PNNL Protein Complex Characterization Efforts Richard D. Smith, Joshua N. Adkins, Gordon A. Anderson, Deanna L. Auberry, Yuri A. Gorby, Eric A. Hill, Kim K. Hixson, Chiann-Tso Lin, Mary S. Lipton, Lye Meng Markillie, Uljana Mayer, Matthew E. Monroe, Ronald J. Moore, Sewite Negash, Ljiljana Paša-Tolić, Liang Shi, David L. Springer, Thomas C. Squier, Eric F. Strittmatter, Keqi Tang, and Nikola Tolić Biological Sciences Division, Pacific Northwest National Laboratory, Richland, WA 99352 Himadri B. Pakrasi, Department of Biology, Washington University, St. Louis, MO 63130 Identifying protein complexes using Higher characterization throughput at lower cost AM T tags A component of the PNNL program is to develop an approach that provides both increased confidence, higher P rotein synthesis Nucleoba se synthesis 96% 97% Am ino acid biosynthesis throughput, and a quanti tati ve tool for characterizing protei n complexes. We have ini ti all y expl ored the utili ty of The database of Shew anell a accurate mass and time (AMT) 96% Othe r categories Transcription 94% 40% characterizing protein complexes at the peptide level using AMT tags with Q-TOF instrumentation as an alternative tags provi des the basi s for hi gh throughput characteri zati on of Cen tral intermediar y metabolism 94% to much less sensitive and lower throughput approaches based upon tandem MS (e.g., using ion trap mass protein complexes at either the peptide level or intact protein Ener gy metabolism 94% Prot ein fate spectrometers) or more expensive FTICR instrumentation that is needed for much more demanding “whole level. The intact protein level analysis is enabled by the Fa tty acid and phospholipid peptide level approach (by providing their initial me tabolism 94% proteome” analyses. T ransport and binding pr oteins 93% identifications) and also complements the information Cov erage This initial evaluation examined the highly active oxygen-evolving photosystem II (PSII) complex purified from the

Hypothetical pro teins DNA metab olism 9 3% obtainable at the peptide-level with additional information on65% HT-3 strain of the cyanobacterium Synechocystis sp. PCC 6803. This initial study used high pressure capillary LC- protein modifications (e.g., chemical modifications, protein Regulator y functions 93% Q-TOF instrumentation and used AMT tags that were generated from capillary LC-MS/MS analyses. The table

truncation). Regardless, the use of AMT tags can greatly Cell envelope 93% below shows a partial listing of >120 proteins that were identified, along with a measure of their relative abundances speed the analysis and potentially allows characterization S ignal transdu ction 92% based upon the integrated peak intensities for the corresponding peptides. The proteins highlighted in yellow were Cellular pro cesses times of <5 minutes per complex. Proteome coverage by 91% previously identified by Pakrasi and coworkers (Kashino et al., Biochemistry 2002, 41, 8004 – 8012). All proteins B iosynthesis of cofactors Unknown function 90 % AMT tags is indicated by the pie chart (right). 90 % that were assi gned based upon more than one peptide are done so wi th very hi gh confidence.

Value-added protein complex characterization at the intact protein Mass Tag Average Approx. Reference Description Descr iption fr om fromFASTA FASTAGene GeneCount Ab un Count da nc e otherwise) AbundaSLR0906 ncephotosystem MW II core light harvesting protein psbB 26 23386 45 00 0 level – initial demonstration SL L1 13 0 unknown protein 7 2 31 13 10 00 0 SL L1 63 8 hypothetical protein psbQ 12 14362 11 00 0 T he se re su l ts sh o w: SLR0144 hypothetical protein 4 1 36 78 24 00 8 To evaluate our approach, we have initially studied the well characterized yeast large ribosomal SL L0 42 7 photosystem II manganese-stabiliz ing polype ptide ps bO 19 12461 31 00 0 • Q-TOF instrumentation augmented SL L0 25 8 c yt o ch r o me c5 50 p s bV 11 11983 16 00 0 SL L1 41 8 s imilar to p ho to syst em I I o xyg en - evo lvin g comp le x 2 3K pr ote in Psb P p sb P 2 78 78 16 82 8 subunit. The 43 proteins in this complex were previously identified at the peptide level using tandem SL L1 39 0 hypothetical protein 7721321 00 0 by the use of LC elution time SLR1181 photosystem II D1 protein psbA1 4 7098 32161 MS, providing an expected set of tentative molecular weights. The constrained level of complexity SL L1 09 9 e lo ng atio n fa cto r T u tu fA 22 67 91 35 47 2 information provides sufficient SL L1 19 4 photosystem II 12 kDa extrinsic protein psbU 12 67 41 10 00 0 associated with most protein complexes (ignoring obviously low-level contaminants) allows the SLR0172 hypothetical protein guaB 3667515 00 0 specificity for application of AMT tag SSR0692 hypothetical protein 3 6445 4746 detected masses to be assigned to the various proteins, as well as (in most cases) assignment of SL L0 85 1 photosystem II CP43 protein psbC 22 62 67 34 00 0 approaches. SLR1604 cell division protein FtsH ftsH 21 61 82 57 00 0 their modification states. SL L1 41 4 hypothetical protein 7617724 00 0 SL R0 01 2 r ib ulo se bis ph os ph at e c ar bo xyla se sm a ll s ub un it r bc S 4 61 58 10 73 8 • The AMT tag approach with SLR1051 enoyl acyl-carrier-protein 7 6001 22407 Partial 2-D display SSL0426 putative transposase ISY100_t 1 5806 8867 LC-Q-TOF analysis provides SLR0228 cell division protein FtsH ftsH 19 56 46 59 00 0 Capillary LC-FTICR of SML0007 photosystem II protein Y psbY 1549236 00 suffi cient speci fi city for protei n L6B SL L1 86 7 photosystem II D1 protein psbA3 6548327 00 0 yeast ribo somal comp lex L6A SLR1311 photosystem II D1 protein psbA2 6548327 00 0 L17AB Acetylated SL L1 39 8 photosystem II reaction center W protein (Psb28 b?) psbW, psb13, ycf79 5527710 00 0 complex characterization, along with L14B SLR1128 hypothetical protein 14 50 96 28 97 8 Acetylated SL L1 02 9 c ar bo n dio xid e con ce nt ra tin g m e ch an ism p r ote in Ccm K cc m K 5 49 95 98 16 high throughput, and preliminary Acetylated L14A SL L0 84 9 photosystem II reaction center D2 protein psbD 9475329 00 0 L11A/B L25 L28 SLR0927 photosystem II reaction center D2 protein psbD2 9 47 53 29 00 0 quantitation. L35AB L32 SLR1622 soluble inorganic pyrophosphatase ppa 6 4707 15482 SLR0151 unknown protein 13 46 93 28 32 0 SLL1694 pilin polypeptide PilA1 pilA1 2 4478 14254 • The use of quantitative information Methylated SL R0 00 9 r ib ulo se bis ph os ph at e c ar bo xyla se la rg e sub u nit r bc L 11 43 37 42 57 6

Mass L23 L31A/B SL L1 46 3 c ell divis ion p ro te in F t sH ft sH 21 42 20 55 31 7 and multiple analyses (e.g., using L38 SL L1 54 5 g lu tat hio ne S- tr an sfe ra se 8 4113 24143 L36A/B SSL2245 unknown protein 1 4087 7467 SLL1106 hypothetical protein 5 3985 14625 di fferent wash condi ti ons) wi ll be SLR0670 hypothetical protein 11 39 72 26 50 4 SLL1543 hypothetical protein 1 3921 13433 needed with this approach to better SLR1645 photosystem II 11 kD protein (Psb27 b?) psbZ 7378191 00 0 200 400 600 800 1000 1200 1400 SLR1165 sulfate adenylyltransferase 13 37 24 35 44 2 qualify which proteins are part of the SMR0007 photosystem II PsbL protein psbL 1368046 00 Spectrum Number SSL 25 98 photosystem II PsbH proteinpsbH 3360957 00 complex in contrast to being non- SLR1855 unknown protein 20 34 92 56 78 0 SSR 34 51 cytochrome b559 alpha subunit psbE 3332378 00 specifically associated. SLR0752 enolase 13 28 77 37 74 0 Large Riboso mal Su bu nit Coverage by In tact Prot ein An alysis Spectrum number SLR1218 hypothetical protein YCF39 ycf39 1 2874 14339 SL L1 02 8 c ar bo n dio xid e con ce nt ra tin g m e ch an ism p r ote in Ccm K cc m K 5 28 69 90 31 SLL1212 GDP-mannose 4,6-dehydratase 5 2866 33526 rpL1 rpL2 rpL3 rpL4 rpL5 rpL6 rp L7 rpL8 rpL9 rpL10 SLL1835 hypothetical protein 2 2852 23386 AB AB A/B A/B A/B A/B A/ B SL L0 08 0 N -a ce tyl- ga m m a- glu ta m yl-p ho sp ha te r ed uc tas e a rg C 6 28 42 30 99 2 Ac & 1 Me 1 Me Ac SLL0680 phosphate-binding periplasmic protein precursor (PBP) 10 2770 32463 Loss of Met Lo ssof Met Loss of Met Lo ss o fMe t Lo ss of M et L os s o f Me t Losso f M et L os s of M et L os s of M et Advantages of intact protein proteomics: SSL1498 hypothetical protein 2 2728 5191 rpL11 rpL12 rpL13 rpL14 rpL15 rpL16 rp L1 7 rpL18 rpL19 rpL20 SLR0149 hypothetical protein 5 2717 14913 A/B AB A/B A/B AB A/B A/B AB AB A/B SLR1619 hypothetical protein 3 2716 22510 Ac 6 Me Ac Ac • Augments pepti de level anal yses SL L1 45 0 n itr at e/n itr ite tr an sp or t s yste m s ub str at e- bin din g pr ot ein n rt A 13 26 78 39 71 7 Loss of Met Lo ss of M et Lo sso f M et L os s of M et Lo ssof Met Loss of Met Lo ssof Met Lo ss o fMe t Loss of Me t SLL1654 hypothetical protein 1 2430 11805 rpL21 rpL22 rpL23 rpL24 rpL25 rpL26 rp L2 7 rpL28 rpL29 rpL30 SL L1 04 3 p o lyrib on uc leo tid e nu cleo tid yltr an sf er as e 18 24 14 63 13 0 A/B A/B AB A/B A/B A/B • L e ss co m pl e xi ty SLR0476 unknown protein 1 2405 11675 Ac & 4 Me SL L1 85 2 nucleo side diphosphate kinase 3 22 97 13 53 9 Loss of Met Lossof Met Loss of Met Lo ss of M et L os s o f Me t Lossof Met Lossof Met Lossof M et L os s of M et rpL31 rpL32 rpL33 rpL34 rpL35 rpL36 rp L3 7 rpL38 rpL39 rpL40 SL R1 21 6 M g 2+ t ra ns po rt pr ot ein 5 2280 41448 A/B A/B A/B AB A/B A/B AB • Much more information on protein SL L1 55 5 t wo -co m po n en t h ybr id se ns or a nd re g ula tor 4 22 59 33 63 9 SLL1641 glutamate de ca rboxylase 4215143022 Lo ssof Met Losso f Met L os s of Met Lo ss o fMet Loss ofMe t L os s o fMe t Losso f M et L os s of M et Lo ssof Met Fragment, 77- 1 28 modification states SLR0723 hypothetical protein 5 2128 33603 rpL41 rpL42 rpL43 SLR1618 unknown protein 3 2123 23523 AB AB AB • 42 of the 43 core l arge ribosomal subunit proteins (58 of SLL0290 polyphosphate kinase ppk 13 2115 67003 2 Me 1 Me the 64 possible core larg e subunit pro tein iso forms) are • Potenti all y more quanti tati ve, faster, and SL L0 01 8 f ru cto se -b isp ho sp ha te a ldo las e, cla ss II fb a A, f da 8 21 01 31 61 0 Lo ssof Met Lo ssof Met i den ti fied in a si ngl e an aly sis. SLL0735 hypothetical protein 5 2056 50350 • Av erage mass mea surement ac curac y : 2.5 ppm more sensitive SLR2033 rubredoxin rub 1 2042 10196 SL R1 98 4 n u cleic a cid- b ind ing p ro tein , 30 S r ibo so ma l p ro te in S1 ho m olo g n bp 1, rp s1 b 3 20 00 27 41 1 SLR1609 long-chain-fatty-acid CoA ligase 8 1909 62994 SLL1911 hypothetical protein 2 1821 7269 : Ob ser ved w/ M od ifi cati on s : Obs erv ed w/o Mod ifi cat ion s Or A : No t Ob ser ve d Proc. Natl. Acad. Sci. 99, 5942-5947 (2002)

Complex generation and isolation Tagged proteins generated to date for pull-down studies at PNNL Using recombinant proteins Biochemical enrichment Automation (capture column) Gene Description Annotation EExpre xpre ss ss rereco com mbi bin na an nt t proteip rotei n n inin E.co li li S.S.Oneidensis One ide nsi s CultureCu lture S.S. OnOneidensis ei den sis culturecul tu re withwi th taggedta gg ed YFP hydB periplasmic Fe hydrogenase small subunit SO3921 LoLoad ad BEABEADS DS hydA periplasmic Fe hydrogenase large subunit SO3920 P rotei n pu rifica ti on Wash intoin to columnc olu mn Ce ll lysi s napA peri plasmic nitrate reductase SO0848 CultureCu lture S. PurPuri ified fi ed protep rotei in n SS.Oneidensis . One ide nsi s CeCel lls ls Wa sh CellCe ll lysatesl ysates omcA decaheme cytochrome C SO1779 oon nei eid de en nsi sis s Ad d ta gg ed protei n LL oa oad d ananti ti bo bod dy y omcB deceheme cytochrome C SO1778 Peri pl asmi c p rep ara ti on Flow cell lysate to cell lysate onto BEABEADS DS ho xK Quinone-reactive Ni/Fe hydrogenase small subunit precursor SO209 9 onto co lu mn petA ubiquinol-cytochrome C reductase iron-sulfur subunit SO0608 Lyse EnEnriched rich ed pperiplasmic eri pla smic pproteins rotei ns flavocytochrome C flavin subunit SO3301 MMixture ixture oof f cecell ll lysatel ysate with w ith tagged ta gg ed proteinp rotei n ProteinProte in bindingb ind in g toto theth e antibodyan ti bo dy Sal t p recip itati on Gfo/Idh/MocA family oxidoreductase SO3120 In cub at e Wa sh oxidireductase molybdopterin-binding SO0715 ReductionR edu ctio n ofof complexitycomp lexi ty of IsolationIsol atio n ofof complexcomp le x on columnco lumn nrfC formate-dependent nitrite reductase SO0483 FormaFo rma tion ti on of coco mp mpl lex ex wwi ith th ta gg gg ed ed proteip rotei n n pperiplasmic eri pla smic pproteins rotei ns ptpA phosphotyrosine protein phosphatase SO2208 Affi ni ty pu rifica tion Size excl usio n fractio na ti on Elute ptpB Tyrosine-specific protein phosphatase SO3124 an d activ ity assay Isolated complexcomplex EElute lute complex fromfrom columncolumn cpxP Spheroplast protein y precursor SO4476 EnrichedE nric hed proteinprote in complexesco mpl exe s with activityac ti vity msrA methionine sulfoxide reductase (isoform A) SO2337 Tryptic digest Tryptic digest msrB methionine sulfoxide reductase (isoform B) SO2588 Tryptic digest Enolase PePeptide ptid e mixturesmi xtu res PeptidePep ti de mixturesmi xtu res en o SO3440 rnlB ATP-dependent RNA helicase SO0407 PePeptide ptid e MixturesMix tu res Mass sp ectrom etry Mass sp ectrom etry rpoD RNA polymerase sigma-70 factor SO1284 an d ICR2 LS an d IC R2 LS Mass sp ectrom etry Cytochrome c3 SO2727 rpoA DNA-directed RNA polymerase alpha subunit SO0256 Pe ptid e an d pro tein ide ntific ation s an d IC R2 LS PePep ptid ti de e aan nd d ppro rotei te in n idide enti ntifica fi catio tion ns s Pe ptid e an d pro te in ide ntifica ti on s rpoZ DNA-directed RNA omega subunit SO0360 PePe ptid ptid e e anan d d propro tein te in ideiden ntifica tifica tion ti on s s hepA RNA polymerase-associated protein SO0575

Initial results Initial attempt of pull-down with

rpoA Average Q T OF Pe ptid es Avg Peptide Reference Description MW(kDa) Abundance Observed Pr op he t 3, 00 0 SO3237 f lagellin 28 8635 9 1.00 ptpA SO1797 DNA-binding protein HU family 9 6399 11 0.99 Initial attempt of pull-down with SO 24 01 in te gr atio n ho st fac tor beta su bu n it 11 6 26 1 5 0. 99 112 protei ns identifi ed SO 39 27 r ibo so m al p ro te in L 9 16 4804 9 0.95 ptpA Average QTOF Peptides Avg Peptide SO 02 57 r ibo so m al p ro te in L 17 15 4695 5 1.00 Reference Description MW(kDa) Abundance Observed Pr op he t SO 02 17 t ra nsla tio n e lon ga tio n f ac tor T u 43 4 29 0 2 6 0. 98 SO 39 30 r ibo so m al p ro te in S6 15 4860 7 0.98 SO 02 35 r ibo so m al p ro te in S1 9 10 3267 7 0.97 SO 36 52 r ibo so m al p ro te in L2 1 11 3361 4 1.00 SO 09 33 f ru cto se -b isph os ph ate a ldo las e c lass II, Ca lvin cycle su bt ype 38 2 75 9 4 1. 00 SO 02 34 r ibo so m al p ro te in L2 30 3221 7 1.00 SO 12 07 r ibo so m al p ro te in S1 5 10 2721 4 0.99 SO 02 23 r ibo so m al p ro te in L7 /L1 2 13 3057 3 0.97 SO 02 47 r ibo so m al p ro te in L 18 13 2519 4 1.00 SO 02 17 t ra ns latio n elo ng atio n fac to r Tu 43 2228 20 0.98 SO0704 chaperonin GroEL 57 2150 26 0.97 SO 37 65 c on se rve d hyp ot he tica l prot ein 25 1549 5 0.96 SO 02 21 r ibo so m al p ro te in L 1 25 1939 12 0.96 SO 39 30 r ibo so m al p ro te in S6 15 1781 6 1.00 SO 02 47 r ibo so m al p ro te in L1 8 13 1344 6 0.99 1, 70 0 SO 02 55 r ibo so m al p ro te in S4 23 1344 9 1.00 Mass SO1126 chaperone protein DnaK 69 1425 32 1.00 SO 39 40 r ibo so m al p ro te in L 13 16 1395 4 1.00 SO 02 35 r ibo so m al p ro te in S19 10 1227 4 0.91 SO 20 87 in te gr at ion ho st fac to r a lph a sub un it 11 1 18 4 5 0. 98 SO 02 27 r ibo so m al p ro te in S7 18 1375 10 1.00 SO1931 2-oxoglutarate dehydrogenase E2 component, dihydrolipoamide succinyltransferase 43 1208 8 1.00 SO 39 27 r ibo so m al p ro te in L9 16 9 94 7 0. 94 SO 02 56 D NA- dir ect ed RNA po lym er as e a lph a sub un it 36 9 81 1 2 0. 98 SO 02 34 r ibo so m al p ro te in L 2 30 1169 14 1.00 SO 21 12 r ibo so m al p ro te in L 25 17 1000 5 0.98 SO 24 02 r ibo so m al p ro te in S1 61 9 51 1 1 1. 00 SO 12 07 r ibo so m al p ro te in S15 10 9 23 3 1. 00 SO 13 60 r ibo so m al p ro te in L 19 13 98 2 6 0. 99 SO 02 45 r ibo so m al p ro te in S8 14 95 3 5 1. 00 SO0704 chaperonin GroEL 57 9 22 1 9 0. 99 SO 02 21 ribosomal protein L1 25 837 4 0. 97 SO 02 23 r ibo so m al p ro te in L 7/ L1 2 13 91 1 1 0 0. 98 SO 34 40 e no las e 42 89 6 1 6 0. 94 SO 02 57 r ibo so m al p ro te in L1 7 15 7 24 3 0. 95 SO 02 36 r ibo so m al p ro te in L 22 12 88 6 5 1. 00 SO 39 39 r ibo so m al p ro te in S9 15 6 58 6 0. 97 SO 44 10 g lut am in e s ynth et as e t ype I 52 87 5 8 1. 00 SO 21 12 r ibo so m al p ro te in L2 5 17 5 61 3 0. 99 400 SO 02 48 r ibo so m al p ro te in S5 18 82 8 1 0 0. 99 SO 02 30 r ibo so m al p ro te in S10 12 5 23 1 4 1. 00 SO 31 46 D NA-b ind ing p ro te in H -NS fam ily 15 82 3 6 1. 00 SO 02 22 r ibo so m al p ro te in L1 0 18 4 82 8 1. 00 30 165 36 0 SO 17 93 t rig ge r f ac tor 48 80 3 2 1 1. 00 SO 21 78 c yto chr o me c5 51 p er ox ida se 36 4 76 9 0. 99 Time (minutes) SO 02 30 r ibo so m al p ro te in S1 0 12 77 5 1 4 1. 00 SO 23 02 r ibo so m al p ro te in L2 0 14 4 75 5 1. 00 SO 20 87 in te gr atio n ho st fac tor alph a sub un it 11 59 2 4 0. 98 SO 02 50 r ibo so m al p ro te in L1 5 15 4 65 7 0. 90 SO 02 31 r ibo so m al p ro te in L 3 23 58 7 1 2 1. 00 SO 02 27 r ibo so m al p ro te in S7 18 4 57 9 1. 00 SO 14 90 a lco ho l de hy dr og en ase II 40 57 1 9 0. 98 SO 39 40 r ibo so m al p ro te in L1 3 16 4 52 5 0. 98 SO2644 phosphoenolpyruvate synthase 87 481 20 0.98 SO 02 45 r ibo so m al p ro te in S8 14 4 08 2 0. 97 3,000 SO 47 49 AT P s ynt ha se F 1 a lph a sub un it 55 47 0 1 4 1. 00 SO2644 phosphoenolpyruvate synthase 87 3 59 9 0. 98 SO 23 28 t ra nslation elon ga tion f ac tor P 21 46 9 4 1. 00 SO1126 chaperone protein DnaK 69 3 43 2 3 0. 99 rpoA SO 02 22 r ibo so m al p ro te in L 10 18 45 9 1 1 1. 00 SO 20 16 h ea t sho ck pr ot ein Htp G 72 3 19 8 1. 00 159 protei ns identifi ed SO 23 02 r ibo so m al p ro te in L 20 14 45 6 1 0 0. 99 SO3681 universal stress protein family 16 3 17 3 1. 00 SO0426 pyruvate dehydrogenase complex E3 component, lipoamide dehydrogenase 51 452 15 1.00 SO 44 10 g lut am in e s ynt he tas e t ype I 52 3 15 8 0. 99 SO 21 78 c yto chr om e c5 51 p er oxid as e 36 44 4 1 2 1. 00 SO 35 37 r ibo so m al p ro te in S20 10 3 05 4 1. 00 SO0432 aconitate hydratase 2 93 43 0 1 9 1. 00 SO3237 f lagellin 28 3 03 7 0. 97 SO 20 16 h ea t sho ck pr ote in Htp G 72 39 9 1 7 0. 99 SO3440 enolase 42 2 62 7 0. 98 SO 15 24 h ea t sho ck pr ote in Gr pE 23 39 9 3 1. 00 SO 23 28 t ra ns latio n elo ng atio n fac to r P 21 2 34 3 0. 90 SO4105 MSHA pilin protein MshA 18 377 4 1.00 SO 02 48 r ibo so m al p ro te in S5 18 2 25 1 2 0. 97 SO 08 42 t ra nsla tio n e lon ga tio n f ac tor G 77 34 6 1 3 1. 00 SO4105 MSH A pilin protein MshA 18 2 23 4 0. 96 SO 47 51 AT P s ynt ha se F 0 B sub un it 17 34 1 5 1. 00 SO 02 36 r ibo so m al p ro te in L2 2 12 2 08 6 0. 99 SO0424 pyruvate dehydrogenase complex E1 component, pyruvate dehydrogenase 99 340 12 0.98 SO 04 32 a co nit ate h ydr at ase 2 93 2 08 1 8 0. 99 SO 37 65 c on se rve d hyp oth et ical pr ot ein 25 32 5 8 1. 00 SO 13 60 r ibo so m al p ro te in L1 9 13 2 05 4 0. 99 1,700

Mass SO 02 43 r ibo so m al p ro te in L 5 20 31 2 1 3 0. 99 SO 02 42 r ibo so m al p ro te in L2 4 11 1 99 1 0 0. 93 SO 02 53 r ibo so m al p ro te in S1 3 13 27 4 6 0. 99 SO 02 43 r ibo so m al p ro te in L5 20 1 96 7 1. 00 SO 06 91 h yp oth et ical pr ote in 15 27 2 4 1. 00 SO 27 76 3 - oxo ac yl-( ac yl-c ar rie r- pr ote in) r ed uc tas e 26 1 85 4 0. 96 SO 02 56 D NA-d ire ct ed RNA po lym er ase a lph a s ub un it 36 23 5 1 5 0. 97 SO 04 26 p yr uva te de hy dr og en ase c om ple x E3 c om po n en t, lip oa m ide de hy dr og en as e 51 1 85 8 1. 00 SO 02 20 r ibo so m al p ro te in L 11 15 23 5 8 0. 98 SO 02 54 r ibo so m al p ro te in S11 14 1 59 6 1. 00 SO 36 81 u niv er sal str es s p ro tein fa m ily 16 22 1 5 1. 00 SO 14 90 a lco ho l d eh ydr og en as e I I 40 1 37 7 1. 00 SO 08 74 D na K su pp re sso r pr ote in 17 20 6 2 1. 00 SO 19 31 2 - oxo glu ta ra te de hyd ro ge na se E2 co mp on e nt, dih ydr olip oa m ide su cc inyltr an sf er ase 43 1 15 7 1. 00 SO 24 02 r ibo so m al p ro te in S1 61 18 6 1 8 1. 00 SO 31 46 D NA- bin din g p ro te in H -NS fa mily 15 1 01 4 0. 97 SO 16 29 r ibo so m al p ro te in S2 27 18 1 3 0. 96 SO 24 01 in te gr at ion ho st fac to r b et a s ub un it 11 1 00 4 0. 97 SO1807 phage shock protein A 26 17 4 4 1. 00 SO 17 93 t rig ge r fac tor 48 95 9 0. 99 SO 02 37 r ibo so m al p ro te in S3 26 14 7 1 4 1. 00 SO 00 52 p r ote in- ex po rt pr ot ein Sec B 17 88 2 0. 99 SO 34 66 r ibo fla vin syn tha se be ta su bu nit 17 13 4 2 0. 96 SO 39 88 a er o bic re spir at ion co nt ro l pr ot ein Arc A 27 84 3 0. 98 40 0 SO 23 45 g lyce ra lde hyd e 3- ph os ph ate deh ydr og en as e 36 94 7 1. 00 SO 06 91 h yp oth et ical pr ot ein 15 73 2 0. 97 30 165 36 0 SO 02 50 r ibo so m al p ro te in L 15 15 79 6 1. 00 SO 02 37 r ibo so m al p ro te in S3 26 64 8 1. 00 SO 47 47 AT P s ynt ha se F 1 b et a s ub un it 50 69 7 0. 99 SO 47 49 AT P syn tha se F 1 a lph a su bu nit 55 63 7 1. 00 Ti me (mi nutes) SO 00 52 p ro te in- ex po rt pr ote in SecB 17 63 2 0. 94 SO 15 24 h ea t sho ck pr ot ein GrpE 23 61 2 1. 00 SO 02 24 D NA-d ire ct ed RNA po lym er ase b eta su b un it 15 0 52 1 6 0. 98