CAC L Q AI RPY L DW V5UF45 GL TFEWTYR V PRKAV V PELYDDVELCR...D.MPA V L ATG Y LA GI LE CAC L Q AI RPY L DW V2GN25 GL AFTWRYT V PPKA TV PNLYDDVPGCP...E.MPD V L ATG Y MV GI ME CAC L Q ML REY L DW Q476Z6 GL TFTWEYP V PPKA TV PRLYDDIAMCT...E.MPD V L ATG Y MV GI ME CAC L Q AL RDY L DW B2AGA5 GL AFSWQYT V PPKA TV PRLYDDIPGCP...E.MPD V L ATG Y LV GI ME CAC L Q ML REH L DW R7XDM1 GL TFSWQYT V PPKA TV PRLYDDIPGCP...E.MPD V L ATG Y MV GI ME CAC L Q ML REH L DW Q0KF81 GL TFSWQYT V PPKA TV PRLYDDIPGCP...E.MPD V L ATG Y MV GI ME CAC L Q ML REH L DW G0ET77 GL TFSWQYT V PPKA TV PRLYDDIPGCP...E.MPD V L ATG Y MV GI ME CAC L Q ML REH L DW U3QKD1 GL TFRWQYT V PPKA TV PQLYDDIPCCP...E.MPD V L ATG Y MV GI ME CAC L Q ML RDH L DW E8WN56 GI GTTLKFS V PVEK TV PCLYPESALFR...E.MPE V F ATG Y LV GF IE WAC M E AL APY L EE B5EG61 GL KHTFSYL V PKER TV PFLYPESSYFQ...V.MPE V F ATG Y MV GF ME WAC M D AL APY L DE Q2RWF7 GM GETLSFE V PREK TV PFLYPESAEFQ...A.IPE V F ATG Y MI GL ME WCC V RS L APA L ED V5SDR4 GA TARLTFT V TAEK TV PNLYPESADFR...A.MPG V F ATG F MV GL ME WAC L E VL KPH L DA D6V9R7 G STHRFTYR V PENK TV PHLFPEARDFQ...I.MPH V F ATG Y MV GL ME WAC M D MI RPH L EE K8NP64 G STHRFTYR V PENK TV PHLFPEARDFQ...V.MPH V F ATG Y MV GL ME WAC I D MI RPH L ED B6JGW2 GA THRFTYR V PENK TV PDLFPEAHDFQ...I.MPH V F ATG Y MV GL ME WAC M D LI RPH L ED J5PI50 GL THRFSYT V PETK TV PCLYPEAPEFS...A.MPK V F ATG F MV GL ME WTC V R LM APH L DA F8J907 GA KTQFTYR V PATK TV PHLYPEAHEFQ...L.MPT V F ATG Y MV GL ME WTC L H II APH L DK D8JZE0 GA RTEFSFR V PATK TV PYLYPEAREFQ...L.MPT V F ATG F MV GL ME WTC L H IL EEH L DQ N0B387 GA RTQFSFR V PATK TV PHLYPEAREFQ...L.MPT V F ATG F MV GL ME WTC L H II DPH L DP A7HFD1 GV SLTFRYQ V PETK TV PHVFPESPRFV...E.MPQ V F AT AF MV GL LE WAC I E AM QPH L DG F2NC79 GL AYEFRYQ V PLER TV PHLLPESEEFQ...Q.MPK V L ASG F MV GL IE WAC I Q AI KPF L DW I4CEW2 GL TFRFRFM V PDNK TV PHLFPESSEFQ...V.MPK V L ATG F MV GL VE WAC I R AI NPH I DW M1SXE6 E I KYEYKFM V TDAQ TV PAMYPGSKEAA...V.RPK V F ATG F LV GF LE LAC V K AI ASH L DW D8FEX3 GI EHTFSFG I TASKI V PALYPESDEFQ...A.MPN V F ATG Y LV GF IE WTC I Q AL KPH L DW D9SHK4 GI RFEHKYL V PANK TV PALYPESPEFL...A.MPE V F ATG F MV GF LE WAC I M AI KPH L DW D5CM24 GI RYEHRFL V PSSK TV PALYPEAEEFL...A.MPE V F ATG F LV GF LE WAC I KC I NPH I DW W6M343 GL TGEFEFV V PPSK TV PHLYPEALEFQ...A.MPE V F ATG F MV GL IE WTC I Q AL KPH L DW S7VEV0 GL TFEFHYK V PEDK TV PRLFPEFPEGQ...I.MPN V F ASG Y MI GLF E FAC I K AV KPH L DW F4GGF8 Q I SFDEVYV V PSHQ TA RSLFTQLPHGRGYAETLIECI ATG Y LV AV VE SIC I KE M QRH V DH C5CWG3 E I CFEETYA V SHDQ TA RSLFARLPHGREYAQTLIECL ATG Y LV AV VE SIC V RE M QMH I DA D8NFT6 G DTACATTI V TGAEL A DVLS...... QTAEDAFPP V Y AT SR MV GL ME LAA A R VM RQA L A. A8ET68 G TKATIDYK V LNKDL A ANLQ...... ISKDDAFPE V F AT AR MV AL ME CSA A K MM LPL L K. D5V0F7 G TKDSIEFK V EDKDL A KNLQ...... ISPEDNFPE V F AT AR MV AL ME CSA A K IL IPF L E. F7ZKG2 G QSHTLTST V DAAR TA EAL...... AAQGEALPE V F ATP F MI AD LE RAC A A LL GPL L S. B2JRL7 G FTTSLTVT V TEEHL A SRLD...... IDAGERFPN V LS TP W LI AH LE RAA A K AL HPL L Q. K0DQR3 G FTTSLTVT V TEEHL A SRLD...... IDAGERFPN V LS TP W LI AH LE RAA A K AL HPL L Q. J7J7X3 GA TATLKRR V AVQDL A SAFG...... NDSDETYPD V MS TP A ML GL ME RAC A E IM RGE L E. K8RJQ7 G RAATLTHT V TSADL A SAHA...... RDADERYPD V LS TP A LL SL IE RAS A D VL RDA I G. G8MI72 G RAATLTHT V TSDDL A SAHA...... RGAGERYPD V LS TP A LL AL IE RAC A D AL RDA V G. R4X3Z9 G RAATLTRT V TAADL A SAYA...... QAEGERYPD V LS TP A LL AL IE RAC A D AM RDA V G. I5CHG7 G QAATLTHT V AAADL A SAHA...... HAAGERYPD V LS TP A LL TL IE RAC A G LM HDA L R. W6X1M9 G QAATLTHT V AAADL A SAHA...... HAAGERYPD V LS TP A LL AL IE RAC A G LM HDA L R. U3QHT6 GA TCSRTLT V DRQR TI DFLG...... E...S..LR I Y ATP E LV RD IE QTC L DF L QGY L E. B3R275 GA TCARTLT V DRQR TI DFLG...... E...S..LR I Y ATP E LV RD IE QTC L DF L QGF L E. F8GSV9 GA TCARTLT V DRQR TI DFLG...... E...S..LR I Y ATP E LV RD IE QTC L DF V QGF L D. J3G8Y6 G SATERRLT V DKER TI SFLG...... E...D..LR I Y ATP R LV DD IE QTC L DY L LTF L D. J2WAB7 G NATERQLI V DKER TI SFLG...... E...D..LR I Y ATP R LV DD IE QTC L DY L LTY L D. U7DRC6 G TATERRLI V DQPR TI SFLG...... E...D..LR I Y ATP R LV DD IE QTC L DY L LTF L D. J3H1U4 G SATERPVI V DKQR TI SFLG...... E...D..LR I Y ATP R LV DD IE QTC L DY L LTF L D. J3GW14 G SATERPLI V DKQR TI SFLG...... E...D..LR I Y ATP R LV DD IE QTC L DY L LTF L D. R4X4Z9 GA HYSRTIE V DVDR TI SFLG...... D...D..LR I Y ATP K LI DD IE RTC L DY L LDF V D. G8MJ21 GA THSRTIE V DRER TI SFLG...... E...D..LR I Y ATP K LI DD IE RTC L DY L LDF V D. Q13AZ6 GL SASHRIE I DPAR SI DFLG...... E...T..LR V Y ATP E LV RDF E TVCRE LI LAHTD. H6SJ72 GL CLTRHLS V DAAR TI TFLG...... D...S..LR V Y AT YQ II SD IE YACRD LL LAH V D. M2Y638 G QSATRRWV I DEGR TI GILG...... P...E..SR V Y ATP Y LV YD LE TTCREF L LEH L A. Q2W7Y1 GL TATRRYE V DKGR TI GFMG...... D...E..AR V YN TP A LI YD ME VTARD LL LEHSD. M2ZCB4 GL TATRRYE V DKGR TI GFMG...... D...E..AR V YN TP A LI YD ME VTARD LL LEHSD. W0SEE6 GL SATRRVE I DRDR TI SFMG...... D...D..CR V YS TP N LL YD IE MACRD LL LEH I D. N6YXR0 GL SLTRKYD I DAAR TI AFMG...... D...E..CR V YS TP A LL YD IE VCCRD LL LQH I G. S6H1G1 G HRHHCAFVPGLQQ TA AAIG...... N...A.GVQ V VS TP A LI AL LE ETAGSG V QAFCV. W9B1R2 G YPHTCVFTPGPHQ TA AAIG...... N...T.GVK V VS TP A LI AL LE ETS A D AV AKD L P. R4YBG9 G YPHTCVFTPGPHQ TA AAIG...... N...T.GVK V VS TP A LI AL LE ETS A D AV AKD L P. H3MVK1 G YPHTCVFTPGPHQ TA AAIG...... N...T.GVK V VS TP A LI AL LE ETS A D AV AKD L P. X7FEJ4 G YPHTCVFTPGPHQ TA AAIG...... N...T.GVK V VS TP A LI AL LE ETS A D AV AKD L P. K8P2Z0 GI PGETVVT V THDM TV QHFL...... S...T.MPP V Y ATP V MI LH ME KACTAS I AGL L P. F7QFC8 GI PGETVVT V THDM TV QHFL...... S...T.MPP V Y ATP V MI LH ME KACTAS I AGL L P. K8P616 GI TGETVVT V THDM TV QHFL...... A...T.MPP V Y ATP V MI LH ME EACTAS I AGL L P. U1GWE8 GM TAEKVVV V TPEM TV GHVV...... P...G.MPA V YG TP M MI LH ME MASGS AI QPF L P. G7DNP5 GL MAEKLVT I TPEM TV GHVV...... P...G.MPE V YG TP M MI LH ME MAAGS AI QPS L P. Q89XN0 GM TAEKLVT V TSEM TV GHVV...... P...G.MPA V YG TP M MI LH ME MAAGS AV QPF L P. I0G1C1 G TTAEKLVT V TPEM TV GHVV...... P...G.MPE V YG TP M MI LH ME MAAGS AV QPS L P. U1HEJ6 GM SAERMLV V PPER TV GHFV...... P...G.MPM V Y ATP M MI LD ME MASGD AI RGA L Q. W1K0M8 GM NADRTLV V PTER TV GHFV...... F...G.MPM V Y ATP M MI LE ME MTAGD AI RAA L Q. I2QHF9 GM SAERTLV V PVER TV GHFV...... P...G.MPM V Y ATP M MI LE ME MTSGD AI RSV L Q. Q89DI4 GM SAERTLV V PVER TV GHFV...... P...G.MPF V Y ATP M MI LE ME MTSGD AI RPA L Q. G7D4M7 GM SAERTLV V PAER TV GHFV...... P...G.MPF V Y ATP M MI LE ME MTSGD AI RAS L Q. H5YA88 GM SAERTLV V PVER TV GHFV...... P...G.MPM V Y ATP M MI LE ME MTSGD AI RAA L Q. J2VKT2 GM SAERTLV V PVER TV GHFV...... P...G.MPM V Y ATP M MI LE ME MTSGD AI RAA L Q. I0GET7 GM SAERMLV V PAER TV GHFV...... P...G.MPM V Y ATP M MI LE ME MTSGD AI RAA L Q. H0TP42 GL RAERILV V PPER TV GHFL...... A...H.MPM V Y ATP M MI LE ME MTSGD AI SAA L P. H0S5U0 GM RAERLLV V PPER TV GHFV...... P...D.MPM V Y ATP M MI LE ME MTSGD AI KPM L P. H0SDP2 GL RAERLLV V PPER TV GHFV...... A...H.MPM V Y ATP M MI LE ME MTSGD AI KEV L P. H0SXL7 GM RAERLLV V PPER TV GHFV...... P...H.MPM V Y ATP M MI LE ME MTSGD AI NPV L P. A4Z0L3 GM RAERSLV V PPER TV GHFV...... P...H.MPM V Y ATP M MI LE ME MTSGD AI NPV L P. M4ZEE9 GM RAERLLV V PPER TV GHFV...... P...H.MPM V Y ATP M MI LE ME MTSGD AI NPV L P. A5ECQ7 GM RAERLLV V PPER TV GHFV...... P...H.MPM V Y ATP M MI LE ME MTSGD AI NPV L P. Q13BL0 GM SAERQIT V TPEQ TV QHFV...... P...Y.MPA V F ATP L MI LE ME MASGE AV HPK L P. E6VGD3 GM SAERLTT V TQDV TV QHFV...... P...Y.MPA V F ATP L MI LE ME MASGE AI MPK L P. Q6NAK8 GM SAERLTT V TQDI TV QHFV...... P...S.MPA V F ATP M MI LV ME MTSGD AI APK L P. I3TU50 GL EGRMTHL V RSQDL A DAWQ...... G...E..VS A L AS AP VI WL AE LAC M R AV AAA L P. U2HCP0 GL RARATHR V DTISL A DQWG...... G...E..AH A L ASP I MI IF IE QTC M Q A TDHL L G. C5AK92 GL CARATHR V DTISL A DQWG...... G...E..AH A L ASP I MI IF IE QTC M Q A TDHL L G. F2NH42 GM INELRQKTGPEH SA RRFF...... P...D.LPD A F ATP F LV GL ME GVS A D LM AKH L Q. A0LL45 GM SHELKIKSLPEH SA RRFY...... P...N.LPD V LG TP I L GGL ME RVS A E LI NAH L S.

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! 178! C3X5Q5 GM AEKTELI V TEEQ TA RYWG...... S...G.KSD I L ATP A LV AV ME ATTQK I TDGG L P. G7D213 GA KGTSTLR V QPEHL A NRFK...... D...ALLPQ V L ATP V MI LV ME NAA L N AM RPYFD. B1M8N7 GA KGSFAML V GPSHL A SQFK...... D...NILPP V F ATP M MV LI ME NAA L N AV RQY L D. K8NJU9 GA KGRFGLE V TSQHL A SQFK...... D...PMLPP V F ATP M MI LA ME NAA L N AI RAY L E. W4LKY3 GL SAEITTE V DDSLV V KHVG...... G...... DG V LS TP S MI GL ME RAG I Q AV QPH L H. B6WPN7 GM MGLWETT V EEGML A AAVG...... S...G.EVR V L ATP M MI MG ME RAA M E AI RPC L P. B8J205 GL SGTLEIT V SEAML A CNVG...... S...G.LVD V FS T AM MI AW ME ATA V AT V QEI L DH G1UVA4 GL KGQSETV V GKELL A SEVG...... S...G.LVT V YS T AM MI AG ME GTA V AS V QDA L E. D9YAY2 GL KGQSETV V GKELL A SEVG...... S...G.LVT V YS T AM MI AG ME GTA V AS V QDA L E. E8RIW0 GI KGKEELV V TFEE TA AKYG...... S...G.LVE V Y ATP A MV AL ME KTC L KS V LPY L P. D8JR26 G SKGTSSAL V VEQRL A PAVG...... S...G.IAP V F ASP M LI AL ME AAA V DC I EAH L P. N0B8B7 GL RGSASAL V VEQRL A PAVG...... S...G.SAP V F ASP M LI AL ME AAA V DC I EAQ L P. F8J518 GL SGTASML V TDERL A TRVG...... S...G.NVP V F ASP M LI AA IE AAA V DC L DGF L P. A0LP78 GI KGTSRTS V DAGI TA LSMG...... S...G.EIE V L ATP A MA AL ME AAA V NC V KPY L P. V5SDQ6 GL EGRAETV V THAL TA AALG...... S...G.TAD V Y ATP A MI AL ME AAA V A AI EKH L A. V4RI11 GL TGRAKMV V GTND TA PRVG...... S...G.HVH V L ATP V MI GL ME EAA L A AV EHL L P. J6UG66 GA KGHAALT V TEAE SA PRIG...... S...G.TIA V L ATP V MI RL ME AAA L D AV EDS L P.

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! 179! β2 β3 β4 β5 F8JPF9 ...T TT .. TT 70 80 90 100 110 F8JPF9 ... G EG SL G TA I C V TH T A A TP P G LT V T V T A E L RS V .. EGR R L S W R V S A H D GVDE I GS G T H E6VPY1 ...QQR TV G TH V N V SH L A P TP V G MSFE A D V K L VE I ..NSNS L L F T V SCR D EVGL I GE G T H I4JL97 ...EQR TV G TH V D M SH T A A TP V G MT V T A T V E L VG I .. EGK A L L F N V QCR D EHGV I GE G T H N6ZAE8 ... G QH TV G IH V D V GH V A A TP V G MK V T A E V E L IE I .. EGK A L L F K V ACR D EGGL I GE G T H V2TPW7 ...SDK TV G TH V N L SH S A A TP I G LR I K A E V I L IN I ..NK K K L T F S V YCT D GLDI I CE G T H Q8RS32 ...SQR TV G TH V N L SH S A A TP V G MQ V T A E V E L IE V .. EGK R L T F K V LCR D EVDV I CE G R H E3BLC4 ..PSQQ SV G VGFN L TH ISP TP PDFT I K V D I E L VE M ..KKNR L R F K I I A H D GVDV I SE G V H R7WV65 ..PREQ SL G TL V SFS H L A A TP A G DT L R I QGK L VD V ..S GR T L R F E V V A W D GLDK V SA G T H V5PXQ4 ..PREQ SL G TL V SFT H L A A TP A G DT L R I RGT L VD V ..A GR T L R F E V E A W D GLDK V SA G T H V5UF45 ..PREQ SL G TL V SFT H L A A TP A G DT L R I RGT L VD V ..A GR T L R F E V E A W D GLDK V SA G T H V2GN25 ..PREQ SL G TL V SFT H L A P TP A G MT V T V KGV L TA I .. EGR K L R F E L S A W D GEDK I SE G V H Q476Z6 ..PREQ TL G TL V SFS H L A A TP P G MT I T V KGQ L VE V .. DGR R L R F E V S A W D GEDK I TE G V H B2AGA5 ..PREQ SL G TL V SFS H L A P TP P G MT V T V KGE L VE V .. DGR R L R F Q L S A W D GEDK I SE G V H R7XDM1 ..PREQ SL G TL V SFS H L A P TP P G MT V T V KGE L VA V .. DGR R L R F Q L S A W D GEDK I SE G V H Q0KF81 ..PREQ SL G TL V SFS H L A P TP P G MT V T V KGE L VA V .. DGR R L R F Q L S A W D GEDK I SE G V H G0ET77 ..PREQ SL G TL V SFS H L A P TP P G MT V T V KGE L VA V .. DGR R L R F Q L S A W D GEDK I SE G V H U3QKD1 ..PREQ SL G TL V SFS H L A L TP P G MT V T V KGE L VE V .. DGR R L R F E L S A W D GEDK I SE G V H E8WN56 ...DER SV G TM I N V TH S A A TP P G ME V T A Q V RCVE V ..T GK RTV W E I E V H D QADL I SK G T H B5EG61 ... G ER TV G TM I N V TH E A A TP A G ME V T A T V T L VE V .. DGK RTV W E I E A R D EVEV I GR G R H Q2RWF7 ... G EG SL G IA I N V SH L A P TP P G AR V V V E A K I IA I .. DGR K V S W H V V A R D EVDL I GE G TI V5SDR4 ... G EG SL G IH V D V SH SGA T VP G QV V T V D A TCTS V ..T GR R I A F D V V A H D GVEV I GK G R H D6V9R7 ... G EG TL G TL I N V NH T A A TP P G LT I H V D V ECTD V ..K GK L L R F N V K A H D GVDV I GE G R H K8NP64 ... G EG TL G TL I D V DH T A A TP P G LT I N V D V ECTE V ..K GK L L R F N V K A H D GVDV I GK G R H B6JGW2 ... G EG TL G TL I D V NH T A A TP P G LT I N V D V ECLE V ..K GK WSR F K V K A H D GIDV I GE G F H J5PI50 ... G EG SL G VH V D V DH T A A TP P G LT V T V D A ECVA V .. DGR R L T F Q V T A H D GVDT I GK G R H F8J907 ... G EG SL G VH I N V SH L A A T VP G QT V T V D A ECTK V ..A GR R L Y F H V K A H D GIDL I GE G E H D8JZE0 ... G EG SL G VH I N V SH V A A T VP G QT V T V E A ECTK V ..A GR R I F F H V K A H D GIDL I GE G E H N0B387 ... G EG SL G VH I N V SH V A A T VP G QT V T V E A ECTK V ..A GR R I F F H V K A H D GIDL I GE G E H A7HFD1 ... G EQ SV G TG I W V TH G A A TP P G FT V T V D V A V TK V .. EGR R L T F S V R A H D GVDA I CE G T H F2NC79 ..PREQ TV G IGFQ L SH S A P TP P G HT V T I R V E L SQ V .. EGR K L T F M V Q A A D ESEL I SQ G V H I4CEW2 ..PEEQ TV G TG I T L TH S A A TP P G LL V T I HGK L EQ V .. EGR K L V F SFS A E D GVDS I SE G T H M1SXE6 ..PEEQA VG TF I S V TH E A A TP P G ME V T A K V E L TE V ..R GK K L I F S V E A Y D DAEL V SK G S H D8FEX3 ..PQEQ TV G TH V D V SH M A A TP P G LE V T A N V R L VE V .. DGR R L L F E V E V Y D DVDL V SR G K H D9SHK4 ..PEEQ SV G TH I N V SH E A A TP P G LE V T A S V E L TI V .. DGR R L T F A V S A H D GVDT I AR G T H D5CM24 ..PAEQ TV G TH I N V SH Q A A TP P G LE V T A L V E L VE V .. DGR K L V F Q V E A H D GVEV I SK G R H W6M343 ..PHEQ SV G TH V D L SH T A A TP P G FT V T V K V R L VEK.. DGK K L K F A V E A H D GIDS I CQ G T H S7VEV0 ..PDEQ TV G IGFQ L NH T A A TP P G FT V T V K V K L TA V .. EGK K L T F A I E A F D GIDR I SR G T H F4GGF8 ..EREVV VG RS I H L EH RGPI P P G AE I R L SGW V ER L ..G PR S V T F N V R A H D SHEL V CE G HV C5CWG3 ..MMEVV VG RT V H I EH LGPI P PTTR L R L RGW V EG L ..GE R S A T F R V Q A Y D DHEL V LD G TV D8NFT6 ..A G EL SV G VT V E V SH M A A TP I G VE V T A E A RYVGQ.. DGK LFV F E V S A R D RGGE I GR G M H A8ET68 ..D G EL SV G VE V N I KH L A P T LE G DT A IST A TFVG M .. EGK LFK F E I E V V D SGGV I GN G T H D5V0F7 ..E G QL SV G VE V N I KH L A P T LS G DV A IST A TFEG M .. EGK LYK F S L E V V D SGGV I GT G T H F7ZKG2 ..D G EV SV G AR I E V SH T A P TP I G GQ I S A T A TFTEQ..V G ALYW F D V V A A D AAGQ I GK G RI B2JRL7 ..D G QL SV G SR V VFD H F A P TP L G ATFT A Y A KFTG M .. EG PLYL F D L W A A D AGGA I GK G T H K0DQR3 ..D G QL SV G SR V VFD H F A P TP L G ATFT A Y A KFTG M .. EG PLYL F D L W A E D AGGA I GK G T H J7J7X3 ..D G QL SV G VTTQ L TH S A P TP VDAQ V S A T A SFRGR.. EG SLFV F D V V V H D PKGQ V GS G T H K8RJQ7 ..D G QL SV G VTTH L NH L A P TP L G AE V S A T A TFRD I .. DG ALYW F D I V A T D AGGP I GTAS H G8MI72 ..D G QL SV G VTTH L HH L A P TP P G VE V S A T A TFRG I .. EG SLYW F D V A A S D PGGP V GTAS H R4X3Z9 ..AAQL SV G VTTH L NH L A P TP L G AD V S A T A TFRG I .. EG ALYW F D V V A A D AGGP V GTAS H I5CHG7 ..D G QL SV G VTTH L SH L A P TP P G ID V S A T A TFRG V .. EG ALYW F D I V A A D AGGP V GTAS H W6X1M9 ..D G QL SV G VTTH L SH L A P TP P G IN V S A T A TFRG V .. EG ALYW F D I V A A D AGGP V GTAS H U3QHT6 ..D G ES SV G AE V S V QH SGA TP L G GE V R I S A E V TA I .. DGR S V T F S V T A E D AAET V CS G T H B3R275 ..D G ES SV G AE V S V QH TGA TP L G GE V R I H V E V TA I .. DGR S V T F G V T A D D AVER V CS G T H F8GSV9 ..D G ES SV G AE V S V QH TGA TP L G GE V R I H A E I TA V .. DGR S V T F S V T A D D AVER V CS G T H J3G8Y6 ..E G EN TV G AA V D I RH VGA T LL G MS V N I A A T V TR I .. EGR S V T F N V E V R D DVEL V ATAA H J2WAB7 ..D G EN TV G AA V D I RH VGA T LL G MS V S I V A T V TR I .. EGR S V T F N V E V R D DVEL V ATAA H U7DRC6 ..D G EN TV G AA V D I RH VGA T LL G MS V S I V A T V TR I .. EGR S V T F N V E V R D DVEL V ATAA H J3H1U4 ..E G EN TV G AA V D I RH VGA T LL G MS V N I V A T V TR I .. EGR S V T F N V E V R D DVEL V ATAA H J3GW14 ..E G EN TV G AA V D I RH VGA T LL G MS V N I V A T V TR I .. EGR S V T F N V E V R D DVEL V ATAA H R4X4Z9 ..DEEN SV G TA V D I AH TGA T LL G MT V R I E V S V AS I .. EGR A V K F D V T V F D DIEQ V CT G S H G8MJ21 ..A G EN SV G TA V D I AH SGA T LL G MS V R I E I K V AS I .. DGR A V N F D I A V F D DIEQ V CA G S H Q13AZ6 ..P G ED SV G TG I A I SH GGA T LC G MH V D I T V T V SK I .. EGR L V T F E L R A N D AIEE I SR G S H H6SJ72 ..S G WD SV G TA V G L SH V A A TP L G AR V N I T V R V AE I .. EG SR V R F E A E V R D TAEV V AR G T H M2Y638 ..P G ED TV G TR V E M DH A A P TP L G MW V D I T V T V AA V .. EGR A V TLAFT A A D ALDR V AS G R H Q2W7Y1 ..A G ED SV G TR V E V DH L A P T LL G QW V E I T V T I EE V ..K G PG V T F SFT A K D SLDN I GK G V H M2ZCB4 ..A G ED SV G TR V E V DH L A P T LL G QW V E I T V T V EE V ..K G PG V T F SFT A K D SLDN I GK G V H W0SEE6 ..P G KD SV G TR V E L DH VGA T LL G MW V E I T V T L TA V ..N G GA V S F DFT A R D SVEE V AR G K H N6YXR0 ..E G KD SV G TR V E L DH VGA T LL G MW I E I T V K L VQ V .. DG PA V V F EFS A R D ALEE V AR G R H S6H1G1 ..P G WG TV G TK V D I AH LSPAWP G IE V QCE A V L EST..K GR T L S F A V S A SQQGRL V MR G H H W9B1R2 ..S G WV SV G TH V D V HH HKPAWPYQE V RCT A T L IS A .. E E K R L Q F F V Q A HQAGAL I MS G K H R4YBG9 ..S G WV SV G TH V D V HH HKPARPYQE V RCT A T L IS A .. D E K R L Q F F V Q A HQAGAL I MS G K H H3MVK1 ..S G WV SV G TH V D V HH HKPAWPYQE V RCT A T L IS A .. D E K R L Q F F V Q A HQAGAL I MS G K H X7FEJ4 ..S G WV SV G TH V D V HH HKPAWPYQE V RCT A T L IS A .. D E K R L Q F F V Q A HQAGAL I MS G K H K8P2Z0 ..D G HV SV G ME V N V RH L A A TP I G RT V R V TSR V KQ I .. D A K S V L F E V E A W D GSRK I GD G T H F7QFC8 ..D G HV SV G ME V N V RH L A A TP I G RT V R V TSR V KQ I .. D A K S V L F E V E A W D GSRK I GD G T H K8P616 ..E G YV SV G ME V N V RH L A A TP V G RT V R V SSR V RQ I .. DPK S V L F D V E A W D GSRK I GD G T H U1GWE8 ..V G YV SV G MM V N I RH L A A TP I G RT V R A I A R A VG V .. E A K S I L F E V E A W D GDRK I GD G T H G7DNP5 ..A G YV SV G MM V D V RH L A A TP V G RT V R A V A R V VA V .. E A R S V L F E V E A W D CGRK I GD G T H Q89XN0 ..T G HV SV G MM V N I RH L A A TP V G RT V R A I A R V VA V .. E A R S V L F E V E A W D GDRK I GD G S H I0G1C1 ..A G HV SV G MM V N I RH L A A TP V G RT V R A I A Q V VA V .. E A R S I L F E V E A W D GNRK I GD G T H U1HEJ6 ..P G WV TV G TE V D I RH L A AALV G AT V RTTSK V VV V .. E R R V I R F E V E A F E GTRK L GE G R H W1K0M8 ..P G WV TV G TE V D I RH L A AALV G AT V RTT A Q V VA V .. E R R V I R F E V E A F E GTRK L GE G R H I2QHF9 ..P G WV TV G TE V D I RH L A AALV G AT V RTT A K V VA V .. E R R V I R F E V E A F E GSRK L GE G R H Q89DI4 ..P G WV TV G TE V D I RH L A AALV G AT V RTT A K V VA V .. E R R V I R F E V E A F E GTRK L GE G R H G7D4M7 ..P G WV TV G TE V D I RH L A AALV G AT V RTT A K V VA V .. E R R V I R F E V E A F E GMRR L GE G R H H5YA88 ..P G WV TV G TE V D I RH L A AALV G AT V RTS A K V FA V .. E R R V I R F E V E A F E GSRK L GE G R H J2VKT2 ..P G WV TV G TE V D I RH L A A S LV G AT V RTT A K V VA V .. E R R V I R F E V E A F E GSRK L GE G R H I0GET7 ..P G WV TV G TE V D I RH L A AALV G AT V RTT A K V VA V .. E R R V I R F E V E A F E GPRK L GE G R H H0TP42 ..E G WV TV G TE V E V KH L A AA P V G AT I RTT A E V IE V .. E R R V I R F A V T A FHGDLK I GE G R H H0S5U0 ..P G WV TV G TE V N I RH L A A SP V G AT I RTV A E V IE V .. E R R V I R F S V E A YHGDVK I GG G R H H0SDP2 ..P G WV TV G TE V N V RH L A A SP V G AT I RTV A E V IE V .. E R R V I R F S V E A FHGEVK I GD G R H H0SXL7 ..P G WV TV G TE V N V RH L A A SP V G AT I RTV A E V IE V .. E R R V I R F A V E A FHGEVK I GD G R H A4Z0L3 ..P G WV TV G TE V N I RH L A A SP V G AT I RTV A E V IE V .. E R R V I R F A V E A FHGEVK I GD G R H M4ZEE9 ..P G WV TV G TE V N V RH L A A SP V G AT I RTV A E V IE V .. E R R V I R F V V E A YHGATK I GD G R H A5ECQ7 ..P G WV TV G TE V N V RH L A A SP V G AT I RTV A E V IE V .. E R R V I R F S V E A YHGETK I GD G R H Q13BL0 ..D G WV TV G TG V D I RH LTPALV G HV V RTVSR V TA V .. E Q R T V S F E V ACYVGTRR V GD G R H E6VGD3 ..A G WI TV G SE V D I RH L A PAFV G QT V RTT A T V SA V .. E R R A I R F E V ASYVGDRK I GD G R H Q6NAK8 ..P G WV TV G SE V D I RH L A PAFV G QT V RTT A V V SA V .. E R R V I R F D V ASFLGDRK V GD G R H I3TU50 ..P G HT TV G VGFD L RH L A P TP A G VT I E I T A R L NR A ..I GR S L L F E V RGT D GAGI I LD G RM U2HCP0 ..ADLM TV G YNFE I KH L A P TP PDWE V T V D A E L IS V .. EGR M M T Y K V S V R D AAGV V GE G I H C5AK92 ..ADLM TV G YHFE I KH L A P TP PDWE V T V D A E L IS V .. DGR M M T Y K V S V S D AAGV V GE G T H F2NH42 ..S G EQ SV G IG M N L KH T A P TP L G ME V R V VTE L IG I .. EGK K L T F K L E A F D EKEK I GEAT H A0LL45 ..P G EQ SV G IS M N L KH T A A TP L G ME V R V RTE V IA V .. EGR K L T F R L E A F D EAEK I GEAT H

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β1 η1 α1 F8JPF9 TT 1 10 20 F8JPF9 ...... MK.....DG M RV G ERFTHDFV V PPHKT V RH U5RWS7 ...... MDFSKLFKV G STYVSEYI V KPEDT A NF D8GUT2 ...... MDFSKLFKV G STYVSEYI V KPEDT A NF R5XE94 ...... M..KE I QLNAEASVSLE V TDDNT A VA R6J0V9 ...... M..KT A VGEVQGEALTL V NTTNT A IA F2I7N4 ...... MDTYFT V LPHQL A NQ H3NJZ3 ...... MTAIYTKNYE V TPEQA A SA M2DSM2 ...... MSIYSKSYVTTPQHS A MN J3F9A6 ...... MSIYSKSYVTTPEHS A MN G5KAY0 ...... MAIYSKVYETSTEHS A KY B9DSI3 ...... MTIFSKIFDTEEKHS A SQ F5ZJ64 ...... MTKYTHQFDTKSEHS A KH C0MC17 ...... MSIYSQLYETKSKHS A QA G5K657 ...... MSIYSQIYETRSEHS A KA K4N6V7 ...... MTIYSHTYETRPNHS A KA E7PZY6 ...... MTIYSHTYETDNQHS A KA I7MYH5 ...... MTIYSHIYETNAQHS A KA R6GEG5 ...... M G IKGELNYT V QEEDT A KA V6QBM2 ...... MSVFNYQ A EAQHT A SA J1HAQ4 ...... M..NE L KI G QTISQDYT V TSADL A SK U2QSW7 ...... MN I KLNKLFEKNFL V EKQHS A KN C5NYW0 ...... MN I EINKEFKQNYL V QDNNT A KF E5V1L6 ...... MN I ELNKIFETHYT I KGENS A KS F3A7K0 ...... MN I QLDKVFEESYV V KNENS A EN F2BXC3 ...... MKEEK..MN L KT G LNATSTFK I KSTET A KF W1U6V8 ...... MSMI G TKGSFTRV I AATDT A AA S4BZU7 ...... MESKKEFI V GQQDT A AV J0XJB8 ...... MESKKDFL V DLQDT A AV T2NPG1 ...... MESKKDFL V DLQDT A AV G5IUE9 ...... MIEKEFL V TEEDT A LA C8ZYY8 ...... MIEKEFL V TEEDT A LA T0V7E2 ...... MIEKEFL V TEEDT A LA K8E3X9 ...... MITKLSKTFI V KEEQT A IE R2SKA9 ...... MDKQILSYD V AENTT A KN R2VCM1 ...... MKEITKKYL V KQEQS A LK R2PBH5 ...... MKKITKQYK V SEDQT A LA R2R932 ...... MKKLTEYYQ V TKDQT A LS S0RW90 ...... MKKLIQLYQ V KEEQT A LK E4KRB3 ...... MKEIEKEFQ V TPELS A AK R2S8F8 ...... MDQTFAKEYT V TADLT A EA K8F818 ...... MPTEMFTKEFL V KESQT A KM D4MBC5 ...... MPTEMFTKEFL V KESQT A KM R3WMJ9 ...... MKKFIQHFV V AENQT A YA S0KCS8 ...... MIQKDFI V TKELT A AT R2TEP8 ...... MEEFSKTFV V GSKDT A KE R2TK84 ...... MEEFLKEFV V GSKDT A EE R3WR68 ...... MEEFSKEFV V GAKET A KE W8U886 ...... MKDV..KP G VKASVQRV V SFEDT A AA G8TZK5 ...... MTQEFLPHV G MEGQWSWV V GHEHS A DA F8I584 ...... MTQEFLPHV G MEGQWSWV V GHEHS A DA F4X9G0 ...... M.EK P TEYTLTWE V TDEMT A KR V2XUL5 ...... M.DK P TQHSLTWT V DDTMT A KR R6NZJ4 ...... MEEKTLTKTIK V EEEQL A VS R5DLE4 ...... M.SEINTHSVTIK V EEENL A VS D4L561 ...... M.SEINTYSVTIK V EEENL A VS R9LQS4 ...... MMNTKVEFERV V EEDGL A SS R5ZKL6 ...... MELKSVTIERK V ERANL A SV U2R6S0 ...... MIPFKMCLEDFI.MEQKTIKIERV V EKQHL A SI B7CCX0 ...... MELKTIRIQRV V EQQNL A SV J8HC92 ...... ME L IA G LKSKFTHE I SENEL A TN J8I2Q3 ...... ME L IA G LKSKFTHE I SENEL A TN J8HQS7 ...... ME L IA G LKSKFTHE I SKHEL A TN J8HXW9 ...... MNFQIDSLGKYEYV V QEKDL A SN C3GXB1 ...... MNFQVDSLGKYEYV V QEKDL A NN J8GWT9 ...... MNFQVDSLGKYEYV V QEKDL A NN J8LFQ4 ...... MNFQVDSLGKYEYV V QEKDL A NN J8AI75 ...... MNFQIDSIGKYEYI V KEKDL A SN H5XWA2 ...... MK.....ST L QS G LYYEFKFT V PENKT V PY G7W6G7 ...... MK.....ST L QS G LDYEFKFT V PDNKT V PY J7IYH8 ...... MK.....ST L QS G LSYEFKFT V PEDKT V PY D4J4P3 ...... ME M KA G MKFETEII V NEGML A NH U2L2J7 ...... M L KE G MKYREEVE V NREMT A ER Q8EMG8 ...... MK.....EG L NV G YQSTLEIE V TEDMF A QF T0JGA7 ...... MK.....EG L CI G DTKTITVE V TREMF A QF Q2B862 ...... MK.....PG L KE G AAASIHLT V GSEMF A QF U5L8R5 ...... MK.....PG L KE G AAASIHLT V GSEMF A QF U6BAI1 ...... MK.....LG L AV G DTAEIQAV V SEDMF A RF B1YII8 ...... MK.....NG L SL G DSAVIETT V TEEMF A QF K0A8N8 ...... MK.....NG L SL G DTAVIEAI V TEDMF A QF W7LB64 ...... MK.....PG L QI G NRAVIRAV V SPEMF A QF E5WMY4 ...... MK.....PG L QI G NQAVVHAV V SPEMF A QF S2XPF3 ...... MK.....RG L EL G REESIEII V TEDMY A AF W4RK43 ...... ML.....DG L IA G RTASIGFI V TPDMF A RF V6T386 ...... ML.....DG L IA G RTASIGFI V TPDMF A RF I8UG48 ...... MK.....PG I KI G DEAVITAT V TPEMF A QF A6CP21 ...... MVFKRRVNMK.....SG M KV G QTASVTVT V TPDMF A QF F8CXF7 ...... MK.....PG M KV G DTAVVEAV V TPDMF A QF E3IAI3 ...... MK.....PG M KV G DTAVVEAV V TPDMF A QF K6PLM5 ...... MCEVMPVK.....PGFVP G LRAEVEAT V DAGMQ A AF E6SLA6 ...... MK.....PGFVP G LRAEVEAT V DAGMQ A AF G8TSI3 YGIALGTLVMAQRIPNPRLDSRRLSKEEPPMR.....EG L VP G TKATVTTT V TEQMV A RL F8I237 YGIALGTLVMAQRIPNPRLDSRRLSKEEPPMR.....EG L VP G TKATVTTT V TEQMV A RL V9W1G5 ...... MRPAF L5MUG4 ...... MK.....EG L QP G TTAEFTVT V TEDMLPKF J2HJM9 ...... MK.....EG L QP G TTAEFTVT V TEDMLPKF C0Z567 ...... MI.....DR L QP G TTEEFTVT V TKDMLPVF J2G9M0 ...... MI.....DR L QP G TTEEFTVT V TKDMLPVF C8WU92 ...... MR.....EG L AP G QQKSWEIT V TEDMCPSF F5SL03 ...... MK.....PG L KP G HREQMQVT V TGEMT A SF D5WXP8 ...... MQEGVEGVK.....PG L NV G HRETMSIV V ASDMCPAF R7G5H6 ...... MKE I KINSAFTQEMI V SKEQL A SH A8RFU2 ...... MKE I KINSAFTQEMI V SKEQL A SH H1BNL1 ...... MKE V QLHAKCQEEVI V DEAKL A CN

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! 182! U5F4M4 ...... MKE V QLHAKCQEEVI V DEAKL A CN E2SKL5 ...... MNVKGRYVYERNQTAYKAHKKIT V EESML A SH H1B0W4 ...... M.KE I KLHTKLTKKIT V EESML A SH E4LXV6 ...... M.....KHKKEGTFM.KE I KLHTKLTKKIT V EESML A SH T4NF97 ...... M.KE I KLHTKLTKKIT V EESML A SH N9WK99 ...... M.KE I KLHTKLTKKIT V EESML A SH R6UIH5 ...... M.KE I KLHTKLTKKIT V EESML A SH G1VX76 ...... M.KE I KLHTKLTKKIT V EESML A SH H1BDX8 ...... M.KE I KLHTKLTKKIT V EESML A SH E1L909 ...... M I QV G QFATATVT V TESNI A KT S3A0K0 ...... M I QV G QSATATVT V TESNI A KT J5AQJ6 ...... M I QV G QSATATVT V TESNI A KT X8HFB3 ...... M I QV G QSATATVT V TESNI A KT W3Y8I0 ...... M V SA G QTATATVV V TESNI A RT D1BM20 ...... M V SA G QTATATVT V TESNI A KT D6KMG8 ...... M V SA G QTATATVT V TESNI A KT D6KHB2 ...... M V SA G QTATATVT V TESNI A KT E4LB70 ...... M V SA G QTATATVT V TESNI A KT W1UP58 ...... M V SA G QTATATVI V TESNI A KT C4FPZ7 ...... M V SA G QTATATVT V TESNI A KT W1W8P8 ...... M V SA G QTATATVT V TESNI A KT W1X196 ...... M V SA G QTATATVT V TESNI A KT U2BAI1 ....MGNC...... EG.RE.....PLQKGKKRGTRM L EA G TEGYQELV V TMEET A LA U2SW53 ...... M L QK G IKNQIERM V TETIT A EH R9IHN6 ...... M L ET G MKGYQEMT V TTADT A KV R9KEX9 ...... M L EA G IKGYQELT V TEGDT A KA S0J977 ...... M L ET G IKGHQELT V TAEDT A KV R5HR96 ...... M L TP G TALELTFP V TETLT A KT R6WU27 ...... MTN L HT G LTGTANVT V TEANI A AT F0T2R2 ...... M EI G AKGKSETV V TEKVT A KT D4J9J7 ...... M EA G IKNEKSIV V TEDVT A SK D4J2H1 ...... M L EI G MKGNAETV V TIDNT A KA R6GT38 ...... M EV G IKGKRERI V PYEQT A EY V2YEB4 ...... MS V TV G LKGRAETV V SDSNT A QA F4X8V5 ...... MS V EV G IKGRAEDV V SEKNT A QA A6NV90 ..MLTQP...... E.....PQFFREDEVSMS I EI G MTGRAECA V TEQNT A LA R5B634 ...... MS I SV G AVGTASTV V TPENT A AA U2BG97 ....MGL...... F.....QKKLERRNQFMS V TV G SKGRAACV V NDQNT A AA G9YX66 ....MGL...... F.....QKKLERRNQFMS V TV G SKGRAACV V NDQNT A AA H1C7K7 ...... MS V TV G SKGRAACV V NDQNT A AA D4JRV3 ...... M L TT G TKGKAEVK V DDTNT A VT R6RPT5 ...... M L TT G TKGKAEVK V DDTNT A VT F7K912 ...... MLLEEREKV L KT G IKGFRELM V TGENT A SA R6QC10 ...... MT L TT G IHGEQSVA V TNENT A KT E2ZHD1 MWYLIKVF...... KNKMR.....HTARKRRGKYMT L ET G IRGEQSVL V TAANT A KT R7C7Z2 ...... MS L EV G IKGTCTVT A DESNS A KT R6IH13 ...... M EI G IKGKVETV V DGSNT A KF R5CSL0 ...... M EI G IKGRAETV V NHENT A KF I5AU99 ...... M L EP G IKGCVERV V TEEMS A LR R6BVQ8 ...... M L EA G IKGQHTII V EKENT A AV G2T231 ...... M EA G ITGTQTIT V TEEKT A QA R5Q2H0 ...... M EA G IKGRKETL V TEENS A QA D7GQ38 ...... M L KT G IKGRQEVV V SDANS A KT R5FXP5 ...... M L ET G IKGRQEVV V SDANS A KT R6RV13 ...... M L TV G IKGKEEIV V TDANS A KT R6ZN81 ...... M L EA G IKGREEVM V TEANS A AA D6DGH0 ...... MNM L EA G IKGREEVM V TEENS A AA R5M2P2 ...... M L EV G IKGREEVM V TEENS A AA D4CH55 ...... MNM L EV G IKGREEVM V TEENS A AA D4MPY0 ...... M L EV G IKGREEVM V TEENS A AA R7B681 ...... M L EA G IKGRQEVM V TDENS A AA G5FFC4 ...... M L KA G IQGRQEVM V TEDNS A SA U2D4S0 ...... MRKEVEKM L KA G IQGRQEVM V TEDNS A SA R8VSU0 ...... M EL G IKGYKELT V TEDLL A KN B8I4C0 ...... ME L KV G LTGNAEVL V SESNT A KK U4R6H3 ...... ME L EV G ITGSAQVL V SEANT A KT H2JF67 ...... ME L KV G ITGSTEVL V SEANT A KT L1Q3R4 ...... M I KV G ITGETKDV V NESNI A KT R6KH82 ...... M I KV G IIGEVKDK V SENNT A KT R6FUY0 ...... M I KV G IIGEVKDK V SENNT A KT N2BI76 ...... M L TV G ISKELNRS V TKETT A AA E6LQN3 ...... M L EV G IKGQRETV V TKDKT A TG H1LX05 ...... M L EV G IKGQRETV V TKEKT A TG W2VGZ8 ...... M L EV G IKGQRETI V TKENT A AG K0XJJ2 ...... M L EV G IKGQRETI V TKENT A AG J4TCP2 ...... M L EV G IKGQREII V TKQNT A AG F3B6C0 ...... M L EV G IRGQKETI V TKQNT A AG R5RJN6 ...... M V KK G IKGHGEIV V TAALS A EK R6LSC1 ...... M L KT G IKGHQELV V TQELT A KN R6VIH0 ...... M L EV G IKGHQEMV V TDRYT A KT R7JYS9 ...... M L EV G LKGHQEMT V TDQFT A KT F7V6W4 ...... M I ET G IKGTLTDT V TKEKT A AA U2PAA7 ...... MMNFRGGHKM L KA G IKGEQSVE V VYENT A TA R5TLI5 ...... M L EA G IKGEQSVE V VYENT A TA R6P4I1 ...... M L NT G IKGSASEI V TNEKT A VA C0CNB6 ...... MY...... DES.....TENELLTGGFSM L KA G IKGTQEVL V TEANT A KT R6DLF5 ...... M L EK G IRGEQTVV V TKELS A KE R5SHR8 ...... M L KK G IKGTGRQV V TEKLT A KE R5ZA51 ...... M L KE G IKGTQEVV V SEENT A KT C4Z997 ...... M L QT G IKGKKEIT V TLDKT A KA E4Q5E0 ...... MKKPE L QV G LNFKVEEE V TDNML A SY D9TGD2 ...... MKKPE L QV G LRFEIEEV V SDSML A SH B9MM60 ...... MKKPE L QV G LKFKIEEV V SDNML A SH E4SE45 ...... MKKPE L QV G LKFEIEEV V NDNML A SH G2PV11 ...... MKKPE L QV G LKFEIEEV V SDNML A SH E4S8L3 ...... MKKPE L QV G LKFEIEEV V SDNML A SH E4QCS6 ...... MKKPE L QV G LKFEIEEV V SDNML A SH B0TBP0 ...... MEN I KI G LKGFKEEK V TADNT A IK D5X9M4 ...... MDIN L QT G IKGESKVV V SENNT A IA F6DMU1 ...... MTTE L KP G LTREVETI V DETNT A IA A4J0S5 ...... ME L KT G LIHETSIM V EESNT A IA K8DZX3 ...... ME L TV G MCHETSTT V TENQT A IA F6B4J8 ...... ME L KV G LIHEASIT V EDSNT A IA

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! 183! A5D1P6 ...... MARE L SF G LEGEARTT V DENNT A IA F6CP13 ...... MAGE L KI G LRGEAVTR V TEDNT A LA R7AYY7 ...... M A EKLQEYSLTWE V TDEMT A KR D7GRB2 CP...... A.....CTAVAAILADKADLYAPTYSLTWT V TDDMT A KR R5G526 CP...... A.....CTAVAAILADKADLYAPTYSLTWT V TDDMT A KR R7NT78 ...... MENFVIEKV V TEDML A VN R7AWH4 ...... MEQKTIKIERV V EKQHL A SI R5RY61 ...... M QI G DTYEYKNV V TKEIT A GA A1HRR7 ...... MALAQA L TV G LRGEVTIK V TPDNT A ER F7NH57 ...... MGQEQTVKP L SV G LRGEAESK V TSELT A ER H1HSX5 ...... M L NP G IKGHQEIT V EEKDL A IH I7K9K8 ...... MKEELFDK I KV G LNSIIETT V QGKDS A AN R7RUE8 ...... MIKVKE I RV G KRHIVERV A EPKDS A IN N1ZJT1 ...... MEF.N L SV G MSMEEEYV V TEADT A VH R7B8B0 ...... MEFNN I VP G MTYEREYI V EESDT A SH R6PY32 ...... MEFNN I VP G MTYEREYI V EESDT A SH R5AF40 ...... MEFKN I VP G MTFEKEFI V EEGDT A SH M1E923 ...... M I EV G LNAEIETI V TDNDT A AF R4KGW9 ...... MSLTKQ I TL G LTGSAQVE V NDQNT A IA A6TLR1 ...... MEFN L KE G MAAQVEIT V AQKDT A QA F4A004 ...... MDQEFH I QI G LCAAVETI V AEKDT A IT G8M073 ...... MENIN L KV G LTASIETK V FNKNS A EE A3DHM5 ...... MENVN L KV G LNASVETW V SQKST A EE W4V2Q9 ...... MKNVN L KV G LNASVETW V SDKST A EE H6LHK5 ...... MKNE L KT G TSYEKEFL V TKEDT A LA E6MGL5 ...... MTQEQISNK L RS G MEMEKDNT V SRELT A RV E3GGD1 ...... MQE L KV G MELKKDYM V TRTET A QA R7K521 ...... MN I EK G LKNTEQFE V TENLT A AS R5AKF1 ...... MMEA L KT G ATAEKLYS V TEQNT A LA B2A5Z4 ...... M QV G DRNEIVKE V SKEFS A KE T0N2A7 ...... MGNK L RE G ISAIIEKV V SENEA A SK A0PXM9 ...... MEFN V HE G TKGIMEMV V EDQHS A KR C6Q0E0 ...... MEFN V RE G MASTREIY V TENDV A SK A5N4K6 ...... MEFN L KE G ITSTMEMH V TDNNT A KK U2D856 ...... MEFN L KE G LSASSEVT V GGNNT A SF K6U6J6 ...... MEFN L KE G SSIVKKII V SDNDL A IK W6N7S3 ...... MEFN L KE G TVFTRKVK V TENDT A LK G7M699 ...... MELN L KE G TSIIKNLK V TENET A IK A6LWA7 ...... MEFN L NE G TSYIKEFK V TENET A IK V8G1J7 ...... MEFN L NE G TSYIKEFK V TENET A IK U5MUT7 ...... MEFN L KE G TSIIKESK V TENET A IK S1MVB3 ...... M L AMNQKAQQTMI V TQKET A QQ R6BIU7 ...... M L QV G LKGKKTFV V TTENT A EH R5GAX2 ...... MA L DT G MKMIKTEK V TVENA A KT R6G283 ...... MS L NT G MKMIKTET V TVENA A KT R6QH26 ...... MK I EI G DKITLSEE V TDELT A AH B0MBZ2 ...... MK I EI G DKITLSEE V TDELT A AH E5VQV8 ...... MK I EI G DKITLSEE V TDELT A AH D4MVS3 ...... MN I HV G DEIELTEE V VPENT A EK B0NZW1 ...... MN I HV G DEIELTEE V VPENT A EK E5VM16 ...... MN I HV G DEIELTEE V VPENT A EK L1PTM0 ...... MN I HV G DEIELTEE V VPENT A EK R5YWL8 ...... MN I HV G DEIELTEE V VPENT A EK C0EF68 ...... MED L KV G LTAAKTVT V GRDDL A VH D1PP05 ...... M L EP G IQGHAALR V TTENT A RA U2KTG5 ...... M I QV G MKHKESVE V TWEMT A EK E0E4V9 ...... M L EV G LKGVEKTV V DKTNT A AA D3MSS8 ...... M L EV G MKHSVELV V DKSKT A AA B1C982 ...... MNSK L TV G LKNERELT V EEKHL A SS G9YHC1 ...... MDFT L KANAAAEITEV V TEDNT A IK E2ZCX0 ...... MMDFT L KEQSSAELTER V TEKNT A IA U7UR89 ...... MEFT L KPQSTAEVSEI V TEDNT A IK R7MZQ7 ...... MEFN I KANSTAEVSEKCTENNT A IK S7HMM5 ...... MEFN I KANSTAEVSEKCTENNT A IK F9MNK7 ...... MDFT I RPNAQAEVSEI V NDNNT A IA F5TG45 ...... MEFT I RPNSQAEASEK V TDKNT A IA D3LTB8 ...... MEFT I RPNSQAEASEK V TDKNT A IA F5TG44 ...... MDFE V QKNSRAEVSEK V TDANT A VA D3LTB7 ...... MDFE V QKNSRAEVSEK V TDANT A VA F9MNK8 ...... MDFE V RKNSQAEVSEL V NDKNN A VS H1D2D9 ...... M I IK G ITAQITRT I TEKDT A EK R5SND6 ...... ME I KI G TAGTASRT V TEKDT A LA R7CTJ9 ...... ME I KE G MTGKAVLT V DTPHT A RV R6A5L1 ...... MS I TI G MKGTATKI V TENDT A KV R9MCN0 ...... MI G KTKEISVT V DREKL A CT B0PBF1 ...... MADEINSISIQ V VPEHL A CA K9CK05 ...... MEFTSK I AP G MTREVRGE V TEKNT A RV J4V0H9 ...... MEFTST I AP G MTHEVQDT V TEEKT A RV E7N2I9 ...... ME.....KELELFMEFTST I AP G MTHEVQDS V TEEKT A RV E4LMB1 ...... ME.....KELELFMEFTST I AP G MTHEVQDS V TEEKT A RV E0NZB5 ...... S.....QKERFAMEFNEK V HV G ATHEVHGA V TEENT A AT D4S5R8 ...... MDFTSS I AV G MTHEAHGK V TEENT A AT F5RQF3 AIDM...G...... EDAVS.....IQRRIYMDYTSK I AV G MTHEAVDT V TDANT A VT U2JVQ3 ...... M...... LSLYD.....KQRSRQMDYTSK I AV G MTHEAVDT V TDVNT A VT J7SJ73 ...... MN.....NRKDFHMDYTSK I AV G MTREAVDT V TDANT A LT C4V4L2 ...... MDYTSK I AV G MTHEAVDT V TDANT A VT L1MYN3 ...... MDYTSK I AV G MTHEAVDT V TDANT A LT G5GQK6 ...... MDYTSK I AV G MTHEAVDT V TDANT A LT C9LW87 ...... MVETK I EV G MKREAAAK V TEENT A IE J6I3C7 ...... MVETK I EV G MKREAAAK V TEENT A IE I0GTL3 ...... MELTD V KV G LQNVVTEE V TQEKT A RA R5EG72 ...... MG I TV G MKGVAETLCEREDT A KE R5VLB0 ...... MQP I TI G TKASASVQ V TEAQL A VT R7H2Y6 ...... MEP I TI G ARGTAQTV A DASKL A NA U2M1B8 ...... MEP I TI G SKGTASVQ V DETNL A VA W7UZ64 ...... MKD I TI G LTGTSELT V SENEL A KN R7MJM6 ...... MKE I II G TKGNAEII A EKSTL A VN D4LA09 ...... MKEFAL G TKGEAETQ V DESRL A CT R7AJW2 ...... MKE L AL G TKGEAETQ V DESRL A CN U2F0D7 ...... MEQENRSYTATEE V TPEKT A KA G4KR18 ...... M V MI G AKCQMEQV V TDKLT A AV U2RDD7 ...... M I LI G TKCRLEQT V TDALT A AN U2QNH9 ...... M I LI G TKCRLEQT V TDALT A AN

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! 184! R6TMK5 ...... M I VI G TKCQLEET V TQELT A AY R9LX73 ...... M I LI G TKCQLEQT V TQELT A AA R6H0F9 ...... M L EI G KSYTIEQV V TPEMT A RA R6D0Y4 ...... M I EV G MTYTAEKT V TPDMT A KA R6UAG6 ...... ME I EI G KKHTEKTR V TDDML A CS B2V410 ...... MNEPKI G QISKRELM V TKDTL A VN B1QTQ7 ...... MGKYEI G KKSKRECL V TKDLL A SS R5KLD7 ...... MKE V KS G AFAEKTLV V NGENT A VA R7BG85 ...... MKEFHGEYKGEALVT V NSTNT A AA R6M9E2 ...... MLT L KT G IKGQAQVK V VEKNT A KI F9N406 ...... MA I EI G QVGMATVV V EANNT A EY K9D8B9 ...... MS V EV G LKGEATVV V NEQNI A KT I4D8E6 ...... MA I SI G MRGYAETN V TFSNT A KS G2G1T3 ...... MN L QL G VRGQAQTI V TSLNT A KA J7IWS0 ...... MD L KV G RKGRAQTT V STLNT A KT G7WFQ0 ...... MN L IV G MKGEAKTT V TLSNT A KA H5XVU6 ...... MT L NV G IKGQAQTT V TVSNT A KA W0EBY5 ...... MD L KV G LKGRAENV V YEANT A KS L0FAB3 ...... MGE L TL G AKGRAEKL V DQTNT A KT B8G028 ...... MAE I TI G VKGRAEKI V DQTNT A KT I4AA80 ...... M G LKGRAEKI V DQTNT A KT Q67JY1 ...... MS L AP G LTATAETV V TPENC A RA Q3A9K1 ...... MAES L TE G LEGTAKTT V SLEKT A KA R6J6I5 ...... MKVE L RP G LQGRAEAI V DTTNI A AV R6I9I9 ...... MLQE L KP G LTGTAETV V RETNT A LA U2U2T3 ...... MTEFRI G SAAEATVM V MHTNT A KA G4Q7C4 ...... MTEFRI G SAAEATVM V MHTNT A KA C0WAV5 ...... MTEFRI G SAAEATVM V MHTNT A KA S2Z443 ...... MTEFRI G SAAEATVM V MHTNT A KA R7M286 ...... MKEFRI G AVGEATDT V LHTNT A AA D2RNA4 ...... MKEFRI G AVGEATDT V LHTNT A AA C0GGI3 ...... M L KQ G LTGEAAMK V AKGNT A ME C9R9F2 ...... MS L SV G LRGTATWV V REEDT A IV

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! 185! α2 α3 η2 β2 F8JPF9 . TT . 30 40 50 60 70 80 F8JPF9 L YPESPEFAEFPE V F ATG F MV G LM E WACVRA M AP.Y L .E..P G EG SL G TA I .C V TH T A A T U5RWS7 IG ...... N NG V V M LS TP A MI KY ME YTTLHI V DN.V I ..P.KNYRP VG TK I .D V EH IKPI D8GUT2 IG ...... N NG V V M LS TP A MI KY ME YTTLHI V DN.V I ..P.KNYRP VG TK I .D V EH IKPI R5XE94 AG ...... SG S L R V LG TP F MIALM E KATCNA V HE.F L ..E.F G ET TV G TC I .S V SH DKA S R6J0V9 A E...... SG S L P V LG TP F MLALM E KATCEA V AD.L L ..E. D DET TV G TA M .N I TH IKA S F2I7N4 VG ...... SG S L E V LS SP W LL GYF E NA A VRF L KD.H L ..E. E EET TV G TY A .Q L EH L A P S H3NJZ3 VG ...... SG D L P V L ATP A LIALM E NACYTYSQE.V L IED.PQQT SV G AK I .E V NH L L A S M2DSM2 W G ...... SG S L N V LS TP S MIA F IE NA A FLYCEE.D L .D..VSKT TV G AQ I .T V QH L A A T J3F9A6 W G ...... SG S L N V LS TP S MIA F IE NA A FLYCEE.D L .D..VSAT TV G AQ I .S V QH L A A T G5KAY0 IG ...... SG E L E V LS TP S LV GF LE NA A CLL A KE.K I .QN. D LLT TV G AK M .T I DH LKA S B9DSI3 MG ...... SG G L S V LS TP S LVA F ME NA A YHLGET.L L .QD.PKQS TV G SH I .S I QH LKA S F5ZJ64 LG ...... SG S L E V LS TP S IVA F ME HT A YLY A QE.T I .KD.SDYT TV G TE I .V I SH L A A S C0MC17 MG ...... SG G L E V L ATP A LVA F ME HA A FTF L QS.Q L .K.. D KQT TV G SE I .A I QH L A A S G5K657 MG ...... SG G L E V L ATP A LV TF ME NA A FLF A QE.H L .E..T G FT TV G SE M .A I QH ISA S K4N6V7 MG ...... SG T L N V L ATP A LVA F ME NASYLF A QE.Q L .D..SDFT TV G SE I .A I QH L A A S E7PZY6 MG ...... SG T L D V L ATP A LVA F ME NASYLF A QE.S L .E..T G LT TV G SE M .A I QH L A A S I7MYH5 MG ...... SG T L D V L ATP A LV SF ME NA A YLF A QE.S L .E..T G LT TV G SE M .A I QH L A A S R6GEG5 MG ...... TG D L D I FS T SK MIAML E LT V CKS L EK.H L ..N.KEIT TV G KST.S I QH FCP T V6QBM2 IG ...... SG E L E V V ATP A LIA F AE NACHQL V ...D L ..A.A G ET SV G TF I .E L NH LKA S J1HAQ4 LG ...... SG S V D V L ATP R LVALM E GVCARM L NT.Y L ..P. EG IT SV G SE V .H I KH L A P T U2QSW7 M K...... SG D L E V L ATP S LVA F ME QVSKEY L NT.F L ..E. DG CG SV G IN I .N I NH I A P T C5NYW0 MG ...... SG D L E V L ATP S LVA F ME NT A KNY L NT.F L ..S. D ELG SV G SN I .N I NH L A P T E5V1L6 MG ...... SG D L E V LS TP S LVA F ME NS A KNY L NN.F L ..N. E ELG SV G SN I .N I NH L A P T F3A7K0 MG ...... SG D L A V LS TP S LVA F ME NA A KNY L NK.F L ..P. E EMG SV G SN I .N I NH I A P T F2BXC3 I K...... SG S L P V L ATP V L C A Y IE ETCVKASEN.H L ..P.K G FT TV G FH I .E L DH I A P S W1U6V8 VG ...... SG T I D V C ATP V IAAAM E AA A VAA L AP.H L ..N. D DET TV G TR I .S L TH T A P S S4BZU7 LG ...... SG G L D V L ATP R LVAMV E NTCWEA L EP.T L ..D.K G FT TV G TKF.S I NH LKP S J0XJB8 LG ...... SG G L A V L ATP R LVAMV E NTCWEA L EP.T L ..D.K G FT TV G TKF.S I DH LKP S T2NPG1 LG ...... SG G L A V L ATP R LVAMV E NTCWEA L EP.T L ..D.K G FT TV G TKF.S I DH LKP S G5IUE9 VG ...... SG G L P V L ATP R LAAMV E AVCFEA L ES.R I ..A.S G KT TV G TQF.T I EH R A A S C8ZYY8 VG ...... SG G L P V L ATP R LAAMV E AVCFEA L ES.R I ..A.S G KT TV G TQF.T I EH R A A S T0V7E2 VG ...... SG G L P V L ATP R LAAMV E AVCFEA L ES.R I ..A.S G KT TV G TQF.T I EH R A A S K8E3X9 M K...... SG D L A V L ATP A LVAML E ET A KDS I LE.L L ..N. EG ET TV G IE V .N L KH VRP S R2SKA9 VG ...... SG D L S V LS TP A LLAMM E NC A KEL L SQ.Q L ..S. E QET SV G FA V .E L KH L A P S R2VCM1 MG ...... SG D L E V L ATP A LVAMM E NCCKVL L SS.Q L ..S.SEQT SV G FK M .N V KH LYP T R2PBH5 MG ...... SG D L D V L ATP A LVAMM E NC A KIL L AP.E L ..S.SVQT SV G YK M .D L KH L V P T R2R932 MG ...... SG D L E V L ATP A LVAMM E NC A KRL L AP.E L ..S.SNYT SV G FT M .N L KH I A P T S0RW90 MG ...... SG D L E V L ATP A LVAMM E NC A KQL L AP.E L ..S.SEQT SV G FK M .T L KH L V P S E4KRB3 MG ...... SG Q L E V L ATP A LIAMM E NT A QDL L NG.V L ..E.A G QS SV G TQ I .QCN H L I A S R2S8F8 MG ...... SG G I P V L ATP A MVAMV E NACYGE L QK.E L ..K.AEET SV G GF L .S I EH L A P S K8F818 LG ...... SG D L E V LG TP A LAAMI E QT A KEA V KD.Q L ..S.V G ET TV G TV L .E L RH LYP S D4MBC5 LG ...... SG D L E V LG TP A LAAMI E QT A KEA V KD.Q L ..S.V G ET TV G TV L .E L RH LYP S R3WMJ9 LG ...... SG E L K V L ATP A LAAMI E NTCMNS V KN.E L ..S. D SET TV G TL L .S L TH QRP S S0KCS8 IG ...... SG G L A V L ATP A VV Q MV E NTCYEY L QQ.K L ..N. E QQT TV G TK I .E L RH VKP S R2TEP8 MG ...... SG D L D V L ATP V MIAMV E NT A KEY L HK.E L ..A.SEET SV G TV I .N A KH LRP S R2TK84 IG ...... SG D L A V L ATP V MIAMV E NT A KEY L HK.E L ..A.LEET SV G TI I .N A KH LRP S R3WR68 IG ...... SG D L D V L ATP V MIAMV E NT A KEY L HK.Q L ..A.PEET SV G TV I .E A KH LRP S W8U886 LG ...... N EG I K V LS SP S MILLM E LASSKV L EP.L L ..E. E DER TV G ISF.D I KH M A P T G8TZK5 LG ...... N AG V M V L ATP M VL D LM E IASVQA I LP.A L ..P. D DWI SL G IH A .D L RH LQA T F8I584 LG ...... N AG V M V L ATP M VL D LM E IASVQA I LP.A L ..P. D DWI SL G IH A .D L RH LQA T F4X9G0 IG ...... S AGSK I LS TP N MVALM E GA A LEL A KA.Y L ..D. EG MT TV G AE I .HCR H L A P T V2XUL5 IG ...... T AG A K I LS TP N MLALM E AC A LEL A QE.Y L ..D. EG MT TV G AE A .H V RH L A P T R6NZJ4 MG ...... SG G L A V L ATP A VVALM E NA A FELCEEILN..N. D ELT TV G TA I .S I EH TSP T R5DLE4 MG ...... SG S L C V L ATP C VVALM E NA A AAL A DKVLD..N. D ELT TV G TM I .S I EH TSP T D4L561 MG ...... SG S L C V L ATP C VVALM E NA A AAL A DKVLD..N. D ELT TV G TM I .S I EH TSP T R9LQS4 M K...... SG T V N V L ATP Q MIA W ME EA A SQ...CLN L ..N. D DLT SV G TK I .N V SH IKA S R5ZKL6 M K...... SG S L D V L ATP Q MIA W ME EA A CL...CLD L ..E. DG QT SV G IS M .NTS H D A P S U2R6S0 M K...... SG S L D V L ATP Q MIA W ME EA A CL...CLE L ..E. EG KT SV G TL M .N V SH D M A S B7CCX0 M K...... SG S L D V L ATP Q MIA W ME EA A CL...CLD L ..D.Q G KT SV G IS M .N V SH D M A S J8HC92 ...... WRND V P V L ATP V LL W L TE LSCMKT I EQ.SE.LS.TNQM TV G YAH.E M KH L A A T J8I2Q3 ...... WRND V P V L ATP V LL W L TE LSCMKT I EQ.SE.LS.TNQM TV G YAH.E M KH L A A T J8HQS7 ...... WRND V P V L ATP V LL W LA E LSCMKT I EQ.CE.LG.TNQM TV G YAH.E M KH L A A T J8HXW9 ...... WEND V E V L ATP V LL W LA E VACMEA I KN.Q..IE. E NYM SL G LGHYE M KH L A P S C3GXB1 ...... WEND V E V L ATP V LL W LA E VSCMDA I KN.Q..IE. E NYM SL G LGHYE M KH L A P S J8GWT9 ...... WEND V E V L ATP V LL W LA E VSCMDA I KN.Q..IE. E NYM SL G LGHYE M KH L A P S J8LFQ4 ...... WEND V E V L ATP V LL W LA E VSCMDA I KN.Q..IE. E NYM SL G LGHYE M KH L A P S J8AI75 ...... WEND V D V L ATP V LL W LA E VSCMDA I KD.Q..IE. E NYM SL G LGHYE M KH L A P S H5XWA2 L YPESEEF Q AMPK V FG TG F MV G L FE WACIKA I NP.H L .DW.PNEQ TV G TD V .K L SH L A A T G7W6G7 L YPEAEEF Q AMPK V FG TG F MV G L FE WACIKA I NP.H L .DW.PNEQ TV G TD V .K L SH I A A T J7IYH8 L YPESEEF Q AMPK V FG TG F MV G L FE WACIKA I NP.H L .DW.PNEQ TV G TD V .K L SH I A A T D4J4P3 NDPQLPA.IELPA V FS TP S MI N LM E LTCAKL M DN.C L .E.. EG KG SV G MS V .E V RH L A S T U2L2J7 LG ...... N KG V P V FS TP E MLA F LE RTCRHC V EP.E L .E..Q G LG TV G IS V .E L NH L A S T Q8EMG8 GD...... EVVHP A YS T AS MV YH ME WVSRDL L LP.F L ..K. E QEE SV G AA V .K L KH I A P S T0JGA7 G G ...... EVVHP V YS T AS MV YH ME WVSRQC I LP.Y L ..E. E NEEA MG AA V .A L NH I A P S Q2B862 G G ...... Q IVHQ L FS T AS MV YY ME WASRKV I LP.F L ..E. E SEEG MG NA V .S V KH L A PA U5L8R5 G G ...... Q IVHQ L FS T AS MV YY ME WASRKV I LP.F L ..E. E SEEG MG NA V .S V KH L A PA U6BAI1 E G ...... Q LVHP A YS T VS MV YH ME WA A RQL I LP.Y L ..E.A G EEG VG GA V .S L KH LGMA B1YII8 E G ...... VVVHP A YS T VS MV YH ME WA A RKL I LP.Y L ..E. E QEEG VG GA V .S L KH VGMA K0A8N8 E G ...... VIVHP A YS T VS MV YH ME WA A RKL I LP.Y L ..E. E QEEG VG GA V .S L KH VGMA W7LB64 E G ...... EIIHP V YS T VT MI YH ME WASRLL I TP.Y L ..D.KDEEG MG GA V .S A RH LSP S E5WMY4 E G ...... EVIHP A YS T VT MI YH ME WASRLL I IP.Y L ..D. E EEEG IG GA V .S A RH LSP S S2XPF3 E G ...... EVVHP V YS T VA M TYH ME WVSRKI I LP.F L ..E. E HEEG MG AS V .Q L KH I A A S W4RK43 E G ...... KVVHP V YS T VS MV YH ME WVSRQI I VP.F L ..E. D HEEG MG GA A .T V KH I A PC V6T386 E G ...... KVVHP V YS T VS MV YH ME WVSRQI I VP.F L ..E. D HEEG MG GA A .T V KH I A PC I8UG48 E G ...... S LVHP A YS T VS MV YH ME WA A RQI I LP.F L ..E. EG EEG IG GG V .S A KH L A P T A6CP21 D G ...... EVVHP V YS T VS MV YH ME WASRKI I LP.F L ..E. E NEEG MG GA V .S M KH I A P S F8CXF7 E G ...... N IVHR A YS T VS MV YH ME WASRKI I LP.Y L ..E. E HEEG MG AA V .F V KH I A P T E3IAI3 E G ...... S IVHR A YS T VS MV YH ME WASRKI I LP.Y L ..E. E HEEG MG AA V .F V KH I A P T K6PLM5 D G ...... RVIHP L YS T WS LV HH LE HA A RRV L EP.Y L ..E.PHEEG VG YA V .E V RH L A P T E6SLA6 D G ...... RVIHP L YS T WS LV HH LE HA A RRV L EP.Y L ..E.PHEEG VG YA V .E V RH L A P T G8TSI3 G G ...... Q KVHP V L AT AQ MI EW ME WAGRKL I LP.Y L ..E. D DEDA VG YA V .D I VH L A P T F8I237 G G ...... Q KVHP V L AT AQ MI EW ME WAGRKL I LP.Y L ..E. D DEDA VG YA V .D I VH L A P T V9W1G5 D G ...... KVVHE V MS T VM MI YY ME KAGREM I VP.F L ..E. EG EEGS G YA L .D I KH VGPA L5MUG4 E G ...... EVVHP V MS T VS MI YY ME WAGRQV I LP.Y L ..E. E DEEGS G FA V .D I RH VGPA J2HJM9 E G ...... EVVHP V MS T VS MI YY ME WAGRQV I LP.Y L ..E. E DEEGS G FA V .D I RH VGPA C0Z567 E G ...... Q VVHP V MS T VS MI YY ME WVGRRV I LP.Y L ..E.ADEEGS G FA V .D I KH VGPA J2G9M0 E G ...... Q VVHP V MS T VS MI YY ME WAGRRV I LP.Y L ..E.ADEEGS G FA V .D I KH VGPA C8WU92 LG ...... KRVHP V LS T AT MI SY ME WV A RRT I LD.Y L ..E. DG EEG IG HS V .S V RH L A PA F5SL03 G G ...... KEVHPTLS T VA MI YY ME WVGRQV I LP.F L ..E. EG EEG VG SE I .S I QH R A PA D5WXP8 G G ...... VQVHPTLS T VS MI YY ME WVGRRV I LP.F L ..E. EG EEG VG AA I .S V EH R A PA R7G5H6 VG ...... SG V V D V F ATP M MIA F ME NT A VKC L EP.F M ..E. EG ET TV G IC M .NST H D A A T A8RFU2 VG ...... SG V V D V F ATP M MIA F ME NT A VKC L EP.F M ..E. EG ET TV G IC M .NST H D A A T H1BNL1 VG ...... SG I V S V Y ATP M MIAAM E HA A VQC L QP.F L ..E. EG ET SV G VL M .HTT H D A A T

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! 186! U5F4M4 VG ...... SG I V S V Y ATP M MIAAM E HA A VQC L QP.F L ..E. EG ET SV G VL M .HTT H D A A T E2SKL5 VG ...... SG I V N V Y ATP M MIALM E NT A ASC L DQ.F L ..D. EG ET SV G VM M .NTT H D A A T H1B0W4 VG ...... SG I V D V Y ATP M MIALM E NT A AAC L NQ.F L ..E. EG ET SV G VM M .RTT H D A A T E4LXV6 VG ...... SG I V D V Y ATP M MIALM E NT A AAC L NQ.F L ..E. EG ET SV G VM M .RTT H D A A T T4NF97 VG ...... SG I V D V Y ATP M MIALM E NT A AAC L NQ.F L ..E. EG ET SV G VM M .RTT H D A A T N9WK99 VG ...... SG I V D V Y ATP M MIALM E NT A AAC L NQ.F L ..E. EG ET SV G VM M .RTT H D A A T R6UIH5 VG ...... SG I V D V Y ATP M MIALM E NT A AAC L NQ.F L ..E. EG ET SV G VM M .RTT H D A A T G1VX76 VG ...... SG I V D V Y ATP M MIALM E NT A AAC L NQ.F L ..E. EG ET SV G VM M .RTT H D A A T H1BDX8 VG ...... SG I V D V Y ATP M MIALM E NT A AAC L NQ.F L ..E. EG ET SV G VM M .RTT H D A A T E1L909 M K...... SG S L E V F ATP A M C ALM E EA A QAA V QP.E L ..N. DG EG TV G IS L .S I SH D A P S S3A0K0 M K...... SG S L E V F ATP A M C ALM E EA A QAA V QP.E L ..N. DG EG TV G IS L .S I SH D A P S J5AQJ6 M K...... SG S L E V F ATP A M C ALM E EA A QAA V QP.E L ..N. DG EG TV G IS L .S I SH D A P S X8HFB3 M K...... SG S L E V F ATP A M C ALM E EA A QAA V QP.E L ..N. DG EG TV G IS L .S I SH D A P S W3Y8I0 M K...... SG A L D V F ATP A M C ALM E EA A QAA V QP.H L ..E.A G EG TV G IS L .S I TH E A P S D1BM20 M K...... SG S L D V F ATP A M C ALM E EA A QAA V QP.Y L ..E. DG EG TV G IS L .S I TH E A P T D6KMG8 M K...... SG S L D V F ATP A M C ALM E EA A QAA V QP.Y L ..E. EG EG TV G IA L .S I TH E A P T D6KHB2 M K...... SG S L D V F ATP A M C ALM E EA A QAA V QP.Y L ..E. EG EG TV G IA L .S I TH E A P T E4LB70 M K...... SG S L E V F ATP A M C ALM E EA A QAA V QP.H L ..E. EG EG TV G IS L .S I TH E A P T W1UP58 M K...... SG S L E V F ATP A M C ALM E EA A QAA V QP.Y L ..E. DG EG TV G IS L .S I TH E A P T C4FPZ7 M K...... SG S L E V F ATP A M C ALM E EA A QAA V QP.Y L ..E. DG EG TV G IA L .S I TH E A P T W1W8P8 M K...... SG S L E V F ATP A M C ALM E EA A QAA V QP.Y L ..E. DG EG TV G IA L .S I TH E A P T W1X196 M K...... SG S L E V F ATP A M C ALM E EA A QAA V QP.Y L ..E. DG EG TV G IA L .S I TH E A P T U2BAI1 AG ...... SG G L R V L ATP V M C ALM E KACWKS A AG.L L ..G. EG ED TV G IS L .S V RH V A A T U2SW53 IG ...... SG E L S V L ATP A MIALI E ETSWRS V AA.G L ..A. EG QG TV G TK I .D V AH L A A T R9IHN6 YK...... SG T L D V L ATP R MAALM E ET A WKS V AE.H L ..E.Q G MG TV G IQ L .N L EH L A P T R9KEX9 HK...... SG T L D V L ATP R MAALM E ET A WRS V AG.E L ..E. EG MG TV G IK L .D L GH L A P T S0J977 HK...... SG T L D V L ATP R MAALM E ET A WKS V AE.Y L ..E.P G MG TV G IK L .D L EH Q A P T R5HR96 VG ...... SG T L D V L ATP V MIA R ME QA A WTA V AP.S L ..S.PEEG TV G TW M .N V KH LSA T R6WU27 M K...... SG S L A V F ATP A M C ALM E EA A CAA V NA.H L ..E. EG SG TV G IS L .N I TH DRA T F0T2R2 LG ...... SG T L D V L ATP I MVAIM E NA A TRA..L.D L ..P. EG QT SV G TY L .E I KH L A A T D4J9J7 VG ...... SG L L P V Y ATP S MIALM E GTCAES V QS.E L ..A. EG EG TV G VS V .D I KH I A A T D4J2H1 FA...... SG A L E V F ATP A MIALM E ETCWKM I QP.E L ..E. EG LG SV G TK V .N I SH M A P S R6GT38 I S...... SG L L P V F ATP C MI QF ME ET A RLS V EP.Y L ..A. EG QS TV G TL V .N V KH L A S T V2YEB4 A C...... SG A L P V FG TP F M C ALM E EA A WKS I AP.H L ..E.P G QS TV G TK L .D I TH DSA T F4X8V5 A C...... SG A L P V FG TP F M T ALM E KA A WTS I AP.Y L ..A.P G ES TV G TK L .D V SH DSA S A6NV90 VG ...... SG A L P V F ATP M MAALM E AA A MNA V SA.C L ..E. EG QG TV G TK L .D I TH D A A T R5B634 VG ...... SG A L P V F ATP Y MIALM E NA A CNA I AD.G L ..E. EG QS SV G TK L .D V SH D A A T U2BG97 VG ...... SG L L P V F ATP Q MIALM E NA A VDATQA.S L ..E. EG QG TV G TR L .D V SH D A A T G9YX66 VG ...... SG L L P V F ATP Q MIALM E NA A VDATQA.S L ..E. EG QG TV G TR L .D V SH D A A T H1C7K7 VG ...... SG L L P V F ATP Q MIALM E NA A VDATQA.S L ..E. EG QG TV G TR L .D V SH D A A T D4JRV3 MG ...... SG S L K V F ATP A MIALI E KA A CKA L EG.Q L ..E. DG QT TV G TK L .D V AH V A A T R6RPT5 MG ...... SG S L K V F ATP A MIALI E KA A CKA L EG.Q L ..E. DG QT TV G TK L .D I AH V A A T F7K912 LG ...... SG R L C V L ATP A LA G LV E RTCWES V EP.L L ..E. EG ET TV G TA L .A I KH L A P T R6QC10 MG ...... SG T L D V F ATP A LVALA E KTCWMS V AE.A L ..D. EG CG TV G TK L .E L EH T A P T E2ZHD1 MG ...... SG T L E V F ATP A LVALA E KTCWMS V AD.A L ..G. EG NG SV G TK L .E L EH T A P T R7C7Z2 M R...... SG T L D V F ATP A MIALM E KT A WTS V AP.Y L ..E.P G EG TV G IH L .D I SH D A P T R6IH13 VG ...... SG S L D V F ATP C MV G LM E QA A QMS V AP.Y L ..G. EG EG TV G TK L .E I SH D A A T R5CSL0 VG ...... SG E L E V F ATP Q MIALM E QA A ASS V AG.E L ..E. EG QG TV G TL L .N V SH D A A T I5AU99 MG ...... SG E L M V L ATP C MIAMI E EA A WKS I SA.E L ..E. EG QG SV G TL M .D V KH T A P T R6BVQ8 M K...... SG T L A V F ATP A MIALI E ET A WKS V AD.E L ..E. EG CV TV G TS L .N V KH L A A S G2T231 MG ...... SG T L P V F ATP A MIALM E NT A YKS V AD.E L ..E. EG AG TV G TL M .N V KH V A A T R5Q2H0 MG ...... SG L L P V Y ATP A MVALM E GA A SES V AP.Y L ..E. EG TT TV G TA L .N I KH TSA T D7GQ38 MG ...... SG T L D V F ATP A M T ALM E KT A WMS V AD.Q L ..D. EG CG TV G TL L .N V TH D A P T R5FXP5 MG ...... SG T L D V F ATP A M T ALM E KT A WMS V AD.Q L ..D. EG CG TV G TL L .N V TH D A P T R6RV13 MG ...... SG T L D V F ATP A MIALM E KT A WKS V AP.Y L ..E. EG CG TV G TL L .N V TH D A P T R6ZN81 MG ...... SG L L P V F ATP A MVALM E QTSWKS V AP.Y L ..N. EG EG TV G TS L .N I SH S A A T D6DGH0 VG ...... SG L L P V F ATP A MVALM E QTSWKS V AP.Y L ..N. EG EG TV G TS L .T I SH SSA T R5M2P2 VG ...... SG L L P V F ATP A MVALM E QTSWKS V AP.Y L ..N. EG EG TV G TS L .T I SH SSA T D4CH55 VG ...... SG L L P V F ATP A MVALM E QTSWKS V AP.Y L ..N. EG EG TV G TS L .T I SH SSA T D4MPY0 VG ...... SG L L P V F ATP A MVALM E QTSWKS V AP.Y L ..N. EG EG TV G TS L .T I SH SSA T R7B681 VG ...... SG L L P V F ATP A MVALM E KT A WMS V AP.Y L ..N. DG EG TV G TK L .E I AH N A A T G5FFC4 LG ...... SG L L E V F ATP A MVALM E KT A WMS V AP.Y M ..N. DG EG TV G TM L .N I KH L A A T U2D4S0 LG ...... SG L L E V F ATP A MVALM E KT A WMS V AP.Y M ..N. DG EG TV G TM L .N I KH L A A T R8VSU0 VG ...... SG L V A V Y ATP M MIA G IE GT A AES V AP.Y L ..E. EG RT TV G TQ M .N V SH V A A T B8I4C0 MG ...... SG N L D V F ATP A MIALM E KA A SMA V QP.Y I ..G. E SSS TV G TM I .D V KH I A A T U4R6H3 MG ...... SG S L D V F ATP A MIALM E KA A SLA V QN.Y I ..D. E DSS TV G TM I .N I KH I A A T H2JF67 MG ...... SG S L D V F ATP A MIALM E KA A SMA V QN.Y I ..D. E DSS TV G TM I .N I KH I A A T L1Q3R4 L R...... SG E L K V Y ATP A MVALM E EA A YKS I QD.E L ..E. EG KG SV G IL M .N I KH LTS T R6KH82 L R...... SG T L N V Y ATP A MVALM E EA A YKS I ES.E L ..E. EG QG SV G TS M .N I KH I A S T R6FUY0 L R...... SG T L N V Y ATP A MVALM E EA A YKS I ES.E L ..E. EG QG SV G TS M .N I KH I A S T N2BI76 LG ...... SG L L D V Y ATP A MIALM E ETCMKS V QE.E L ..D. EG CG TV G TG L .T I HH VSA T E6LQN3 IG ...... SG L L E V F ATP V MIALM E ESSFMS V NE.R L ..D. EG FT TV G IA V .D I KH ISA T H1LX05 IG ...... SG L L E V F ATP V MIALM E ES A FMS V NE.I L ..D. EG FT TV G IA V .D I KH I A A T W2VGZ8 IG ...... SG S L E V FS TP M MILLM E ESCFMS V ND.I L ..E. EG FT TV G TC V .N V KH LSA T K0XJJ2 IG ...... SG S L E V FS TP M MILLM E ESCFMS V ND.I L ..E. EG FT TV G TC V .N V KH LSA T J4TCP2 IG ...... SG S L E V FS TP I MILLM E ECCFMS V DD.K L ..E. EG FT TV G IS V .N V KH VSA T F3B6C0 IG ...... SG S L E V FS TP V MILLM E ESCFMS V SD.K L ..D. EG FT TV G IS V .D V RH LSA T R5RJN6 MG ...... SG V M D V F ATP A MLALM E NT A YKS V LP.Y L ..N. EG CG TV G TK V .D I EH M A A S R6LSC1 MG ...... SG V M D V F ATP A MLALM E KT A FTS V AE.Y L ..N. EG CG SV G TK V .D I EH V A S S R6VIH0 MG ...... SG V M D V F ATP A MLALM E KT A FMS V AD.K L ..N. EG CG TV G TK V .E I EH V A S S R7JYS9 MG ...... SG V M D V F ATP A MLALM E KT A FMS V AE.H L ..N. EG CG TV G TR V .E I EH V A S S F7V6W4 VG ...... SG V L E V YG TP V MIALI E KT A CDS V AP.Y L ..E. EG SG TV G TN L .T I NH V A A T U2PAA7 VG ...... SG V L E V FG TP C MLALM E KT A SES V AP.Y L ..E. EG CG SV G TQ V .T I SH V A A T R5TLI5 VG ...... SG V L E V FG TP C MLALM E KT A SES V AP.Y L ..E. EG CG SV G TQ V .T I SH V A A T R6P4I1 VG ...... SG G L S V Y ATP A MI S LM E KS A YES V QS.L L ..E. EG SG TV G TL M .N I QH ISA T C0CNB6 MG ...... SG T L D V F ATP A MIALM E LT A WKS V AP.Y L ..E. EG NG TV G SY L .D I HH N A P T R6DLF5 MG ...... SG E L M V Y ATP A MIALM E KT A YTS V AS.E L ..E. EG MG TV G TL M .N V SH I A A S R5SHR8 MG ...... SG E L A V Y ATP A MIALM E ET A YKS V TA.E L ..E. DG MG TV G TL M .N V KH VSA S R5ZA51 MG ...... SG A L D V F ATP A MIALM E KT A WES V QP.Y L ..E. EG SG TV G TL M .N V KH V A A S C4Z997 MG ...... SG T M D V F ATP A MIALM E NT A FES V AP.E L ..E. EG SG TV G TA L .D I KH VSA T E4Q5E0 FQ...... SG L L D V F ATP S MIALM E KT A LLC V ES.Y L ..E. EG YT TV G SK V .D V LH L A P T D9TGD2 FQ...... SG L L D V F ATP S MIALM E KA A LLC V ES.Y L ..E. EG YT TV G SK V .D V LH L A P T B9MM60 FQ...... SG F L D V F ATP S MIALM E NA A LLC V EN.Y L ..E. EG YT TV G SK V .D V LH L A P T E4SE45 FQ...... SG L L D V F ATP S MIALM E KA A LLC V EN.Y L ..E. EG YT TV G SK V .D V LH L A P T G2PV11 FQ...... SG L L D V F ATP S MIALM E KA A LLC V EN.Y L ..E. EG YT TV G SK V .D I LH L A P T E4S8L3 FQ...... SG L L D V F ATP S MIALM E KA A LLC V EN.Y L ..E. EG YT TV G SK V .D I LH L A P T E4QCS6 FQ...... SG L L D V F ATP S MIALM E KA A LLC V EN.Y L ..E. EG YT TV G SK V .D I LH L A P T B0TBP0 Y G ...... SG G V E V Y ATP A MI G LM E GA A LAA V DP.L L ..P. EG MA TV G TY I .A V SH L A A T D5X9M4 Y G ...... SG G V E V F ATP A MIALM E NA A LNS V EK.F L ..P.P G HT TV G IN I .QSS H L A A T F6DMU1 Y G ...... SG G V N V F ATP A MI G LM E KA A LEL A DS.Y L ..P. EG QT TV G TL V .N I KH T A A T A4J0S5 Y G ...... SG G V R V F ATP A MI G LM E KA A LEL A DQ.Y L ..P. EG ET TV G TL V .N I KH T A A T K8DZX3 Y G ...... SG G V R V F ATP A MI G LM E KA A LEL A DQ.C L ..P.A G QT TV G TL V .N V KH T A A T F6B4J8 Y G ...... SG G V R V F ATP A MI G LM E KA A LEL A DQ.Y L ..P. EG QT TV G TE V .N V KH T A A T

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! 187! A5D1P6 H G ...... SG SSR V F ATP A MI G LM E KA A LSS V DP.L L ..E.A G LT TV G IR V .D V KH K A A T F6CP13 Y G ...... SG G A R V F ATP A MI G LM E KA A LSS V DP.L L ..E.P G QI TV G IR V .D V EH L A A T R7AYY7 IG ...... S AGSQ I LS TP N MVALM E AA A LEL A KS.Y L ..E. EG QT TV G AE I .HCR H L A P T D7GRB2 IG ...... S AGSK I LS TP N MVALM E DA A LEL A KS.Y L ..E. EG QT TV G AE I .HCR H L A P T R5G526 IG ...... S AGSK I LS TP N MVALM E DA A LEL A KS.Y L ..E. EG QT TV G AE I .HCR H L A P T R7NT78 VG ...... SG S L R V L ATP T VVALM E EASTKL A DT.F L ..D. EG LT TV G TM V .E I QH ISP S R7AWH4 M K...... SG S L D V L ATP Q MIA W ME EA A CLC..L.E L ..E. EG KT SV G TL M .N V SH D M A S R5RY61 LG ...... SG G L D I F ATP F LI CW ME AA A YYY M KE.Q L ..P. EG KS SV G TM V .N V RH VSP S A1HRR7 F G ...... N AG A A V F ATP M LVALM E QA A IAA V AG.C L ..A.P G EG TV G TR V .D V QH L A A T F7NH57 F G ...... N AG A P V Y ATP M LAALM E QA A IAA V SQ.A L ..Q.P G QG TV G TR L .D I SH L A A T H1HSX5 VG ...... SG T V H V L ATP M MIA N ME YTSAAS V EE.L L ..G. EG KT TV G VK V .D V SH V A A T I7K9K8 YL...... NG F I D V LS TP S LI GF LE CA A KNA V DL.H L ..P.T G YS TV G IK V .D V KH L A A T R7RUE8 YA...... SG G V D V YS TP A MI G LM E CA A KEC L DE.F L ..D. EG YS TV G IS L .N I KH L A A T N1ZJT1 F G ...... SG T V T V L ATP K MIA W ME GVSLNA V LP.F L ..P.K G YD TV G TA V .E I KH S A A T R7B8B0 F G ...... N DK V P V F ASP K LL SW IE GT A IGT V AP.F L ..P. DG WE TV G TTF.DFK H L A A T R6PY32 F G ...... N DK V P V F ASP K LL SW IE GT A IGT V AP.F L ..P. DG WE TV G TTF.DFK H L A A T R5AF40 F G ...... N DK V P V F ASP R LI SW IE GT A IGT V AP.F L ..P. EG WE TV G TTF.D L AH L A A T M1E923 Y G ...... SG L V N V LS TP R IV S LL E GA A VEA I KN.N L ..A.S G ET SV G TL I .N I EH I A P T R4KGW9 Y G ...... SG D I S V F ATP A MI G LM E KA A LSS V DP.L L ..P. EG YT TV G IK V .D V EH V A A T A6TLR1 F G ...... SG G V E V L ATP M MI G LM E RA A LTA V DP.H L ..P. DG FA TV G TH L .N V KH MGA T F4A004 F G ...... SG A I S V F ATP S MIALM E KA A LSA V DL.H L ..P.K G YA TV G TR I .E V EH I A A T G8M073 MG ...... SG D L D V Y ATP A MI C LM E RS A SSA V SL.H L ..P.K G YT TV G TA I .N V KH M A A T A3DHM5 Y G ...... SG N M D V Y ATP A MI G LM E RA A LSA V AL.H L ..P.K G YT TV G TA V .N I KH I A G T W4V2Q9 Y G ...... SG D M D V Y ATP A MI G LM E RA A LSA V SL.H L ..P.K G YT TV G TS V .N I KH I A G T H6LHK5 MG ...... SG G A E V L ATP R LVA W ME NV A FEG V EA.AQ..Q.P G NT TV G TF I .E L KH L A A S E6MGL5 VG ...... SG G L D V L ATP A LAA W IE NA A YEM A DL.W L ..P. E EET TV G AN I .N L DH M A P T E3GGD1 MG ...... SG G L E V L ATP I LVA W AE NA A YEM A EL.C L ..P. D EQT TV G VN I .N L NH I A A T R7K521 LG ...... SG L L P V LS TP A MIA F ME GVCQHS V QP.Y L ..S. EG YG TV G AE L .N V RH VSA T R5AKF1 LG ...... SG D L P V L ATP A LIA FF E NA A KDS V AS.M L ..P.GSAT TV G TA I .S V SH R A P T B2A5Z4 F G ...... SG S V D V L ATP A MIA F ME EA A LKL V DS.Q L ..D. E DRA TV G SL V .N V KH L A P T T0N2A7 Y G ...... ATG I E V Y ATP A MIALM E NT A KSA V DL.H L ..S.Y G YT TV G TE V .C I KH LKA T A0PXM9 VG ...... SG L V D V F ATP S MIALM E NTSQAS V KD.S L ..P. EG YA TV G ID I .S V KH MKA T C6Q0E0 LG ...... SG N I D V FS TP S MIALM E NTSKSC V DL.H L ..P.F G YT TV G IE V .N V KH IKA S A5N4K6 LG ...... SG N L D V F ATP A MIALM E NTSKNS V DL.H L ..P.T G YT TV G IE I .N I KH IKA S U2D856 Y G ...... SG S L E V F ATP A MIAIM E NA A LKA V QS.E L ..P. E DFT TV G IE V .S V KH IKS T K6U6J6 IG ...... SG D L E V Y ATP A MIALM E NA A KSL V VN.E I ..P.NEFT TV G IE I .N V KH IKS S W6N7S3 MG ...... SG D L E V Y ATP S MIALM E NA A KSS V AK.D I ..P. E DCT TV G IE M .N V QH VKS S G7M699 MG ...... SG D L E V Y ATP A MIALM E NA A KDI A SP.E L ..P.A G FT TV G IE M .N V KH IKS S A6LWA7 MG ...... SG D L E V Y ATP A MIALM E NVSKSL V IE.E L ..P.S G YT TV G ID M .S V KH IKS S V8G1J7 MG ...... SG D L E V Y ATP A MIALM E NVSKSL V IE.E L ..P.S G YT TV G ID M .S V KH IKS S U5MUT7 MG ...... SG D L D V Y ATP A MIALM E NV A KSL V IE.Q I ..P.QNYT TV G IE I .N V QH IKS S S1MVB3 V E...... SG E L A V F ATP S LLALV E KT A WQS I AS.C L ..S.AEQT TV G TN I .V L DH L A P T R6BIU7 M L...... SG L L P V Y ATP S LIA F ME YTCSET V RP.F L ..D.A G MS TV G TL V .N I KH TSA S R5GAX2 LG ...... SG S L L V YG TP A MLLLV E KT A VAL L DG.H L ..D. EG MT TV G TN L .N V DH VSA S R6G283 LG ...... SG S L L V YG TP A MLLLV E KT A VAL L DG.H L ..D. EG MT TV G TN L .N V DH VSA S R6QH26 W G ...... SG S L P V Y ATP A M T LLV E RA A VKL L EG.K L ..D. EG MT TV G TN L .N I SH VSA T B0MBZ2 W G ...... SG S L P V Y ATP A M T LLV E RA A VKL L EG.K L ..D. EG MT TV G TN L .N I SH VSA T E5VQV8 W G ...... SG S L P V Y ATP A M T LLV E RA A VKL L EG.K L ..D. EG MT TV G TN L .N I SH VSA T D4MVS3 W G ...... SG S L P V Y ATP A MI S LI E HS A VDL L AG.K L ..D. EG QT TV G TN L .N I AH VSA T B0NZW1 W G ...... SG S L P V Y ATP A MI S LI E HS A VDL L AG.K L ..D. EG QT TV G TN L .N I AH VSA T E5VM16 W G ...... SG S L P V Y ATP A MI S LI E QS A VNL L AG.K L ..E. EG QT TV G TN L .N I AH VSA T L1PTM0 W G ...... SG S L P V Y ATP A MI S LI E QS A VNL L AG.K L ..E. EG QT TV G TN L .N I AH VSA T R5YWL8 W G ...... SG S L P V Y ATP A MI S LI E QS A VNL L AG.K L ..D. EG QT TV G TN L .N I AH VSA T C0EF68 VG ...... SG D A E V Y ATP K LV N LI E SVSASL V LE.H L ..SDPELT SV G TL V .N I SH L A A T D1PP05 LG ...... SG E L D V L ATP A VAALI E KACWKS V AG.E L ..D.M G TG TV G TS L .S L VH T A P T U2KTG5 LG ...... N EG V I V FS TP E ML SF LE ITCRHC V EP.H L ..K. EG EG TV G IS V .D L KH M A S T E0E4V9 IG ...... SG G L E V FS TP S MI S LM E CTCKLC A QE.H L ..E. EG LG TV G IS I .STN H K A A T D3MSS8 AG ...... SG G L E V FG TP Y MIALM E CTCKEL A QK.E L ..D. D SQG TV G IS I .STN H K A A T B1C982 K G ...... SG A A E V FS TP D MLLLM E GTCFKL A EE.Y L ..D. EG ES TV G TA A .NFK H L A A T G9YHC1 YA...... SG T A P V Y ATP A LV G LM E HA A VKA I GD.Q L ..P.Q G YS TV G IS M .N I KH TSA T E2ZCX0 YA...... SG T A P V Y ATP A LV G LM E HA A VKA V DS.Q L ..P. EG FA TV G IS M .N I KH TSA T U7UR89 FA...... SG T A P V Y ATP A LV G LM E NA A VHA V GS.Q L ..P. EG YG TV G IS M .N V KH LSA T R7MZQ7 LG ...... SG KSP V Y ATP A LVALM E NA A INACDP.Q L ..P. EG YN TV G IA M .N V KH V A A T S7HMM5 LG ...... SG KSP V Y ATP A LVALM E NA A INACDP.Q L ..P. EG YN TV G IA M .N V KH V A A T F9MNK7 MG ...... SG KSP V Y ATP A MV S LM E TA A IHA I DP.L L ..P. EG YN TV G IG I .K V KH MSA T F5TG45 MG ...... SG KSP V Y ATP A MV S LM E TA A IKA I DP.Q L ..P.P G YN TV G IG I .K V KH LSA T D3LTB8 MG ...... SG KSP V Y ATP A MV S LM E TA A IKA I DP.Q L ..P.P G YN TV G IG I .K V KH LSA T F5TG44 M S...... C G KSP V Y ATP A MVALM E MA A IHA V DP.Q L ..P. EG TN TV G IA I .D V KH LSA S D3LTB7 M S...... C G KSP V Y ATP A MVALM E MA A IHA V DP.Q L ..P. EG TN TV G IA I .D V KH LSA S F9MNK8 M S...... C G KSP V Y ATP A MVALM E MA A IHA V DP.Q L ..P. EG TN TV G IA I .D V KH ISA S H1D2D9 V R...... SG S L P V L ATP V L S AAM E EA A CKA L AP.H L ..T. EG ET TV G GS I .S L IH K A P T R5SND6 V K...... SG S L Q V L ATP V L S ALM E EA A VAA L AP.C L ..L.PKQT TV G GF I .A V RH K A P T R7CTJ9 M K...... SG S L D V L ATP I L S ALM E EA A CAA I AE.A L ..D. EG MT TV G GS I .S L VH K A P T R6A5L1 V K...... SG S L P V L ATP V L S ALM E EA A CDA V KE.G L ..A. EG ET TV G GF I .G L AH KQP T R9MCN0 VG ...... SG S L E V F ATP M MIAAM E QA A CTL L QE.F L ..E. EG QT SV G TM M .H V AH S A A T B0PBF1 VG ...... SG D L D V L ATP M MIAAM E QA A CGL L AR.F L ..E.P G QT SV G TA I .D I KH T A A T K9CK05 VG ...... SG S L D V Y ATP C MAALM E RA A AELCQC.EC..P.A G WT TV G TA L .S I SH R A A T J4V0H9 VG ...... SG S L D V Y ATP C MAALM E RA A AELCQR.EC..P.V G WT TV G TA L .S I SH R A A T E7N2I9 VG ...... SG S L N V Y ATP C MAALM E RA A AELCQR.EC..P.A G WT TV G TA L .S I SH R A A T E4LMB1 VG ...... SG S L N V Y ATP C MAALM E RA A AELCQR.EC..P.V G WT TV G TA L .S I SH R A A T E0NZB5 VG ...... SG S L A V Y ATP A MAALM E RA A AELCAA.GC..P. EG WT TV G TE I .A I RH K A A T D4S5R8 VG ...... SG S L A V Y ATP A MAALM E RA A AELCQR.EC..P. DG WT TV G TE L .H I KH R A A T F5RQF3 VG ...... SG S L A V Y ATP A MAALM E RA A AELCQM.NC..P. EG WT TV G TE L .H I KH R A A T U2JVQ3 VG ...... SG S L A V Y ATP A MAALM E RA A AELCQQ.EC..P. EG WT TV G TE L .H I KH R A A T J7SJ73 VG ...... SG S L A V Y ATP A MAALM E RA A AELCQE.EC..P. EG WT TV G TE L .H I KH R A A T C4V4L2 VG ...... SG S L A V Y ATP A MAALM E RA A AELCQE.EC..P. EG WT TV G TS L .N I AH R A A T L1MYN3 VG ...... SG S L A V Y ATP A MAALM E RA A AELCQS.EC..P. EG WT TV G TS L .N I TH R A A T G5GQK6 VG ...... SG S L A V Y ATP A MAALM E RA A AELCQQ.EC..S. EG WT TV G TS L .N I AH R A A T C9LW87 M K...... SG S L P V F ATP A M T ALM E QA A AEL V DE.L V ..P.SDWT SV G IS L .N I RH K A A T J6I3C7 M K...... SG S L P V F ATP A M T ALM E QA A AEL V DE.L V ..P.SDWT SV G IS L .N I RH K A A T I0GTL3 MG ...... SG S L P V Y ATP A M TC LM E KA A TET L ES.L V ..P. EG WT TV G IS L .H V AH K A A T R5EG72 VG ...... SG D L L V Y ATP C MVALM E GA A CEA V AE.G L ..E. EG QT TV G TA L .N I EH ISA T R5VLB0 VG ...... SG S L R V F ATP M LAALM E KT A CAA V AE.F L ..E. DG ET TV G TA L .D L AH T A A T R7H2Y6 VG ...... SG S L P V F ATP M M T ALM E AA A CDA V AA.Y L ..E.PTET TV G TA L .H I TH D A A T U2M1B8 VG ...... SG S L P V F ATP M MAALM E EA A CNA V AP.F L ..S. EG ET TV G TK L .D I SH D A A T W7UZ64 VG ...... SG S L E V F ATP V MIMLM E KA A CSC I TE.Y L ..E.GDET TV G TE M .N V KH TSA T R7MJM6 VG ...... SG S L E V F ATP M MAMLM E KS A CNA L AD.F M ..E.NDET SV G TE L .N I KH ISA T D4LA09 VG ...... SG S L P V Y ATP M MVALM E QA A CVA V RD.A L ..E. EG ET SV G TA L .N I LH T A A T R7AJW2 VG ...... SG S L P V Y ATP M MVALM E QA A CAA V RD.A L ..E. EG ET SV G TA L .N I LH T A A T U2F0D7 VG ...... SG E L E V Y ATP A MLALM E QA A AAC L AP.L L ..K. EG ET SV G AQ I .GST H LSP S G4KR18 VG ...... SG A L A V FG TP F MIAMM E NA A LTC L QP.G L ..E. EG KG SV G VH M .D V SH D A P T U2RDD7 VG ...... SG A L P V FG TP Y M S ALM E NA A MTC L QS.F L ..E. EG QG SV G TH L .D I SH D A P T U2QNH9 VG ...... SG A L P V FG TP Y M S ALM E NA A MTC L QS.F L ..E. EG QG SV G TH L .D I SH D A P T

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! 188! R6TMK5 VG ...... SG A L P V FG TP Y M S ALM E NA A QTC L QS.F L ..E. EG QG SV G TH I .A I SH D A P T R9LX73 VG ...... SG A L A V FG TP F MAAMM E NA A LTA L QN.F L ..E. EG QG SV G TR L .D I HH D A P T R6H0F9 IG ...... SG G L E V FG TP F MMAMM E HA A MDC V QP.D L ..P. EG KG TV G VD I .QSS H L A P T R6D0Y4 IG ...... SG G L E V FG TP F MM G LM E YA A MQC V QP.E L ..P. EG KG TV G VE I .SSS H L A P T R6UAG6 VG ...... SG S V Q V Y ATP M VVALM E KA A SAL A QQ.Y V ..P. EG CT TV G TN I .S I DH L A A T B2V410 VG ...... SG S V E V F ATP M VAAIM E GA A ADL A QT.F L ..E. D IYT TV G TK I .T V NH L A A T B1QTQ7 VG ...... SG S V D V L ATP I VAAIM E GA A SDL A QT.F L ..D. D DYT TV G TK I .I V EH LNA T R5KLD7 AG ...... SG S L P V LG TP Y MIALM E NATCAA V EP.Y L ..E. EG ET TV G TR I .D V TH DRA S R7BG85 VG ...... SG S L P V Y ATP M ML S LM E EATCQA V SP.L L ..D. DG ET TV G TK I .D I TH DKA S R6M9E2 M K...... SG T L S V F ATP A MVALM E EASCMA L KD.Y L ..E. EG EG TV G IK L .D I SH I A P T F9N406 MG ...... SG T L P V F ATP A MVALM E KA A YTG L QG.D L ..G. DG EG SV G IR I .ETS H E A A S K9D8B9 M K...... SG A L E V F ATP A M C ALM E EA A VAA L TG.K L ..P.T G DG TV G IS L .S I SH DKA S I4D8E6 MG ...... SG N L E V F ATP A MVALM E QA A VNA L E... L ..P. EG QS SV G TS L .T I NH I A A T G2G1T3 MG ...... SG H L E V F ATP A MVALM E QA A VNA L N... L ..P.A G QS SV G TA L .N I SH T A A T J7IWS0 MG ...... SG Q L E V F ATP A MIALM E QA A VNA L E... L ..P. EG QS SV G TS L .S I KH N A A T G7WFQ0 MG ...... SG Q L E V F ATP A MVALM E EA A VNS L Q... L ..P. EG QS SV G TS L .S I KH N A A T H5XVU6 MG ...... SG Q L E V F ATP A MVALM E QA A VNA L E... L ..P.Q G QS SV G TS I .T I KH T A A T W0EBY5 M R...... SG S L D V F ATP A LVALM E AA A VNA L Q...F..E.A G ES SV G TA L .E I IH S A A T L0FAB3 MG ...... SG S L D V F ATP S LVAMM E EA A VGA L A... L ..E. DG QS SV G VS I .E I KH T A A T B8G028 MG ...... SG S L D V F ATP A LVAMM E EA A VSA L T... L ..S. E EQS SV G VS L .D I KH T A A T I4AA80 MG ...... SG S L D V L ATP A LVAMM E EA A VRA L N... L ..A. EG QS SV G VS L .N I KH T A A T Q67JY1 VG ...... SG A V D V F ATP M MIALM E EA A ARA V EP.F L ..G. EG ET TV G TR V .D V EH L A A T Q3A9K1 VG ...... SG S L E V F ATP M MVALM E KA A VNA V AV.A L ..S.P G ET TV G TK I .E V SH IKA S R6J6I5 MG ...... SG D L D V F ATP A MIALM E QA A CNA I LP.C L ..D.SSTS SV G IK M .DTT H E A A T R6I9I9 MG ...... SG S L H V F ATP S MIALM E QA A CNA V AA.C L ..D. E EST SV G TF V .N I TH D A A T U2U2T3 M K...... SG S L D V F ATP A LVALM E QA A CNL V EP.L L ..D. E DTT SV G IS L .N I SH D A A T G4Q7C4 M K...... SG S L D V F ATP A LVALM E QA A CNL V EP.L L ..D. E DTT SV G IS L .N I SH D A A T C0WAV5 M K...... SG S L D V F ATP A LVALM E QA A CNL V EP.L L ..D. E DTT SV G IS L .N I SH D A A T S2Z443 M K...... SG S L D V F ATP A LVALM E QA A CNL V EP.L L ..D. E DTT SV G IS L .N I SH D A A T R7M286 MG ...... SG S L D V Y ATP A MLALM E KA A CNI V NP.C L ..D. E DTT SV G IG V .N L SH D A A T D2RNA4 MG ...... SG S L D V Y ATP A MLALM E KA A CSI V NP.C L ..D. E ETT SV G IG V .N L SH D A A T C0GGI3 VG ...... SG S V P V F ATP M LVALM E NA A INA L KG.Q L ..E. DG QT TV G MK V .D V AH T A A T C9R9F2 Q G ...... SG T V P V L ATP R LLALM E AA A VAA V AG.A L ..P.PEAT TI G VK A .E I EH L A P T 1

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! 189! R6TMK5 VG ...... SG A L P V FG TP Y M S ALM E NA A QTC L QS.F L ..E. EG QG SV G TH I .A I SH D A P T R9LX73 VG ...... SG A L A V FG TP F MAAMM E NA A LTA L QN.F L ..E. EG QG SV G TR L .D I HH D A P T R6H0F9 IG ...... SG G L E V FG TP F MMAMM E HA A MDC V QP.D L ..P. EG KG TV G VD I .QSS H L A P T R6D0Y4 IG ...... SG G L E V FG TP F MM G LM E YA A MQC V QP.E L ..P. EG KG TV G VE I .SSS H L A P T R6UAG6 VG ...... SG S V Q V Y ATP M VVALM E KA A SAL A QQ.Y V ..P. EG CT TV G TN I .S I DH L A A T B2V410 VG ...... SG S V E V F ATP M VAAIM E GA A ADL A QT.F L ..E. D IYT TV G TK I .T V NH L A A T B1QTQ7 VG ...... SG S V D V L ATP I VAAIM E GA A SDL A QT.F L ..D. D DYT TV G TK I .I V EH LNA T R5KLD7 AG ...... SG S L P V LG TP Y MIALM E NATCAA V EP.Y L ..E. EG ET TV G TR I .D V TH DRA S R7BG85 VG ...... SG S L P V Y ATP M ML S LM E EATCQA V SP.L L ..D. DG ET TV G TK I .D I TH DKA S R6M9E2 M K...... SG T L S V F ATP A MVALM E EASCMA L KD.Y L ..E. EG EG TV G IK L .D I SH I A P T F9N406 MG ...... SG T L P V F ATP A MVALM E KA A YTG L QG.D L ..G. DG EG SV G IR I .ETS H E A A S K9D8B9 M K...... SG A L E V F ATP A M C ALM E EA A VAA L TG.K L ..P.T G DG TV G IS L .S I SH DKA S I4D8E6 MG ...... SG N L E V F ATP A MVALM E QA A VNA L E... L ..P. EG QS SV G TS L .T I NH I A A T G2G1T3 MG ...... SG H L E V F ATP A MVALM E QA A VNA L N... L ..P.A G QS SV G TA L .N I SH T A A T J7IWS0 MG ...... SG Q L E V F ATP A MIALM E QA A VNA L E... L ..P. EG QS SV G TS L .S I KH N A A T G7WFQ0 MG ...... SG Q L E V F ATP A MVALM E EA A VNS L Q... L ..P. EG QS SV G TS L .S I KH N A A T H5XVU6 MG ...... SG Q L E V F ATP A MVALM E QA A VNA L E... L ..P.Q G QS SV G TS I .T I KH T A A T W0EBY5 M R...... SG S L D V F ATP A LVALM E AA A VNA L Q...F..E.A G ES SV G TA L .E I IH S A A T L0FAB3 MG ...... SG S L D V F ATP S LVAMM E EA A VGA L A... L ..E. DG QS SV G VS I .E I KH T A A T B8G028 MG ...... SG S L D V F ATP A LVAMM E EA A VSA L T... L ..S. E EQS SV G VS L .D I KH T A A T I4AA80 MG ...... SG S L D V L ATP A LVAMM E EA A VRA L N... L ..A. EG QS SV G VS L .N I KH T A A T Q67JY1 VG ...... SG A V D V F ATP M MIALM E EA A ARA V EP.F L ..G. EG ET TV G TR V .D V EH L A A T Q3A9K1 VG ...... SG S L E V F ATP M MVALM E KA A VNA V AV.A L ..S.P G ET TV G TK I .E V SH IKA S R6J6I5 MG ...... SG D L D V F ATP A MIALM E QA A CNA I LP.C L ..D.SSTS SV G IK M .DTT H E A A T R6I9I9 MG ...... SG S L H V F ATP S MIALM E QA A CNA V AA.C L ..D. E EST SV G TF V .N I TH D A A T U2U2T3 M K...... SG S L D V F ATP A LVALM E QA A CNL V EP.L L ..D. E DTT SV G IS L .N I SH D A A T G4Q7C4 M K...... SG S L D V F ATP A LVALM E QA A CNL V EP.L L ..D. E DTT SV G IS L .N I SH D A A T C0WAV5 M K...... SG S L D V F ATP A LVALM E QA A CNL V EP.L L ..D. E DTT SV G IS L .N I SH D A A T S2Z443 M K...... SG S L D V F ATP A LVALM E QA A CNL V EP.L L ..D. E DTT SV G IS L .N I SH D A A T R7M286 MG ...... SG S L D V Y ATP A MLALM E KA A CNI V NP.C L ..D. E DTT SV G IG V .N L SH D A A T D2RNA4 MG ...... SG S L D V Y ATP A MLALM E KA A CSI V NP.C L ..D. E ETT SV G IG V .N L SH D A A T C0GGI3 VG ...... SG S V P V F ATP M LVALM E NA A INA L KG.Q L ..E. DG QT TV G MK V .D V AH T A A T C9R9F2 Q G ...... SG T V P V L ATP R LLALM E AA A VAA V AG.A L ..P.PEAT TI G VK A .E I EH L A P T 1

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HKI RY EFV A YE .GER.Q I AK G SH QR AVV EIDS F LK RI T R3WMJ9 KVG QE I TV ETE I ..SKK..TG. K LI DF SFQ V FM.KDV.L V SE G TH QR AVV QIEQ F LK RI . S0KCS8 KMGA VI TV EVS L ..LKQ.. D F. KKA TF AFSCR D .NEN.L I AT G TH TR VQ V NCEK F LSQ L D R2TEP8 KVGA NI LV KVK V ..DSQ.. E T.S KI NF SFD V FD .NEE.L V AT G IH QR AVI LTDV F LD KL A R2TK84 KVGA KI IV KVK V ..DSQ.. E K.T KI NF SFE A FD .DEQ.L V AT G TH QR AVI LTDV F LD KL A R3WR68 KVGA QI IV KVK V ..DSQ.. E K.T KI NF SFE A FD .NGQ.T I AT G TH QR AVI LTDV F LD KL A W8U886 PAGM SV VA NSE L ..VE A ..KG.I K FK F NV EV LD .DLE.K IG EG TISMA VI KMDR F LEG A I G8TZK5 PLG SR V HA TSR V ..VQ V .. D G.Q KV TF QV AV FD .EWE.K V AE G TH TR YC L PRLE F DR RV A F8I584 PLG SR V HA TSR V ..VQ V .. D G.Q KV TF QV AV FD .EWE.K V AE G TH TR YC L PRLE F DR RV A F4X9G0 PVGM KV SA TAR L ..RS I .. E R. RKM WF DL EV FD .EKG.KC G EG SH LR IMV TPKGIQD KA G V2XUL5 PVGM QV TA TAT L ..RE V .. E R. RKM WF DI EV HD .EKG.KC G EG SH LR IMV PNKDRSQG.. R6NZJ4 PLGA EI SA TAV L ..KEN.. D G. R FFN F EV SA SD .AKG.I I AK G TH TR VS V KAEK F QK KA D R5DLE4 PLGA EV TA TAV L ..KS V .. D G. R MFN F EV FA ED .KKG.E I AR G TH TR VS V KSEK F QM KA D D4L561 PLGA EV TA TAV L ..KS V .. D G. R MFN F EV FA ED .KKG.E I AR G TH TR VS V KSEK F QM KA D R9LQS4 PLGA VI RI VAT I ..TN V V.ND. R TI EF WV QA FQ.GDD.L IG EG SH ER VIV NSEK F VN K TY R5ZKL6 ALGA TI SI TAK M ..TE V ..QG. RKV LY DV EA YD .GKT.C IG KG KH AR FIV DAKK F LS KV Y U2R6S0 PLGA TI EI HAT L ..LKQ.. E N. RKL VF EV QA FQ.NGK.S IG KG QH ER FIV DANR F LE K TY B7CCX0 PLG QT I TI EAK M ..VH V .. E G. RKI DY EV QA FQ.DGK.S IG KG VH SR FVV DAQK F MD K TY J8HC92 PMG HT I NI ESE L ..VE V ..SG. KKL IF KV VGY D .FHD.K V VE G YH TR FII DKEK F LKN L A J8I2Q3 PMG HT I NI ESE L ..VE V ..SG. KKL IF KV VGY D .FHD.K V VE G YH TR FII DKEK F LKN L A J8HQS7 PIG HT I NI ESE L ..VE V ..SG. KKL IF KV VGY D .FND.K V VE G YH TR FII DKEK F LKN L A J8HXW9 LHNEK V TI NSK L ..IK A .. D K.N KL LF EV EA FD .ESN.K I LS G FH ER AVV KKEK F SK KL D C3GXB1 LYNEK V TI HSK L ..IK V .. D K.N KL LF EV EA FD .ESN.K I LS G FH ER AVV KKEK F SK KL E J8GWT9 LYNEK V TI HSK L ..IK V .. D K.N KL LF EV EA FD .ESN.K I LS G FH ER AVV KKEK F SK KL E J8LFQ4 LYNEK V TI HSK L ..IK V .. D K.N KL LF EV EA FD .ESN.K I LS G FH ER AVV KKEK F SK KL E J8AI75 LL NEK I TI HSK L ..IK M .. D K.N KL LF EV EA FD .ESS.Q I LR G FH ER AVV KKEK F LRN L E H5XWA2 P PGL TV TV KLR L ..EK I .. E G. KKL FF HV EA HD .GID.L I SE G TH ER FVI DAAK F NE KV N G7W6G7 P PG FT V TV KLR L ..EK I .. D G. KRL LF HV EA HD .GVD.L I SE G TH ER FII DAAK F NE KV T J7IYH8 P PG FT V TV KLR L ..EK I .. E G. KKL LF HV EA HD .GVD.Q I SE G TH ER FII NAAK F NE KV T D4J4P3 P CG KK V RCEVE I ..TE V V.NG. KKV TF AV NA YD .ENG.L IG TG TH KR AVI NKAA F NM... U2L2J7 PI RM QV RCECE L ..TE I H.N.. KRL VF QV KL FD .EKE.L VG KAK H ER YIV SPDALKA KI A Q8EMG8 ALG TK V KL EAV V ..TS I ..TE. K QV VTK V IA RN.ESV.I IG EG EVKQS IL PKNTMKS KL D T0JGA7 ALG TN V KL TAA V ..SE I ..TK. R MV TTE L IV EN.DNG.R IG EG EVRQA IL PKHIIQE KV R Q2B862 AE G SL L KV TAE V ..VS L .. E R.NL I FTS V KA FH.GDT.V IG KG QVTQA IL PRSKIKRFTE U5L8R5 AE G SL L KV TAE V ..VS L .. E R.NL I FTS V KA FH.GDT.V IG KG QVTQA IL PRSKIKRFTE U6BAI1 AE G TR L IV TAT V ..TA M ..TS. RRV DAS I EV RD .GQT.I IG TG EVTQF IL EKSRIQE KL Q B1YII8 TLG SN I RI EAV V ..SE L ..TP.G RV LAK V EV RH.GNR.V IG LG EVKQI IL NKQLIQE KL T K0A8N8 TLG SN I RI EAT V ..SE L ..TP.D RV VAK V EV RH.GDR.L IG LG EVKQI IL KKQLIQE KL T W7LB64 P EG SS I LI TAT V ..SK L .. D G.NS V YST V TV HN.GEM.L VG EG EVKQV IL KKSRIAKL L K E5WMY4 P EG SS I II TAT V ..SK L .. D G.NS V YST V TV HN.GKI.L AG EG EVKQV IL KKSRIAEM L K S2XPF3 PLG SV V SL TAN V ..IE L ..RD.NF V KTK V TA RN.HLG.V IG KG EVMQV IL SKETIAN KL K W4RK43 TE GA EV TV TAT V ..TG L ..RA.NT I LTN V RA ES.NGR.L IG VG EVKQV VL PKEKITEL L T V6T386 TE GA EV TV TAT V ..TG L ..RA.NT I LTN V RA ES.NGR.L IG VG EVKQV VL PKEKITEL L T I8UG48 P EGM NL TI TAT V ..TE L ..TG. K TV VCK V KV AN.EKM.V VG EG DVTQY IL SKEKITQ KL Q A6CP21 SE G TT V TI TAT L ..TE L ..QS.NI V ITK A EA RN.ENG.L IG IG EVKQV IL PKERIRMMGK F8CXF7 AE G ST V TV TAT V ..TE L ..RD.NV V VTK V EA KN.EVQ.V IG IG EVKQV IL PKQKIDQM L Q E3IAI3 AE G ST V TV TAT V ..TE L ..RD.NV V VTK V EA KN.EVQ.V IG IG EVKQV IL PKQKIDQM L Q K6PLM5 PVGA RV RA VAV L ..EA V .. E G.N RV ICR V EA YN.DLE.K I AE G RQVQV VL ERDA F RR RI E E6SLA6 PVGA RV RA VAV L ..EA V .. E G.N RV VCR V EA YN.DLE.K I AE G RQVQV VL DRAA F RR RI A G8TSI3 LVG ETFS A TAE L ..LA I .. D K.N RI IAR V TA EN.GRG.L IG RG QFTQV LL PKKALED KI R F8I237 LVG ETFS A TAE L ..LA I .. D K.N RI IAR V TA EN.GRG.L IG RG QFTQV LL PKKALED KI R V9W1G5 VVG QT V TFRAVC..SE V ..SP. KRV ICH V TA ET.EYN.P VG TG SFTQA I FDKKAVTQ R FM L5MUG4 VVG QT V TFKAVC..QE V ..TE. KRV VCE V TA DT.ARN.R VG VG TFTQA I FNKHEIRQ R FE J2HJM9 VVG QT V TFKAVC..QE V ..TE. KRV VCE V TA DT.ARN.R VG VG TFTQA I FNKHEIRQ R FE C0Z567 VIG QE V TFRASC..VQ V ..TE. KRV VCE V TA DT.TRN.R VG LG TFVQA I FKKDEIKQ R FE J2G9M0 VI DQE V TFRATC..VQ V ..TE. KRV VCE V TA DT.TRN.R VG LG TFVQA I FKKDEIKR R FE C8WU92 PVG KR V SFYAE V ..TR V .. E G.P RV DCR V WA EH.DKA.R IG EG EVVQY IL PKAKIEA RI R F5SL03 PVG KE V TFTAE V ..IE V ..NP.P KV VCR V RA EH.DKS.L VG AG TFVQA IL PKQGILE RI R D5WXP8 PVG KE V TFTAE A ..VE V ..TP. RRV VCR V WA EH.DKG.I VG EG MFHQA IL PRQKILD RI A R7G5H6 PVGM KV TCTAT I ..TA V .. E R. KKI SF SI VA KD .EKD.V IG KAT H DR FLV IKEK F EA KA K A8RFU2 PVGM KV TCTAT I ..TA V .. E R. KKI SF SI VA KD .EKD.V IG KAT H DR FLV IKEK F EA KA K H1BNL1 PAGM KV RI SAE I ..IA V .. D R. KKV TF SI IA QD .EKD.I IG KAS H ER FVV MKEK F EA KA Q

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U5F4M4 PAGM KV RI SAE I ..IA V .. D R. KKV TF SI IA QD .EKD.I IG KAS H ER FVV MKEK F EA KA Q E2SKL5 PAGM EV EA EVE I ..TA V .. D R. KKV SF HI IA RD .AKD.T IG TAQ H DR FIV KKES F EA RA L H1B0W4 PAGM TV TA EVE I ..TA V .. D R. KKV SF HI IA RD .ERD.L IG TAD H DR FIV KKEA F EA KA L E4LXV6 PAGM TV TA EVE I ..TA V .. D R. KKV SF HI IA RD .ERD.L IG TAD H DR FIV KKEA F EA KA L T4NF97 PAGM TV TA EVE I ..TA V .. D R. KKV SF HI IA RD .ERD.L IG TAD H DR FIV KKEA F EA KA L N9WK99 PAGM TV TA EVE I ..TA V .. D R. KKV SF HI IA RD .ERD.L IG TAD H DR FIV KKEA F EA KA L R6UIH5 PAGM TV TA EVE I ..TA V .. D R. KKV SF HI IA RD .ERD.L IG TAD H DR FIV KKEA F EA KA L G1VX76 PAGM TV TA EVE I ..TA V .. D R. KKV SF HI IA RD .ERD.L IG TAD H DR FIV KKEA F EA KA L H1BDX8 PAGM TV TA EVE I ..TA V .. D R. KKV SF HI IA RD .ERD.L IG TAD H DR FIV KKEA F EA KA L E1L909 PMGA TI TA TAT V ..TA V .. E G. RKI SF TI EA SD .GVG.I IG KG SH ER FVI NNEK F MT KV G S3A0K0 PMGA TI TA TAT V ..TA V .. E G. RKI SF VI EA SD .GVG.I IG KG SH ER FVI NNEK F MT KV G J5AQJ6 PMGA TI TA TAT V ..TA V .. E G. RKI SF AI EA SD .GVG.V IG KG SH ER FVI NNEK F MT KV G X8HFB3 PMGA TI TA TAT V ..TA V .. E G. RKI SF AI EA SD .GVG.I IG KG TH ER FVI NNEK F MT KV G W3Y8I0 PLGA TI TA KAT V ..SA V .. E G. RKI TF DI EA SD .GIG.I IG RG TH ER FII NNDK F TK KV V D1BM20 PLGA TV TA KAI V ..SA V .. E G. RKI TF DI EA SD .GIG.I IG RG TH ER FVI NNEK F IA KV H D6KMG8 PLGA TV TA KAI V ..SA V .. E G. RKI TF DI EA SD .GIG.I IG RG TH ER FVI NNEK F MA KV H D6KHB2 PLGA TV TA KAT V ..SA V .. E G. RKI TF DI EA SD .GVG.I IG RG TH ER FVI NNEK F MA KV H E4LB70 PLGA IV TA KAT V ..SA V .. E G. RKI TF DI EA SD .GVG.I IG HG TH ER FVI NNEK F MA KV A W1UP58 PL SA TV TA KAT V ..SA V .. E G. RKI TF DI EA SD .GVG.I IG RG TH ER FVI NNEK F MA KV H C4FPZ7 PLGA TV TA KAT V ..SA V .. E G. RKI TF DI EA SD .GVG.I IG RG TH ER FVI NNEK F MA KV T W1W8P8 PLGA TV TA KAT V ..SA V .. E G. RKI TF DI EA SD .GVG.I IG RG TH ER FVI NNEK F MA KV T W1X196 PLGA TV TA KAT V ..SA V .. E G. RKI TF DI EA SD .GIG.I IG RG TH ER FVI NNEK F MA KV V U2BAI1 PVGM KV WCKSC L ..TQ V .. E G. RKL TF HV EA YD .ETG.L IG TG EH ER VLI QAQR F QE KA D U2SW53 P CGM KV RCETE L ..VE V .. D G. RRL VF SA TV FD .EMG.K IG EG RH ER FIV DNAK F QA KA D R9IHN6 PVGM KI WCETV L ..KE V .. D G. RKL TF DL TV YD .ETG.K IG AG VH ER FII EEEK F QS KA D R9KEX9 PVGM KV WCESL L ..TG V .. D G. RKL TF SI TV FD .GAG.K IG EG LH ER FII EETK F QA KA D S0J977 PVGM KV WCDSV L ..TE V .. E G. RKL TF SI TA FD .ESG.K IG EG FH QR FII EETK F QS KA D R5HR96 PIGV EV TCRAE L ..TE V .. D G. RRL VF RV TA QD .SQG.L VG EG IH ER VIV QNDR F LT KA Q R6WU27 AMG DS V TA TAK L ..TA V .. D G. RKL TF EV SA AD .SKG.V IG KG TH ER FII DNEK F MA KV N F0T2R2 PVGM KV WA VAE I ..TG I .. E G. RKL SF KI EA YD .EKE.K IG EG EH ER FII NAEK F LE KA Y D4J9J7 PIGM KV RCESL L ..KE V ..NG. KKL VF EV NV YD .EKG.L IG TG TH KR AII NNEA F MA RL A D4J2H1 AIG KT V MCEAT L ..IE M .. D G. RRL TF EV VCS D .ENG.V VG MG TH ER FII NNEK F MA KA N R6GT38 FVG SK V TCETE L ..IE V .. D R. RKL VF SV KV YD .ETE.L VG EG IH ER FII DNRK F ID KL E V2YEB4 PVGM KV WA ESE V ..TQ V .. D G. KRL VLK V AA FD .ERG.P IG QG TH ER FIV TDER F LS K TA F4X8V5 PIGI KV WA ESE V ..TE V .. D G. KRI ELK V AA YD .EKG.M IG HG TH QR FIV TDER F LA K TA A6NV90 PVGM KV WA EAT V ..TA V .. D G. RRI SF TV KA QD .ACG.P IG SG TH ER FVI SNDR F LA R TN R5B634 PVGM HV TA KAE L ..VE V .. D R. RRL VF RV TA ED .DAG.P IG QG TH ER FLI MADK F LA KA E U2BG97 PVGM QV WA EAE V ..TA V .. D G. RKL TF AV TA YD .ETG.P IG KG TH ER FII QNDK F LA KA Q G9YX66 PVGM QV WA EAE V ..TA V .. D G. RKL TF AV TA YD .EAG.P IG KG TH ER FII QNDK F LA KA Q H1C7K7 PVGM QV WA EAE V ..TA V .. D G. RKL TF AV TA YD .EAG.P IG KG TH ER FII QNDK F LA KA Q D4JRV3 PVGM NV TA EAV L ..TE I .. D N. RRL VF EV VA RD .EKD.I IG KG IH ER FIV NAEK F TA K TY R6RPT5 PVGM NV TA EAV L ..TE I .. D N. RRL VF EV VA RD .EKD.I IG KG SH ER FIV NAEK F TA K TY F7K912 PVGM KV WCESR L ..VQ A .. D G. RRM VF QA EV FD .ESG.K IG EG EH ER FLV RAER F QQ KA D R6QC10 PVGM TV TCESE L ..VA V .. E G. RKL VF KV SL HD .EKG.P VG GG TH ER FVI NNAK F AA KA E E2ZHD1 PVGM TV TCESE L ..VA V .. E G. RKL TF KV AL HD .EKG.P VG GG TH ER FVV NDAK F AA KA E R7C7Z2 PLGM TI TCESE L ..VE I .. D G. RKL VF SV SA KD .EKGTV IG RG TH ER FVI LEEK F QE KA N R6IH13 PLGM KV WA ESE L ..IE I .. D R. RRL VF EV KA FD .ECG.Q IG AG RH ER FII NNEK F IA KA N R5CSL0 PLGM KV WA ETE L ..VE V .. D R. RRL VF DV KA YD .ECG.L IG QG KH ER FII QNDK F LA KV N I5AU99 PMGM KV TCETV L ..KE V .. D R. RRL VF EV KA YD .ETG.I IG EG IH ER FLI DNEK F QA KA E R6BVQ8 PVGM KV TCNTT L ..VE V .. D K. RRL VF SV EV ED .ETG.K VG EG TH ER FIV QAEK F QN KA D G2T231 PVGM EV TCETK L ..VE V .. D R. KRL VF EV KA YD .AAG.V IG EG TH ER FII DNER F LA KA E R5Q2H0 P TGM RV YCEST L ..TE V .. D G. RRL VF SV SA YD .ECG.E IG SG TH ER FII KSEK F MA K TN D7GQ38 PLGM TV WCETE L ..VE V .. D G. RRL VF DV AA YD .AKG.K IG GG RH ER FII QNEK F QA KA N R5FXP5 PLGM TV WCETE L ..VE V .. D G. RRL VF DV AA YD .AKG.K IG GG RH ER FII QNEK F QA KA N R6RV13 PLGM KV WCESE L ..TE I .. D G. RRL VF SV AA FD .ETG.K IG GG SH ER FIV PNEK F QA KA D R6ZN81 PLGM KV WCESE L ..TE V .. D R. RRL VF HV TCY D .EAG.V IG EG TH ER FII GNEK F LA KA E D6DGH0 PLGM KV WCESE L ..TE V .. D R. RRL VF HV TCY D .AAG.V IG EG TH ER FIV GNEK F LA KA Q R5M2P2 PLGM KV WCESE L ..TE V .. D R. RRL VF HV TCY D .AAG.V IG EG TH ER FIV GNEK F LA KA Q D4CH55 PLGM KV WCESE L ..TE V .. D R. RRL VF HV TCY D .AAG.V IG EG TH ER FIV GNEK F LA KA Q D4MPY0 PLGM KV WCESE L ..TE V .. D R. RRL VF HV TCY D .AAG.V IG EG TH ER FIV GNEK F LA KA Q R7B681 PVGM QV WCESE L ..VE V .. D G. RRL VF KV EA FD .AKG.S IG GG TH ER FII KNEK F LA KA Q G5FFC4 PAGM IV WCESE L ..KE V .. D G. RRL VF HV EA FD .ESG.K IG EG EH ER FIV KNEK F MD KA L U2D4S0 PAGM IV WCESE L ..KE V .. D G. RRL VF HV EA FD .ESG.K IG EG EH ER FIV KNEK F MD KA L R8VSU0 PAGM KV HFETE L ..VE I APNG. K IL TF KV AA YD .EAG.L IG EG TH QR AIV AKER F EA KA Q B8I4C0 PIGM NV AA RAE L ..IE I .. D G. KKL IF SV EA FD .GKD.K IG EG RH ER FII NTQK F IT KV D U4R6H3 PVGM NV TA RAE L ..AE I .. D G. KRL VF TV EA FD .GKA.K IG EG QH ER FII NAQK F IA RA N H2JF67 PIGM NV TA RAE L ..VE I .. D G. KRL VF TV EA FD .GKD.K IG EG QH ER FII KAAK F IL KA N L1Q3R4 PIGM EV IA KSE L ..IE I .. D R. KRM VF KI EA FD .ERG.K IG EG IH ER FII DNEE F QN K TD R6KH82 PIGM EV IA KTE L ..IE V .. D R. RRL VF KV EA FD .ERG.K IG EG IH ER FII NNED F QN K TN R6FUY0 PIGM EV TA KTE L ..IE V .. D R. RRL VF KV EA FD .ERG.K IG EG IH ER FII NNED F QK K TD N2BI76 PVGM NV RCISK L ..VE V .. D G. RKL VF DV QA FD .DAG.L IG QG TH ER FII ENDK F FK KA E E6LQN3 PVGM KV VA KSE L ..LK I .. D G. R EL TF KV EA YD .EKG.L IG EG IH KR FIV NNDK F QA K TD H1LX05 PVGM KV VA KSE L ..LK I .. D G. R EL TF KV EA YD .EKG.L IG EG IH KR FIV NNDK F QA K TD W2VGZ8 PLGM KV VI KSE L ..IN V .. D G. R AL TF KV EA YD .EKG.L IG EG IH ER FIV NNEK F QA K TD K0XJJ2 PLGM KV VI KSE L ..IK V .. D G. R AL TF KV EA YD .EKG.L IG EG IH ER FIV NNEK F QA K TD J4TCP2 PLGM KV EI KSD L ..IK V .. D G. R AL TF KV EA YD .EKG.L IG EG IH ER FIV NNEK F QA KA D F3B6C0 PLGM KV EI KSE L ..IK I .. D G. R AL TF KV EA YD .EKG.L IG EG IH ER FIV NNEK F QA K TD R5RJN6 PVGM KV ECDTE L ..IE V .. D G. RRL VF KV EA SD .EKG.L IG KG TH ER FII ESEK F RE K TY R6LSC1 PVGM KI TCDSE L ..IE I .. E G. RKL VF KV EA YD .EKG.L IG KG TH ER FIV ENKK F QE K TD R6VIH0 PIGM KI TCDSK L ..VA I .. E G. RKL VF SV EA YD .AKG.L IG KG TH ER FII ESKK F QE K TD R7JYS9 PIGM KI TCDSE L ..TA I .. E G. RKL VF KV EA YD .SKG.L IG KG TH ER FII ESEK F QE K TN F7V6W4 PVGM QV TCETE L ..VE V .. D R. RRL VF QV KV TD .EAG.L IG EG TH ER FIV NNEK F QA KA N U2PAA7 PIGM TV RCETE L ..TE V .. D G. RRL VF KV AA YD .EAG.L IG EG TH ER FIV QNEK F QT KA N R5TLI5 PIGM TV RCETE L ..TE V .. D G. RRL VF KV AA YD .EAG.L IG EG TH ER FIV QNEK F QT KA N R6P4I1 PVGM KV TA ESE L ..IA I .. D G. RKL TF KV TA FD .EVG.K IG EG TH ER FII NNEK F QA K TN C0CNB6 PVGM TV TCESE L ..IQ V .. D G. RKL TF RL TA RD .AAG.V VG EG TH ER FIV DNAR F SQ KA E R6DLF5 PIGM KI RCESE L ..VE V .. E G. RKL TF KV EA YD .ETG.K IG EG KH ER FIV ANEK F QS KA D R5SHR8 PVGM EI TCETE L ..TE V .. D G. RRL VF TV KA YD .TKG.L IG EG VH ER FVI VNEK F QA K TD R5ZA51 PVGM KI TCETE L ..VK V .. D G. R AL TF TV KA FD .EAG.L IG EG EH ER FVV FNDK F QA KA D C4Z997 PVGM KV TCESE L ..VK V .. D G. R AL TF SV KA FD .EAG.L IG EG TH ER FIV FEEK F QK KA D E4Q5E0 P KGM KV KV VAE L ..IS I .. E D. RKL TF KV ETY D .SFE.K IG EG IH ER FIV DRER F LN K TY D9TGD2 P KGM KV KA VAQ L ..IS I .. E D. RKL TF KV EA YD .SFE.K IG EG IH ER FIV DRER F LN K TY B9MM60 P KGM KV KA VAQ L ..IA I .. E D. RKL TF KV EA YD .SFE.K IG EG FH ER FIV NRER F LN KA Y E4SE45 P KGM KV KA VAQ L ..IA I .. E D. RKL TF KV EA YD .AFE.K IG EG IH ER FIV NRER F LN K TY G2PV11 P KGM KV KA VAQ L ..IS I .. E D. RKL TF KV EA YD .SFE.K VG EG DH ER FIV NRER F LN KA Y E4S8L3 P KGM KV KA VAQ L ..IA I .. E D. RKL TF KV EA YD .SFE.K IG EG VH ER FIV NRER F LN KA Y E4QCS6 P KGM KV KA VAQ L ..IA I .. E D. RKL TF KV EA YD .SFE.K IG EG VH ER FIV NRER F LN K TY B0TBP0 PVGM TV RCEAE L ..TE V .. D G. KKL VF KV VA YD .EAE.K IG EG VH HR FVI QTER F LA K QA D5X9M4 PVGM EV IA RSE L ..IR I .. E G. KKL VF KV EA YD .EKD.K IG EG IH ER FII DREK F MA K CE F6DMU1 PLGL GV KA KAE L ..ME I .. D G. KRL VF RV EA FD .ESG.P IG AG VH ER FVI VKDK F IQ KA E A4J0S5 PVGM KV VA RAE L ..IQ I .. E G. KKL SF VV EA RD .ETG.P VG AG KH ER FIV NQEK F LQ RA E K8DZX3 PVGM RV VA RAT L ..TE I .. D G. KRL VF RV EA TD .ETG.P VG EG TH ER FII NLEK F LQ KV E F6B4J8 PVGM KV VA RAE L ..IE I .. D G. RRL VF KV DA SD .EAG.P IG SG TH ER FII NPEK F LQ KV E

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! 192! A5D1P6 PVGM EV VA KSR L ..VE V .. D G. KRL VF EV SA WD .EAG.L IG TG IH ER FIV KHES F IK KA E F6CP13 PIGM EV VA RSE L ..VE I .. D G. KRL VF RV EA RD .EQE.L IG RG TH ER FIV NQER F LS R TA R7AYY7 PVGM KV TA TAK L ..RS I .. E R. RKM WF DI EV ND .EKG.KC G EG SH LR IMV NSKAMSD KA A D7GRB2 PVGM KV TA TAK L ..RS I .. E R. RKL WF DI EV ND .EKG.KC G EG SH LR IIV NSKAMSE KA E R5G526 PVGM KV TA TAK L ..RS I .. E R. RKL WF DI EV ND .EKG.KC G EG SH LR VIV NSKAMSE KA E R7NT78 PIGA KI KV ESK L ..ISN.. D G. R SFK F EV TA YD .NAG.M I AN G TH NR VS V KSEK F QK KA D R7AWH4 PLGA TI EI HAT L ..LKQ.. E N. RKL VF EV QA FQ.NGK.S IG KG QH ER FIV DANR F LE K TY R5RY61 PIGA EI TA KAE L ..IA I SENG. K MYT F KV EA YD .NKG.L IG EG TH ER AII DCER F MK K CL A1HRR7 PVGM TV RA TAE L ..VE V ..AG. KRL VF AV SA YD .DRE.K VG EG WH ER YII KTAS F LE KV A F7NH57 PVGM SV RA VAT L ..TE I ..TG. KKL VF AV EA YD .DLE.K VG QG QH ER YII QTEP F FN KI A H1HSX5 P KGM KV TFDAE L ..LE I SANG. K IL TF HV EA HD .ECG.L IG EG TH QR AVV DRVR F DE K TQ I7K9K8 PIGM KV KA IAE L ..VE V .. D G. KRL IF KV EA YD .EME.K I ME G TH ER FII DVER F MQ RV N R7RUE8 PIGM KV RA EAE I ..VA V .. E G. KKV VF KV EA FD .EVE.K IG EG IH ER YIV DINK F IS RV N N1ZJT1 PVGM KV RV VSE L ..IE I .. D G. K AL TF KV KA YD .EID.K IG SG IH KR AIV ELDR F IK R TK R7B8B0 PVGM KV RV VTE L ..TE V ..NG. K LL TF AV KA YD .EVD.K I CE G TH GR AII DLKK F LS RV N R6PY32 PVGM KV RV VTE L ..TE V ..NG. K LL TF AV KA YD .EVD.K I CE G TH GR AII DLKK F LS RV N R5AF40 PVGM KV RV VTE V ..TE V ..KG. K ML TY SV KA YD .EVD.K I CE G TH GR AII ELDK F LS RV N M1E923 PVGM KV WA SAK L ..IR V .. D G. RRL IF EV LA RD .EVE.Q V AR G TH ER FIV NFDR F IK KA E R4KGW9 PVG GV V RA QSR L ..LK I .. D G. RRL VF EV QA SD .DTR.V VG RG TH QR FIV KVDE F LQ RV G A6TLR1 PVGM KA YA EAK L ..IK V .. E G. KKL TF EI TA RD .EEE.K VG EG TH ER YII PLEK F IA RA K F4A004 PVGM KV RA DAE L ..IE I ..NG. R AL TF KV RA FD .ENE.M IG QG VH QR YIV NIER F IN KA N G8M073 PIGM KV KA VAE L ..IS I .. E G. RKL IF KV EA FD .AKE.K IG EG QH ER YIV ESAR F RD KV Y A3DHM5 PVGM KV KA LAE L ..IA V .. E G. RKL TF KV EA FD .GVE.K IG EG YH ER YIV SSQN F RD KV Y W4V2Q9 PVGM KV KA FAE L ..IA V .. E G. RKL TF KV EA FD .SVE.K IG EG YH ER YIV ASQN F KE KV Y H6LHK5 SVGM KI RI KAT L ..VAT.. E T. R VL NF DI DA WD .GLE.K IG EAI H QR FIV EAKR F NE KA A E6MGL5 PVGM KV RV KVH L ..DA I .. E G. RKL VF SI EA YD .TAQ.Q I AG G TH TR FIV NKAR F MQ KV Q E3GGD1 PVGM KV RI KVV L ..SN I .. E S. RRL DF TV EA WD .TVQ.K IG EG TH QR FVV QKMK F MG KV L R7K521 PVGM RV KA ECE L ..VS V .. D G.NMYT F EV RA FD .ETG.L IG EG TH KR CVI NDEK F MK K TL R5AKF1 VVG KT A RV ITT L ..YE V ..AG. RKL RF RL EA YD .AAG.L I AE G EH ER CLV FSEA F MD RA N B2A5Z4 PVG DK V KV NAV L ..KE V .. D G. KRL VF EV TA HD .SNN.K IG EG EH ER FII NLSK F MN KL S T0N2A7 PVG SK V RCEAV V ..DN I .. D G. KKI LF SV QV WD .GKG.L IG EG TH IR YIV NIEK F MS KL . A0PXM9 PIGM KV RCETK L ..IK V .. D R. KKL VF EV EA YD .EDG.K IG EG TH TR YIV NSEE F VKN V Q C6Q0E0 PIGM KI RCEAS L ..VK V .. D K. KKL VF NV EA WD .EKG.K IG EG NH IR YIV NSEE F MK KV Q A5N4K6 SVGA KI RCEAS L ..KE V ..KG. KKL VF YV EA WD .ESG.K IG EG SH TR YIV NSED F MK KI Q U2D856 P SGM KV KA EAY L ..EK V .. D R. KRL FF KV EA YD .ESG.K IG EG TH IR YIV NSEE F MG K KK K6U6J6 RIGA NI ECKAT L ..VK V .. D G.M KL FF EV EA YD .DNG.I IG KG TH TR YIV NIND F ME K TK W6N7S3 PVGA NI ECKAV L ..TK I .. D R. KKL FF EV EA RD .DKG.V IG KG SH IR FIV NSHD F ME K TK G7M699 PVGA NI ECKAI L ..TK V .. E N. KKL FF DV EA SD .DHG.T IG KG FH IR YIV NSKD F MA R TK A6LWA7 PIGA NI KCKST L ..TK V .. D G. KKL FF DV EA SD .DQG.T IG KG SH IR FIV NSED F MR K TK V8G1J7 PIGA NI KCKST L ..TK V .. D G. KKL FF DV EA SD .DQG.T IG KG SH IR FIV NSED F MR K TK U5MUT7 PVGA NI KCKAT L ..TK I .. D R. KKL FF DV EA SD .DKG.T IG KAT H IR YIV NSQD F ME K TK S1MVB3 PVGL QV TCHTN L ..TT I .. D R. KRL VF SFE V YD .EVD.L I AK G TH ER FIV NRMQ F SE KA Q R6BIU7 PVG ST I TCECE L ..AQ I .. D G. RKL IF NV IA HD .DFG.P VG EG VH ER FVI NNDK F MS KV A R5GAX2 PIG CE V SCEVT L ..TE I .. D R. KKL TF AV EV KD .PAG.V IG KG TH ER FIV DAEK F QN KA N R6G283 PIG CE V SCEVT L ..TE I .. D R. KKL VF AV EV KD .PAG.V IG KG TH ER FIV DAEK F QN KA N R6QH26 PVG CQ V TCQCH L ..TE I ..NR. KKL EF QA EI MD .NKG.R IG IG TH ER YIV AADS F LDN A K B0MBZ2 PVG CQ V TCQCH L ..TE I ..NR. KKL EF QA EI MD .NKG.R IG IG TH ER YIV AADS F LDN A K E5VQV8 PVG CQ V TCQCH L ..TE I ..NR. KKL EF QA EI MD .NKG.R IG IG TH ER YIV AADS F LDN A K D4MVS3 PVG GT I KCHCS L ..TE I .. D R. KRL VF HV EV TD .NKG.R VG IG TH ER FIV ASEP F ME KA A B0NZW1 PVG GT I KCHCS L ..TE I .. D R. KRL VF HV EV TD .NKG.R VG IG TH ER FIV ASEP F ME KA A E5VM16 PVG GT I KCHCS L ..TE I .. D R. KRL VF HI EV TD .NKG.R VG IG TH ER FIV ASEP F ME KA A L1PTM0 PVG GT I KCHCS L ..TE I .. D R. KRL VF HI EV TD .NKG.R VG IG TH ER FIV ASEP F ME KA A R5YWL8 PVG GT I KCHCS L ..TE I .. D R. KRL VF HV EV TD .NKG.R VG IG TH ER FIV ASEP F ME KA A C0EF68 P EGM KV TA EVE I ..TE V .. D G. RRI VF SV TA RD .EKD.L I CK G TH ER FVV ARTK F IS KA K D1PP05 PIGM VV RCDCE L ..TA V ..AD. RRL TF TA RV YD .DVS.E V AHAT H ER YIV DNDR F TH KA S U2KTG5 PIGM NV TCECE L ..IE I .. D G. KRL VF NV EL WD .EGE.K VG KAV H ER FIV DMNKLLK KI D E0E4V9 PIGM EV TCECE L ..VE I .. D R.S RL VF KV RCY D .ELE.E IG VCT H ER FII DNAK F LAN V D D3MSS8 PMGM KV TCEAE L ..VE I .. D R.S RL VF NV VCS D .ELD.V IG EG QH QR FII DNEK F LAG V N B1C982 PLGM KV RCECE L ..IE V .. D R. RRL LF KV DV YD .EQE.K VG EG TH ER FII DQAK F EA KA E G9YHC1 PVGM TV KA KAV L ..IDQ.. D R. RRL TF KI EGY D .DAG.S VG EAV H ER FII EAAP F IE KA N E2ZCX0 PVGM DV TA KAV L ..TEQ.. D R. RKL TF KI TA CD .AAG.P VG EG IH ER FII EAAP F TE KA N U7UR89 PIGL TV TA KAT L ..TAQ.. D R. RKL TF KV EA FD .TAG.A VG EAV H ER FII ESAP F LA KA E R7MZQ7 PIGL TV TA KAT L ..VEQ.. D R. KKL TF KI EA HD .EAG.L VG EAT H DR FII ESAP F LA KA E S7HMM5 PIGL TV TA KAT L ..IEQ.. D R. KKL TF KI EA HD .EAG.L VG EAT H DR FII ESAP F LA KA E F9MNK7 PLGM KV TA KAT V ..VAQ.. D R. KH FDVK I EA FD .EAG.L IG TAE H ER FIV ESAP F LA K TE F5TG45 PL DM TV TA KAT V ..IAQ.. D R. KH FDVK I EA FD .EAG.L IG TAE H ER FII ESQP F LE KA K D3LTB8 PL DM TV TA KAT V ..VAQ.. D R. KH FDVK I EA FD .EAG.L IG TAE H ER FII ESQP F LE KA K F5TG44 PVGM TV RA EAT L ..TAQ.. D R. RHL TF TI EA FD .DAG.L IG TAT H ER FII DSAP F LA KA E D3LTB7 PVGM TV RA EAT L ..TAQ.. D R. RHL TF TI EA FD .DAG.L IG TAT H ER FII DSAP F LA KA E F9MNK8 PIGM KI RA KAT L ..VAQ.. D R. RHL DF KI EA FD .EVG.L IG TAT H ER FII DSAP F LA KA E H1D2D9 LP G HV I TV TAT V ..TE V ..KG. KKI SF HI TA AD .ENG.D IG EAD H DR FLV DGVR F ME KA E R5SND6 PVG QT V KA IAT V ..KA V ..SG. KKI SF TI TA SD .ENG.T IG EAE H TR FLV DEEA F MK KL Q R7CTJ9 LP G DT V TA TAV V ..TG V ..KG. KKI TF TI RA ED .SAG.E VG TAD H TR FIV EADP F MDN A G R6A5L1 LVG ST V RA EAT V ..TV V ..KG. KKI TLH L IA YD .EAG.E IG EAE H IR FVV EAKP F ME KA E R9MCN0 PLGM QV TA AAT I ..TAQ.. E G. RKV EF EV SA RD .ACG.E IG RG TH TR FVV DAAK F LS K TN B0PBF1 P CGM AYT A SAR L ..TGR.. E G. R CA SF EV WA ED .EIG.E IG RG SH TR VVV DAKR F QE KA D K9CK05 PVGL SV RA VAE V ..TA V .. D G. RKI GLK V TA YD .ARE.E IG AG TH ER FAV ASEK F MA KA E J4V0H9 PVGL SV RA VAE V ..TA V .. D G. RKI NLK V TA YD .ARE.E IG SG TH ER FAV ASEK F FA KA E E7N2I9 PVGL SV RA VAE V ..TA V .. D R. RKI NLK V TA YD .ARE.E IG SG TH ER FAV ASEK F FA KA E E4LMB1 PVGL SV RA VAE V ..TA V .. D E. RKI NLK V TA YD .ARE.E IG SG TH ER FAV ASEK F FA KA E E0NZB5 PVGL DV RA VAE V ..TA V .. D G. R AV TLK V TA YD .TRE.E IG AG TH TR FAV AVQK F MA KA E D4S5R8 PVGL AV RA VAE V ..TA V .. D G. R AV ALS V TA YD .ERE.E IG SG TH TR FAV TSDT F MA KA E F5RQF3 P TGL TV RA IAE V ..TT V .. D G. R AI SLK V TA YD .ERE.E IG SG TH TR FAV SVEK F MA K TE U2JVQ3 P TGL TV RA IAE V ..TA V .. D G. R AI SLK V TA YD .ERE.E IG TG TH MR FAV AVEK F ME KA E J7SJ73 P TGL TV RA IAE V ..TA V .. D G. R AI SLK V TA YD .ERE.E IG TG TH TR FAV AVEK F MA KA E C4V4L2 P TGL TV RA IAE V ..TA V .. D G. R AV TLK V TA YD .ERE.E IG AG TH TR FAV AVDK F MA KA E L1MYN3 P TGL TV RA IAQ V ..TA V .. D G. R AI SLK V TA YD .ERE.E IG AG TH TR FAV AVDK F MA KA E G5GQK6 P TGL TV RA IAE V ..TA V .. D G. R AI SLK V TA YD .ERE.E IG AG TH TR FAV AVDK F MA KA E C9LW87 ALG RS V HA EAE V ..KNF.. D G. R QV TF AV KA FD .EAG.E IG EG LH ER VLV NREK F MS KV S J6I3C7 ALG RS V HA EAE V ..KSF.. D G. R QV TF AV KA FD .EAG.E IG EG LH ER VLV NREK F MS KV S I0GTL3 PVGM KV RA EAE V ..TA V .. E G. RKV TF TV RA FD .DAE.E IG VG TH ER FAV QKEK F LS KA Q R5EG72 PVGL EV RA EAE V ..TA V ..AG. K VI TF AV RA FD .EAG.E IG HG TH QR VVV NSQR F LD KA Y R5VLB0 P EGM TV TA TAE I ..TA V ..SG. R EI TF AI TA SD .EAG.E VG HCT H KR FLV KSES F MK KA A R7H2Y6 P QGM TV RA EAE V ..TG V ..NG. R EI SF AV TA YD .EAG.Q IG TG TH KR FLV EQQR F LD KA A U2M1B8 PVGL TV TA TAE I ..TG V ..NG. R EI SY HV TA ED .GVG.V IG SG VH KR FLV DAER F LS KA Q W7UZ64 P KN M KV CA EAE L ..LE V ..NG. R EL IF SV KA YD .EAG.D IG SG IH KR FLV YGDK F IQ K TY R7MJM6 P ES M KV TA SAE I ..TE V ..NG. R EI TF KV EA FD .ESG.K IG EG LH KR FII YGER F TE KA K D4LA09 P PGM EA HA VAE V ..TE V ..SG. R EI TF HV VA YD .RCG.K IG EG MH KR VVV RAER F LE K TN R7AJW2 P PGM EA HA VAE V ..TE V ..SG. R EI TF HV VA YD .HCG.K IG EG MH KR VVV RAER F LE KA N U2F0D7 PLGA TV RA TAT L ..LSF.. D G. R TA RF AV TA ED .DFG.K IG EG EH TR AVV GAQR F LQ RM R G4KR18 PVGM KV WA EAE I TGVSE..NG. K MV DF KV RA WD .EKG.P IG HG TH TR AII NNER F MA K CA U2RDD7 PIGM KV WA EAE I TAVSE..NG. K MV DF AV KA WD .ESS.P IG SG TH TR AIV KNEK F LA K CN U2QNH9 PIGM KV WA EAE I TAVSE..NG. K MV DF AV KA WD .ESS.P IG SG TH TR AIV KNEK F LA K CN

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! 193! R6TMK5 PVGM KV WA EAE I TAVSE..NG. K MV DF AV KA WD .EKG.P IG SG TH TR AII KNEK F LA K CN R9LX73 PIGM KV WA EAE I TGVSE..NG. K MV DF KV SA WD .ERG.P VG GG TH TR AII NCQR F LD K CN R6H0F9 PVGM TV RA TAT V TGVSE..NG. K LI TF RV EA SD .DWG.P IG EG TH TR AVI ANDR F LQ K CN R6D0Y4 PVGM KV TA TAE V TGIS A ..NG. K MI TF KV TA HD .ADG.L IG EG IH TR AVI DNAR F LQ K CN R6UAG6 AS GA EV YA TAE L ..TES.. D G. RR FE F AV EA YD .NAG.L I AK G TH QR FS V KIEK F LA KA E B2V410 AE GV QV FA EAE L ..TA V .. D G. RK YS F SL KA FD .NKG.L I AT G EH ER VC V KKDS F VN KA I B1QTQ7 AE GV NV WA EAE L ..TN V .. D G. RK YT F LL RA FD .NNG.L I AT G EH VR VS V KKDS F LK KA I R5KLD7 KT G ET I TA RAE I ..RE V ..RG. RKI VF SV EA TN.EKGER IG GG TIE R FAV IREK F MA KL G R7BG85 SMG TT V SA VAK L ..EE V .. D G. RRL VF SV SA KD .NDGNV IG KG TIE R FVV LADK F MK KV N R6M9E2 AL DDV V IA IAT L ..DK I .. E G. RKL TF IV EA KD .THK.V IG KG IH ER FII NNEK F MN KL Q F9N406 PLGA TI TA MAT V ..VA V .. D G. RKV TF EV VA KE .GEK.V IG RG TH ER FII NNEK F MA KI V K9D8B9 AVGA QI RA EAE V ..TA V .. D G. RKI SY KV ATY E .GDI.C IG KG TH ER FVI NNEK F MS KL K I4D8E6 TIGV KI TA TAE L ..IE I .. D R. RRL VF TV EA SD .EAG.Q IG AG KH ER FII DIEP F LN KA Q G2G1T3 PLGA KV SA TAE L ..IE I .. D R. RRM VF SV EV YD .EVG.Q IG VG KH ER FII DIDS F LV KA Q J7IWS0 PLGA NV FA IAE L ..VE V .. D R. RKL VF TV EA FD .DAG.Q IG VG NH ER FVI DVDS F IA KA Q G7WFQ0 PIGA LV KA TAE L ..ME I .. D R. RRL VF NV EA FD .EAG.Q IG VG KH ER FII DVDP F LA K TQ H5XVU6 PLGA KV FA TAE L ..LE I .. D R. RRL VF QV EA CD .EAG.Q IG IG NH ER FII DIEP F LA K TQ W0EBY5 PIGM KV WA EAV L ..VE V .. D R. RRL VF EI DA YD .EVE.K IG SG RH ER FII QEDR F LS KA N L0FAB3 PLGM KV WA VAE L ..LE I .. D R. RRL VF KL EA FD .EKE.L IG TG VH ER FLI DAEK F MK K TL B8G028 PLGM KV WA NAE L ..VE V .. D R. RRL VF KL EA YD .DKE.L IG SG TH ER FII DVEK F MI K TQ I4AA80 PLGM KV WA DAE L ..IE I .. D R. RRL VF QI EA YD .EKE.L IG SG TH ER FII DVEK F MT K TQ Q67JY1 P PGM KV RA TAV L ..ER V .. D G. RRL LF RV EA FD .DRE.R VG SG TH ER VIV RMER F LQ RA G Q3A9K1 PVGA LV EA KAK L ..IS V .. E G. RKL TF LV EA FD .EQG.K IG EG RH ER VR V NIEK F LE KV S R6J6I5 LLG EK I IA TAK L ..IA V .. E G. RKL TF KV GA CD .DHG.P IG NG TH ER FII DKAK F IA KL Q R6I9I9 GMG KK V TA KAT L ..TA V .. E G. RKL VF EI TA AD .EDK.Q IG KG TH ER FIV NKEK F MA KL G U2U2T3 AVG RQ V TA KAV L ..VG I .. D G. RKL SF KL TA SD .EHG.I IG QG SH ER FLV KKEK F MN KL K G4Q7C4 AVG RQ V TA KAV L ..VG I .. D G. RKL SF KL TA SD .EHG.I IG QG SH ER FLV KKEK F MN KL K C0WAV5 AVG RQ V TA KAV L ..VG I .. D G. RKL SF KL TA SD .EHG.I IG QG SH ER FLV KKEK F MN KL K S2Z443 AVG RQ V TA KAV L ..VG I .. D G. RKL SF KL TA SD .EHG.I IG QG SH ER FLV KKEK F MN KL K R7M286 AVG KT V TA KAV L ..VG V .. D G. RKL TF KI TV SD .NYG.T IG QG TH ER FLV NKAK F LG KL A D2RNA4 AVG KT V TA KAV L ..VG V .. D G. RKL TF KI TV SD .NYG.T IG QG TH ER FLV NKAK F LG KL A C0GGI3 PI DM TV TA HAE L ..VE V .. E G. RRL RF KV TA VD .DAG.P VG EG TH DR FVV DQAK F MA KV E C9R9F2 PVGM EV TA VAE L ..VE V .. E G. RRL VF RV EA RD .QVE.V IG RG RH ER VLV DVAK F LA KA Q

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! 194! F8JPF9

F8JPF9 QK TPAG...... U5RWS7 ...... D8GUT2 ...... R5XE94 GD...... R6J0V9 GK ...... F2I7N4 DNSSLQ...... H3NJZ3 ANFIDEV...... M2DSM2 ...... J3F9A6 ...... G5KAY0 ...... B9DSI3 ...... F5ZJ64 KS...... C0MC17 ...... G5K657 ...... K4N6V7 E...... E7PZY6 D...... I7MYH5 D...... R6GEG5 VK KAK...... V6QBM2 ...... J1HAQ4 ER AKQ...... U2QSW7 DNN...... C5NYW0 ...... E5V1L6 SK ...... F3A7K0 N...... F2BXC3 NK ...... W1U6V8 AR TRK...... S4BZU7 EK QS...... J0XJB8 ENQS...... T2NPG1 ENQS...... G5IUE9 EK ES...... C8ZYY8 EK ES...... T0V7E2 EK ES...... K8E3X9 K...... R2SKA9 ENHNM...... R2VCM1 ...... R2PBH5 ...... R2R932 ...... S0RW90 ...... E4KRB3 ...... R2S8F8 ...... K8F818 KD...... D4MBC5 KY...... R3WMJ9 ...... S0KCS8 ...... R2TEP8 LR K...... R2TK84 NPK...... R3WR68 NPK...... W8U886 AK KL...... G8TZK5 AK KVRPKPI...... F8I584 AK KVRPKPI...... F4X9G0 Q...... V2XUL5 ...... R6NZJ4 EK FNG...... R5DLE4 SK FDE...... D4L561 SK FDE...... R9LQS4 NFK...... R5ZKL6 PD...... U2R6S0 AK ...... B7CCX0 KK ...... J8HC92 QK ELVK...... J8I2Q3 QK ELVK...... J8HQS7 QK ELVK...... J8HXW9 EK REAVKING...... C3GXB1 EK REAIKVNG...... J8GWT9 EK REAIKVNG...... J8LFQ4 EK REAIKVNG...... J8AI75 EK RKAVTIND...... H5XWA2 RK SKTESVTE...... G7W6G7 RK SEIERITE...... J7IYH8 RK SEIKA...... D4J4P3 ...... U2L2J7 DK IRRHQKEENESEKNGNSGTCC... Q8EMG8 IQIAAK...... T0JGA7 AASIGGNVSISSQLERKPITFL.... Q2B862 KIRLD...... U5L8R5 KIRLD...... U6BAI1 NNVPTK...... B1YII8 PH VF...... K0A8N8 PR VF...... W7LB64 ID...... E5WMY4 RD...... S2XPF3 EASV...... W4RK43 GS...... V6T386 GS...... I8UG48 SMRMENTQK...... A6CP21 ...... F8CXF7 KQQLHS...... E3IAI3 KQQLHS...... K6PLM5 ALQQRLARQDSPAP...... E6SLA6 EVQQRLGTAAAGTPGQGRTSREAPGV G8TSI3 DLSHHVSSDKEPTANE...... F8I237 DLSHHVSSDKEPTANE...... V9W1G5 ELSQKVENRQKAEKQH...... L5MUG4 ALQAEISAEK...... J2HJM9 ALQAEISAEK...... C0Z567 ALQAEINQEASREL...... J2G9M0 ALQAEINQDVNGK...... C8WU92 AMT...... F5SL03 QME...... D5WXP8 SMK...... R7G5H6 QK AMN...... A8RFU2 QK AMN...... H1BNL1 AK LC......

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! 195! U5F4M4 AK LC...... E2SKL5 SK LEK...... H1B0W4 SK LENS...... E4LXV6 SK LENS...... T4NF97 SK LENS...... N9WK99 SK LENS...... R6UIH5 SK LENS...... G1VX76 SK LENS...... H1BDX8 SK LENS...... E1L909 AR KASN...... S3A0K0 AR KASN...... J5AQJ6 AR KASN...... X8HFB3 AR KASN...... W3Y8I0 QR SSSN...... D1BM20 SR ANAN...... D6KMG8 SR ANAN...... D6KHB2 SR ANAN...... E4LB70 SR AKSN...... W1UP58 SR AKSN...... C4FPZ7 SR AKSN...... W1W8P8 SR AKSN...... W1X196 SR AKSN...... U2BAI1 AK RKPEKGKE...... U2SW53 AK KHA...... R9IHN6 RK KEA...... R9KEX9 KK KQA...... S0J977 RK KQG...... R5HR96 KK RNPK...... R6WU27 K...... F0T2R2 AK LS...... D4J9J7 ...... D4J2H1 GLK...... R6GT38 AK KQSLNK...... V2YEB4 KK LEG...... F4X8V5 KK LEG...... A6NV90 QK KEAPHVN...... R5B634 AK KG...... U2BG97 GK KKGN...... G9YX66 GK KKGN...... H1C7K7 GK KKGN...... D4JRV3 GK V...... R6RPT5 GK V...... F7K912 AK GNLQQGKEQEING...... R6QC10 SK KN...... E2ZHD1 AK KG...... R7C7Z2 RALLG...... R6IH13 AK GAK...... R5CSL0 AK GVK...... I5AU99 GK KAGE...... R6BVQ8 AK LNR...... G2T231 AK KN...... R5Q2H0 AK LEK...... D7GQ38 KK ALAE...... R5FXP5 KK AEQ...... R6RV13 KK AQK...... R6ZN81 QK KNASKGE...... D6DGH0 AK KDAEK...... R5M2P2 AK KDAEK...... D4CH55 AK KDAEK...... D4MPY0 AK KDAEK...... R7B681 SK KEN...... G5FFC4 AK KENKQ...... U2D4S0 AK KEK...... R8VSU0 SK LG...... B8I4C0 SK LEG...... U4R6H3 SK LNE...... H2JF67 SK LNE...... L1Q3R4 NK ...... R6KH82 NK ...... R6FUY0 NK ...... N2BI76 KK LEA...... E6LQN3 SK SEK...... H1LX05 SK LEK...... W2VGZ8 SK LDK...... K0XJJ2 SK LDK...... J4TCP2 SK SDK...... F3B6C0 SK SDK...... R5RJN6 NK LKKDAE...... R6LSC1 NK LAK...... R6VIH0 RK LED...... R7JYS9 RK LEQ...... F7V6W4 SK KA...... U2PAA7 NK LAK...... R5TLI5 NK LAK...... R6P4I1 SK K...... C0CNB6 KK LG...... R6DLF5 AK LEK...... R5SHR8 SK KEA...... R5ZA51 AK LQK...... C4Z997 DK AK...... E4Q5E0 QK VR...... D9TGD2 QK VR...... B9MM60 QK VR...... E4SE45 QK AR...... G2PV11 QK VR...... E4S8L3 QK VR...... E4QCS6 QK VR...... B0TBP0 AK KKG...... D5X9M4 GK KQSG...... F6DMU1 SK AKAKE...... A4J0S5 SK LD...... K8DZX3 SK LSR...... F6B4J8 SK KA......

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! 196! A5D1P6 AR LKE...... F6CP13 AK LKTTS...... R7AYY7 SK AE...... D7GRB2 KK AE...... R5G526 KK AE...... R7NT78 EK FE...... R7AWH4 AK ...... R5RY61 TK LED...... A1HRR7 AK AGSGI...... F7NH57 AK GKK...... H1HSX5 AK LENK...... I7K9K8 NK VK...... R7RUE8 NK NSKEE...... N1ZJT1 EK GKKA...... R7B8B0 EK GKK...... R6PY32 EK GKK...... R5AF40 KK GTK...... M1E923 KK KSF...... R4KGW9 SK K...... A6TLR1 EK GEK...... F4A004 SK FKRER...... G8M073 GK LNS...... A3DHM5 KK AENI...... W4V2Q9 KK SDNI...... H6LHK5 KK LESSRQEES...... E6MGL5 DK KNQ...... E3GGD1 QK KNPK...... R7K521 SK LENRKQSF...... R5AKF1 ER AESEA...... B2A5Z4 ...... T0N2A7 ...... A0PXM9 K...... C6Q0E0 E...... A5N4K6 ...... U2D856 ...... K6U6J6 NK ...... W6N7S3 K...... G7M699 DN...... A6LWA7 NC...... V8G1J7 NC...... U5MUT7 NI...... S1MVB3 QK LHQK...... R6BIU7 EK QAALQMHSAE...... R5GAX2 GK FDK...... R6G283 GK FDK...... R6QH26 QK LN...... B0MBZ2 QK LN...... E5VQV8 QK LN...... D4MVS3 KK LED...... B0NZW1 KK LED...... E5VM16 KK LED...... L1PTM0 KK LED...... R5YWL8 KK LED...... C0EF68 AK LN...... D1PP05 AK LNAD...... U2KTG5 KK KEEYCK...... E0E4V9 AK IERAKNK...... D3MSS8 AK IDKAKNK...... B1C982 AK KK...... G9YHC1 AK LKK...... E2ZCX0 SK LK...... U7UR89 GK KK...... R7MZQ7 AK KAAAK...... S7HMM5 AK KAAAK...... F9MNK7 AK LKK...... F5TG45 AK LK...... D3LTB8 AK LK...... F5TG44 AK KKNNTQL...... D3LTB7 AK KKNNTQL...... F9MNK8 AK RNK...... H1D2D9 QR NGKKQ...... R5SND6 K...... R7CTJ9 KR KNKE...... R6A5L1 KR I...... R9MCN0 AK REAAE...... B0PBF1 AK RRLNQ...... K9CK05 GK KG...... J4V0H9 SK R...... E7N2I9 SK R...... E4LMB1 SK R...... E0NZB5 GK KV...... D4S5R8 GK R...... F5RQF3 GK R...... U2JVQ3 GK R...... J7SJ73 GK R...... C4V4L2 GK RS...... L1MYN3 GK R...... G5GQK6 GK R...... C9LW87 AK ...... J6I3C7 AK ...... I0GTL3 AK H...... R5EG72 SK L...... R5VLB0 AK KESL...... R7H2Y6 KR GQQ...... U2M1B8 KR GQ...... W7UZ64 SK I...... R7MJM6 SK LK...... D4LA09 GK MAK...... R7AJW2 GK MAK...... U2F0D7 EK KPL...... G4KR18 AK LDG...... U2RDD7 AK LEKK...... U2QNH9 AK LEKK......

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! 197! R6TMK5 AK LEG...... R9LX73 GK LEP...... R6H0F9 SK LTEQK...... R6D0Y4 DK LAHV...... R6UAG6 ER LKK...... B2V410 ER KNS...... B1QTQ7 ER KNY...... R5KLD7 ...... R7BG85 G...... R6M9E2 KN...... F9N406 TG...... K9D8B9 ...... I4D8E6 KR NREI...... G2G1T3 TR I...... J7IWS0 AR NQGM...... G7WFQ0 TR KQGM...... H5XVU6 NR KQES...... W0EBY5 QK SGNLKNQ...... L0FAB3 SK RSE...... B8G028 AK SGERE...... I4AA80 SK SL...... Q67JY1 EK RLP...... Q3A9K1 SR EK...... R6J6I5 SK R...... R6I9I9 ...... U2U2T3 TR YDR...... G4Q7C4 TR YDR...... C0WAV5 TR YDR...... S2Z443 TR YDR...... R7M286 AK DQK...... D2RNA4 AK DQK...... C0GGI3 QK SG...... C9R9F2 AK RSG......

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! 198! A)1)6(

β1 η1 α1 α2 α3 F8JPF9 TT 1 10 20 30 40 50 F8JPF9 ....MKD GM RV G ERFTHDFV V PPHK TV RHLYPESP E FAEFPE V FA T G FMV GLM E W A CV RA A6L1R5 ...... M ET G LTYTSTVV V S K E NV A ATM...... GSGDLN V FA T PA MV ALM E N A AMS A F0RKI4 ..... MRPIP ENHSETLTIT V T D DMTV N F T..... ELG P LHP V Y ST Y T LAKH FE E AGRKL H8GSG4 ..... MR T MP E GF QQQLVIT V T D EMTV Q F A..... ELG P VHP V YA T YWLARH FE E AGRKL Q9RR87 ..... M QT IP E GF TQTLEVT V T D EMTV D F A..... ELG R VHP V YA T YWLAKH FE E AGRKI K9ZXJ5 ..... MK E IP A GY TQTLTIT V T H EMTV N F D..... ELG P LHP V YA T YWMAKH FE E AGRKI E8U6U0 MLRAL MRPIP DA F TQTLTVT V T D DMTV H F E..... ELG R LHP V YA T YWMAKH FE E AGRKI Q1IWD4 ..... MRPIP A GF TQILTVT V T D EMTV N F G..... ELG A VHP V YA T YWLAKH FE E AGRKI C1CY65 ..... MRPIP A GF TQTLTVN V T E DMTV D F G..... ELG R LHP V YA T YWMARH FE E AGRKI D7CQM9 ..... M N PIP E GY RATFELT V T DA MTV D F EQP.EP ELG K LHP V YA T YWL T KH VE L V S RKI F2NK44 ..... MKPIP I GY QAVFETV V T P EMTV D F E..... ELG K VHP V YA T YWM T KH ME L AGRKI W2U665 ..... MRPIP P GF QAVFETV V T P EMTV D F E..... ELG P VHP V YA T YWMAKH ME L AGRKI E8PPT9 ..... MRPIP P GF QAVFETV V T P EMTV D F E..... ELG P VHP V YA T YWMAKH ME L AGRKI G8NDE7 ..... MRPIP P GY EATFELV V T EA MTV D F E..... ELG P VHP V YA T YWMAKH ME L AGRKI B7A757 ..... MRPIP I GY EAVFETV V T EA MTV D F E..... ELG P VHP V YA T YWMAKH ME L AGRKI K7R513 ..... MRPIP E GY QAVFETV V T E EMTV N F E..... ELG P VHP V YA T YWMAKH ME L AGRKI H7GG95 ..... MRPIP E G XEAXFETV V T P EMTV H F E..... ELG P V Q PV YA T YWMAKH ME L AGRKI Q5SJP1 ..... MRPIP E GY EAVFETV V T P EMTV R F E..... ELG P VHP V YA T YWMVKH ME L AGRKI D3PRJ9 ..... MKPIP P GY QASFETT V T E EMTV D F EHPDDPQ LG K LHP V YA T YWMAKH ME L AGRKI D7BIK7 ..... M QA IP I GY RAEFETV V T A EMTV D F EHPHDPR LG K LHP V YA T YWMAKH ME L AGRKI

η2 β2 β3 β4 F8JPF9 TT TT TT 60 70 80 90 100 110 F8JPF9 MAPY L E PG E GS LG T A I C V TH T A ATP PG L TV T V TA ELRSV EG R RL SWR V SA HDG V . D E IG S A6L1R5 VA DE L P EG STT VG AM M NTT H IKPS A VG DT V S A TA VLKEV EG R KL TFE V RA QDSK.GV IG E F0RKI4 LLPF L E PG E DS IG IQ V ETF H TG SALP G M EV T V TA TLQEVS G R RV VCA L RA V S S LGD E IG H H8GSG4 ILPF L EE G E E GI G T Q L E V FH T ASALP G M RV T V TA TFT R QG G R RI H A D L FA V NELGD A VG H Q9RR87 ILPF L DE G E G GI G S Q V E V RH T ASALP G L AV R V TA SLD R V EG R RV Y A K L RA V NELGD E IG H K9ZXJ5 ILPF L EE G E G GI G S E V S V TH T ASALP G M QV T V TA VFE R R EG R RI Y A R M SA V NELGD L IG H E8U6U0 ILPY L ED G E Q GI G HA V N V TH T ASALP G M TV T V TA TLD R V EG R RV Y V N V RA V NELGD D IG H Q1IWD4 ILPF L EE G E G GI G T D V Q V TH T ASALP G M TV T V TA TFE R M EG R RI VCS L RA V SELGD E IG R C1CY65 ILPF L ED G E G GI G T Q V D V TH T ASALP G M TV T V TA TFE R M DG R RI L A S M RA V NELGD E IG T D7CQM9 ILPF L E PG D A GI G FE V A V TH V ASALP G M TV R L EA TFV R Q E KN RV Y A R V RA W NELGD L IG E F2NK44 ILPF L EE G E D GI G HE V E V RH L ASALP G M RV K I VA EHL R T EG N RV Y A ACR A Y NELGD L IG E W2U665 ILPF L EE G E E GI G S Y V E V RH L ASALP G M RV R I VA RHE R T EG N RV H A S M EA Y NELGD L IG R E8PPT9 ILPF L EE G E E GI G S Y V E V RH L ASALP G M RV R I VA RHE R T EG N RV Y A S M EA Y NELGD L IG R G8NDE7 ILPF L EE G E E GI G S Y V E V RH L ASALP G M RV R I VA RHE R T EG N RV H A T M EA F NELGD L IG R B7A757 ILPF L EE G E E GI G S Y V E V RH L ASALP G M RV R I IA RHE R T EG N RV H A V M EA Y NELGD L LG R K7R513 ILPF L EE G E E GI G S Y V E V RH L ASALP G M RV R V VA RHE K TV G N RV Y A T M QA F NELGD L IG E H7GG95 ILPF L EE G E E GI G S Y V EXR H L ASALP G M RV R I VA RHE K T EG N RV Y A R V EA Y NELGD L IG V Q5SJP1 ILPF L EE G E E GI G S Y V E A RH L ASALP G M RV R V VA RHE K T EG N RV Y A R V EA Y NELGD L IG V D3PRJ9 ILPF L EE G E E GI G S K V S V DH L ASALP G M KV R I VA EHL R T EG N RV H A QCT A W NELGD Q IG V D7BIK7 ILPF L EE G E E GI G S K V S V HH L ASALP G M RV K I VA EHV R T EG N RI YTACQ A W NELGD L IG E 1

β5 α4 F8JPF9 120 130 F8JPF9 G THER AVI HLE K FN A KVRQKTP...AG...... A6L1R5 G THVRY IV DKE K FMSKLS...... F0RKI4 G TAS QLVLP EE KI R A G F EA L RE RF AASRQPS...... H8GSG4 G T T T QVV TAGT RL QDG F AE L RR RW EAARD...... Q9RR87 G S T T QVVLP QAT IDA G F NE L GR RW AESQEGR...... K9ZXJ5 G A T T QVVLP QS RID EN F AQ L AQ RF EEYRAGR...... E8U6U0 G T T T QVIAP QA RLDA G F AD L AA RW HAHQTTHQR...... Q1IWD4 G T T G QLVL SQA KMDA N F ER L RA RW QASRD...... C1CY65 G T T T QMVLP QE KIDA N F DR L RE RW AAHQR...... D7CQM9 G T T T QVIVP QA KLEA G F AA L RE RW RARKEATA...... F2NK44 G R T T QVILP KA KI Q A A F DK L RA RW ESQKTEVNRG...... W2U665 G R T E QVILP KE KVEA L F AR L RE RW EARGRV...... E8PPT9 G R T E QVILP KE KVEA L F AR L RE RW EARGRV...... G8NDE7 G R T E QVILP KA KVEV L F AR L RA RW REGAGPA...... B7A757 G R T E QVILP KA KVEA L F AR L RA RW EAFRQGAVSGDEKRKPEES K7R513 G R T E QVILP KA KVEA L F AR L KA RW QAAREG...... H7GG95 G R T E QVILP KA KVEA L F QR L RE RW EAQRSS...... Q5SJP1 G R T E QVILP KA KVEA L F RR L KE RW EAERSPS...... D3PRJ9 G Y T E QVILP RA KL LRI F EK L QA RW QEQHK...... D7BIK7 G E T E QVILP KE KLE RI F HK L QE RW ASFQQAKS......

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! 199!