BIOSYNTHETIC INCORPORATION OF 7-AZATRYPTOPHAN INTO THE

CATALYTIC DOMAIN OF PSEUDOMONASAERUGINOSA EXOTOXIN A

A Thesis

Presented to

The Faculty of Graduate Studies

of

The University of Guelph

by

FOO-LIM YEH

In partial fulfillment of requirements

for the degree of

Master of Science

December, 1997

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BIOSYNTHETIC INCORPORATION OF 7-AZATRYPTOPHAN INTO THE CATALYTIC DOMAIN OF PSEUDOMONAS AERUGIRrOSA EXOTOXIN A

Foo-Lim Yeh Advisor: University of Guelph, 1997 Dr. A. Rod Merrill

Pseridornonas aeruginosa exotxin A (ETA), is a mono- ADP-ri bosy l trans ferase

(ADPRT) that catalyzes the transfer of the ADP-ribosyl rnoiety of NAD+ to eukaryotic elongation factor-2 (eEF-2). This transfer inactivates eEF-2 resulting in the inhibition of protein synthesis causing host cell death. The use of intrinsic tryptophan fluorescence to srudy toxin- eEF-2 interaction is inherently limited since the spectral properties of the various tryptophan residues in both proteins cannot be distinguished. To aid in the study of this protein-protein interaction by tryptophan fluorescence, we replaced the tryptophans in the catalytic domain of exotoxin A (PE24) with the analogue, 7-azatryptophan (7AW). The red shifi in the absorption and fluorescence emission spectra of analogue-incorporated PE24, upon cornparison to the native protein. confirmed the presence of 7AW. The selective excitation of the tryptophan analogue within the PE24 catalytic domain will facilitate the investigation of the protein-protein interaction between PE24 and eEF-2. 1 would like to thank my supervisor Dr. Rod Merrill for allowing me the opportunity to work under his leadership and guidance. 1 thank him for his encouragement, enthusiasm and patience. 1 thank my advisory cornmittee members, Dr. Allan Mellors and Dr. Robert Keates for their many constructive and helpful comrnents. 1 would like to express rny gratitude to Dr. Dev

Mangroo for his helpful advise throughout the studies within this thesis. 1 like to thank Monica

Tory for performing the ADPRT assay and Gerry Prentice and James Oak for purifying sorne of the eEF-2 protein.

Speciat thanks to al1 the past and present mernbers of the MerriII lab, Alexander, Ariel,

Brian, Bryan, Claire. Danielle, Fouroozan, Ge-, James, Jason, Joe, John C ., John R., Longzhi,

Monica, Sandra and Yolanda for making the Merrill lab a fun place to work. 1 thank my parents

Frank and Linda Yeh and my sisters Shie-Mee and U-Mee for their love and support throughout my student years. Thanks to the Boyd Family for their kindness and support. Finally, 1 am deepIy grateful to my fiancée Yvonne Boyd for her never ending love and support in helping me through the trials and tribulations of pursuing my graduate degree. Ail of the work indicated in this thesis is my own, with the exception of the kinetic data obtained form the ADPRT assay which was perforrned by Monica Tory. In addition, some of the eEF-2 protein was purified by Geny Prentice and James Oak.

2.5.3 E. coli T~yprophanAllnot~oph and Erpression S'stem ...... 33 2.5. J Media usecl for Analogue Incorporation ...... 33

CHAFTER 3 .BIOSYNTHETIC INCORPORATION OF 7-AZATRYPTOPHANNT0 PE24 ...... 35 3.1 Introduction ...... 35 3.2 Materials and Methods ...... 36 3-21Overexpression and Purification of Wild-type PE2-l ...... 36 3.2.2 Consttuction of Twptophan A rrxotroph ...... 39 3.1.2.1 Preparation of P I Lysogens ...... ***..*.....*.*.**..*..39 3.2.2.2 Preparation of Lysare ...... 39 3.2.2.3 Transduction rvith P I Lysates ...... 41 3.2.3 Mini-prep Test for Overexpression of 7A W-incorporated PE2-I ...... -41 3.2.4 Etpression und Purzjkation of 7A W-incorporated PE2-l ...... 42 3.2.5 ADPRT.4ssay ...... 43 3.2.6 Spectroscopie Measurements ...... 43 3.2. 7 Analysis of Analogue Incorporation Using Absorbante Spectra ...... 44

7 3.3 Results and Discussion ...... 45 3.3.1 PI Transducrion...... --.-...... -..-...-.-...... 45 3.3.2 Etpression of 7AW- incorporateci PE2-I ...... 4 5 3.3.3 Pzrrificarion of 7A W-incorportrted PEI4 ...... 47 3.3.4 En-zymatic Activity of 7.4 W-PE2-i...... 5i 3.3. jA bsorbance and Flzrorescence of 7A W-PE2-I...... 5 5 3.3.6 E@ciencv of 7AW Incorporation into PEN ...... 60

CHAPTER4 .SUMMARY. CONCLUSIONS AND FUTURE STL~DIES...... *... 65

APPENDIX A .STRUCTURES OF TRYPTOPHAN ANALOGUES ...... 75

APPEND~XB . BACTERIAL MEDIA ...... 76

APPENDIX C ...... 77 Table 2.1 Proteins e'rpressed with tryptophan analogues ...... 27

Table 3.1 Kinetics of ADP-ribosyl transferase activity for PE24 and 7AW-PE24...... -54

2AW 2-azatryptophan

4FW 4-fluorotryptophan

5FW 5-fluorotryptophan

5HW 5-hydroxytryptophan

5MH 5-methoxytryptophan

6MH 6-methoxytryptophan

6FW 6-tluorotryptophan

7AI 7-azaindo le

7AW 7-azatryptophan

7AW-PE24 7-azatryptophan-incorporated PE24

~~280 molar extinction coefficient at 280 nm brnmzx wavelength em ission maximum

&,max wavelength excitation maximum

A280 absorbance at 280 nm

ADPRT mono- ADP-ribosyl transferase

A~P ampicillin

Cam chloramphenicol

C-term inal carboxy- terni inal

DT diphtheria toxin

DTT dithiothreitol

EDTA disodium ethylenediamine tetra-acetate eEF-2 eukaryotic elongation factor 2

EF-G prokaryotic elongation factor G

vii ETA Pseudornonas aeruginosa exotoxin A

Fl9NMR F 19 nuclear magnetic resonance

Gn-HCI guanidine hydrochloride

HPLC high performance Iiquid chrornatography

IMPACT intein-mediated purification with an affinity chitin-binding tag

IPTG isopropyf PD-thiogalactopyranoside mo dosage that is lethal to 50% of animal lot

LrNCS l inear corn bination of basis spectra

NATrpA N-acetyltryptophanamide

NATyrA iV-acety Ityrosinarnide

N-terminal amino-terminal

OD650 optical density at 650 nrn

P. aertiginosa Pseudornonas aeruginosa

PDB Protein Data Bank

PE24 catalytic domain of exotoxin A

PE40 ETA fragment containing domains Ib, II and II1 so ground state

SI first electronic state s2 second electronic state

SDS-PAGE sodium dodecyl sulphate polyacrylamide electrophoresis t- boc tert-butoxycarbonyl

Tet tetracyc lin

TWS Tris (hydroxymethyl) aminomethane

UV ultra violet 1.1 Pseudornonas aeruginosa Exotoxin A

Psezidomonas aeruginosa is an ubiquitous, gram-negative, opportunistic pathogen. This pathogen is commonly found in soil, water. sewage, and hospital environments. P. aeruginosa has been implicated as the major cause of many nosocornial infections among immunocompromised patients such as those with AIDS, cystic fibrosis, cancer and major burns

(Vasil, 1986). P. aeruginosa synthesizes a number of virulence factors that are believed to be responsible in the pathogenesis of these infections. The extracellular protein, exotoxin A

(ETA)(EC 2.4.2.36) is the most toxic of these secreted proteins, having an LDso of 0.2 pg/per mouse (Liu, 1974; Iglewski and Sadoff. 1979).

ETA is a member of a farnily of related bacterial toxins, which include diphtheria, cholera. pertussis, shiga, and Escherichia coli heat-labile toxin (Middlebrook and Dorland,

1984). Diphtheria toxin (DT) shares many similar characteristics with ETA and has a simiIar mechanism of action (Iglewski and Kabat. 1975; Chung and Collier. 1977: Iglewski et al.. 1977).

The single-chain, 66-kDa ETA protein is classified as a mono-ADP-ribosyltransferase (ADPRT) due to its ability to cataIyze the transfer of the ADP-ribosyl moiety of NAD+ to covalent linkage with the eukaryotic protein, elongation factor 2 (eEF-2) (Iglewski et al., 1977). The result of this covalent modification of eEF-2 is the inhibition of protein synthesis and host ceIl death. I. 1. I E TA Structural Cene

Tox A, the structural gene for ETA, resides as a single copy on the P. aeruginosa chromosome (Vasil. 1986). The structural gene has been cloned and nucleotide sequence determined by Gray et al. (1984) to reveal that ETA initially produces a 638 amino acid precursor protein with a 25 arnino acid signal sequence at the amino-terminal end. This signal peptide is removed upon processing and ETA is secreted as a 6 13 residue mature protein into the extracellular environment (Gray et al., 1984; Fig. 1.1 ).

1.1.2 CrystaI Structure

The 3-0x-ray crystallographic structure of the mature form of ETA was solved to 3.0 A resolution (Allured et al.. 1986). From the amino acid sequence along with the solved structure,

AIlured and CO-workers (1986) proposed a schematic model for ETA (Fig. 1.2). The c~stal structure data show that the ETA rnolecule has three distinct structural domains. Domain 1 is composed of hvo subdomains, la and Ib. comprising of residues 1-252 and 365-404.respectively.

This domain has 17 Pstrands, al1 of which run antiparallel except for the last short strand. The first 13 strands form the structural core of an elongated kbarrel, while the remainder of the peptide chain traverses one face of the barrel, leading into domain II. Although domains la and

Ib are disassociated in the primary sequence. they are closely associated in the 3-D rnolecular model. Also, the crystal structure reveals that domain 1 possesses three disulphide bonds. two in la (Cyslt-CyslJ and Cys197-Cys214) and one in Ib (Cys372-Cys379). Domain Ia is involved in cell recognition, however the function of domain Ib has yet to be determined.

Domain II is composed of residues 253-364, thus separating the two subdomains of domain 1. This second domain is composed of six consecutive a-helices, with one disulphide linking helix A and helix B. Domain II is involved in the translocation of ETA upon infection of a host eukaryotic ceIl from the endosomal compartment and into the cytoplasm of the host cell. 1(Domain Ia) 2 O 4 O

160 180 Blk 1 Bll I Hlb -ESNE MQPTVTQPRSEKRWS 201 1 Hlc 1 1 Blm 1 Bln PLDGJIYNYLA-QQRCNLDDTW EGKNPAKHDLDIK PTVF

PEGGSLAALT AHQACHLPLE TFTRHRQPRG WEQLEQCGYP VQRLAJYLA 301 320 - ----340--

360 365 (Domain Ib) 380 400 l H2g Blo VRQGTGNDEA GAAN~~~VAAGEEAGPADSGD~TGAE~ 401 405 (Domain III) 420 440 LGDGG~VSFSTRGT~~ RLLQAHRQLE ERG~GYHGT~AAQSI~ 4 60 480 500 H3c 1 B3a ( VFGGVRARS~DLDAiWRGE'Y IAGDPALAYG YAQDQEPDAR GR 501 520 540

FIGURE 1.1 The amino acid sequence of mature ETA and PE24 (shown in bofd). The start of each domain is indicated by the amino acid number 1, 253, 365, and 405 for domains Ia, II, (b, and III, respectively. The position of disulphide bonds within the sequence are shown by brackets. The shaded regions indicates the location of the pstrands (dark) and a-helices (light). The alpha-numeric nomenclature indicates the following: B for bstrands and H for a-helices; the second character designates the domain; and finaily, the third character designates the alphabetical sequence of either Estrands or a-helices (a, b, c. etc.) within each domain. FIGURE 1.2 Schematic mode1 of ETA showing the functional domains la, II, Ib, and III. Domain III consists of the C-terminus of the protein. residues 405-6 13. This domain exhibits a

less-regular secondary structure then seen in the previous two domains, however domain III

possesses an extended cleft, where the active site of the enzyme is located. A higher resolution

structure of the C-terminal. enzymatic domain, PE24. has been crystallized in the presence of

NAD+ at 2.5 A resolution (Li et al.. 1995). Although NAD+ was included in the crystallization

solution. it could not be seen intact in the electron density map, presumably due to the observed

NAD+-hydrolase activity of the enzyme (Li et al., 1995).

1.1.3 General Properiies of ETA

As mentioned earlier, ETA is a mono-ADP-ribosy l transferase (ADPRT) that catalyzes

the following reaction (Iglewski and Kabat. 1975):

NADt + eEF-2 t, ADP-ribose-eEF-2 + Nicotinamide + H+

ETA transfers the ADP-ribosyl moiety of NADi ont0 eEF-2, modifying a post-translationally modified histidine residue known as diphthamide (Iglewski et al., 1977: Van Ness et al.. 1980a;

Fig. 1.3). This transfer halts protein synthesis by inactivating eEF-2, rendering it incapable of translocating ribosomes or shifiing the growing peptidyl t-RNA from the acceptor (A) to the donor (P) sites on eukaryotic ribosomes. As a result, polypeptidyl chain elongation is blocked, protein synthesis is inhibited, and the cell dies.

Along with ADP-ribosy 1 transferase act ivity, ETA possesses NAD+-glycohydrolase activity, the slow hydrolysis of NADf to nicotinamide and ADP-ribose in the absence of eEF-2

(Leppla et al.. 1978). ADP-ribosylation can be reversed if the ribosylated eEF-2 is incubated with excess ETA and nicotinamide at a Iow pH, resulting in the production of NAD+ (Chung and

Collier, 1977). FIGURE 1.3 ADP-ribosyl tram ferase reaction scheme. ETA transfers the ADP-ribosy 1 moiety of NAD+ to eEF-2, rnodifjing the post-translationally modified histidine residue, diphthamide. ETA is secreted from its host as an catalytically inactive proenzyme, which is toxic to cells and

animals (Chung and Collier. 1977; Leppla et al., 1978; Lory and Collier, 1980). The active

processed form of ETA contains four disulphide bonds, of which two must be reduced in the

partially denatured molecule for expression of enrymatic activity (Lory and Collier, 1980).

Enzymatic activity can be activated in vitro in two ways. Activation of the toxin can occur by

treatment with a denaturing agent, such as urea. and a reducing agent. such as dithiothreitol

(DTT)(Leppla et al.. 1978: Vasil et al.. 1977). ETA can also be activated by proteolytic cleavage to release a non-toxic. enzymatically active 26 Dapeptide (Chung & Collier. 1977: Vasil et al..

1977: Lory and Collier. 1980). Since the reduction of the 26 kDa peptide is not required, this supports the evidence that none of the disulphide bonds are located within the region of ETA responsible for enzyme activity (Chung and Collier, 1977). In addition, these data demonstrate that the toxin must undergo a conformational change in order to be active in catalysis. This suggests that the active site of the enzyme is buried within the native intact molecule.

The relationship between tie in vivo mechanism of action of ETA and in viim observations discussed above is not fully understood. More recently. Ogata et al.. (1990) analyzed the processing of ETA in vitro. ELA was proteolytically cleaved to produce an N- terminal 28 kDa fragment. and a C-terminal 37 kDa fragment. The latter contains a portion of domain II and al1 of domain III. Initially. these two fragments are linked by the disuiphide bond joining Cys26j-Cysz87 and cleavage occurs at or near Arg279. Subsequently, the disulphide bond is reduced and the C-terminal fragment is translocated across the endosomal or endoplasmic reticulum membrane and released into the cytoplasm. Once in the cytoplasm, the ADP- ribosylating activity afiliated with the C-teminal fragment inactivates eEF-2 (Ogata et al..

1 990). 1.1.4 Mechanism of Action

ETA exerts its toxicity by a multi-step mechanism: 1) binding of the to'tin to eukaryotic

cells via the ubiquitous a?-macroglobulin receptor (Kounnas et al., 1992) and subsequent

intemalkation by receptor-mediated endocytosis; 2) proteolytic processing to activate the

ADPRT domain: 3) insertion into the endosomal membrane lipid bilayer: 4) translocation of the

enzymatic domain across the membrane bilayer into the cytoplasm; and 5) catalysis of the

transfer of the ADP-ribosyI moiety of NAD' ont0 eEF-2 (Figure 1.4).

PE24 is an active fragment of ETA that consists of the catalytic domain of ETA (domain

II 1) and was produced by recombinant techniques (Li et al.. 1995). PE24 has several advantages

over PE4O (ETA fragment containing domains Ib, II and III) or whole toxin, ETA, since it is a

functionally active toxin: 1) without the bulkiness of using a larger protein molecule; 2) with a

more accessible active site; 3) that is safer to use than whole toxin; and 4) unlike whole toxin, does not require activation by urea or DTT to exhibit ADP-ribose transfer activity. For these

reasons. PE24 was used in this study. It is interesting to note that PE24 and other recombinant

forms of ETA have been used as fusion proteins. coupled to various forms of tumor-specific antibodies or to cytokines, for targeted cytotoxicity (Pastan et al., 1992). €TA b 4 ,Receptor

1. Receptor Mediated End~c6k

2,Vesicle Acidification & ETA Unfddlng.

~~~-~ibosyiatioiof-- I eElongation Factor-:! 4. Insertion & Tramlocution of ETA

5.Reduction of Disutilde Bond & Reieuse of 37 kDa Fragment

- -

FIGURE 1.4 The proposed cytotoxic mechanism of ETA. 1.2 EIongation Factor 2

eEF-2 is a single polypeptide of 857 amino acids (95 kDa) which catalyzes the translocation of peptidyl tRNA from the ribosomal A site to the P site during protein synthesis

(Iglewski. 1994). The translocation is followed by the hydrolysis of GTP into GDP and Pi. eEF-

3 is the only known protein which is selectively ADP-ribosylated and inactivated by diphtheria toxin or ETA in the presence of NADf (Iglewski and Kabat, 1975: Van Ness et al.. 1980b). The toxin-catalyzed ADP-ribosylation of eEF-2 occurs at an unique post-translationaIly modified histidine residue. (2-[3-carboxyamido-3-(trimethyl-ammonio)propyl]histidine), termed diphthamide (Fig. 1.3). which is only found in eEF-2 and is conserved throughout eukaryotic evolution (Pappenheimer. 1977). eEF-2 has not yet been overexpressed. however highly purified preparations of eEF-2 have been obtained from rat liver. pig liver, calf brain, and wheat germ

(Chung and Collier. 1977; Brot, 1982; Crechet et al., 1986).

Like its prokaryotic counterpart (EF-G), eEF-2 belongs to the large family of GTP- binding proteins with GTPase activity (Brot, 1982). This protein is Iarge and interacts with a wide variety of partners during the elongation cycle (translation). for exampie guanylic nucleotides, ribosomal proteins and RNA. Thus. eEF-2 is thought to be composed of several domains. Although the crystal structure of eEF-2 has not yet been solved, the 3-D structure of

EF-G from Thermus tlzrrmophiltu has been solved and was show to be composed of five domains (PDB accession numbers 1 EFG and 1 ELO) (Czworkowski et a[.. 1994; Aevarsson et al.,

1994).

Dumont-Miscopein et al. (1995) treated rat liver eEF-2 with endoproteinase Glu C to investigate the roles of the different domains. Two fragments were obtained, a large N-terminal,

6 1 kDa fragment (F(jl) and a smaller C-terminal fragment that was 34 kDa (F34). F61and Fjl were both unable to catalyze protein synthesis. However, Fol was found to be able to interact with GTP and GDP and Fs4 was ADP-ribosylated in the presence of NADf and diphtheria toxin.

FbI corresponds essentially to domains 1, 2 and 3, whiIe F34 corresponds mostly to domains 4 and 5. which was deduced afler alignment of the eEF-2 sequence with the domains of EF-G

(Dumont-Miscopein et al., 1995). Domain 4 contains the diphthamide residue, which strongly suggests that this domain, and possibly domain 5, is involved in ADP-ribosylation. Currently, the overexpression of a protein that includes domains 4 and 5 of rat liver eEF-2 is being attempted in Our laboratory. If the protein is found to be functionally active, this would give us a less bulky and srnaller substrate for ETA studies.

1.2.1 Interactions between ETA and eEF-2

There have only been a few studies exarnining the interactions between ETA and eEF-2.

The roIe of Tyri81 in ETA was tested using site-directed mutagenesis (Lukac and Collier, 1988).

The investigators replaced the tyrosine residue with phenylalanine and then examined ADPRT and NAD+-glycohydrolase activity and cytotoxicity. The substitution of Tyri81 with phenylalanine resdted in a 10-fold reduction in both ADPRT activity and cytotoxicity. Lukac and ColIier (1988) proposed that the phenolic hydroxyl of Tyfi81 may be involved in eEF-2 binding or rnay alter the orientation of the attacking diphthamide residue of eEF-2.

Recently. active site mutations perforrned on HisJlo of ETA suggest that this residue rnay also be involved in the transfer of the ADP-ribose moiety to the eEF-2 substrate (Han and

Galloway, 1995). This residue is positioned at the bottorn of the hydrophobic active site cavity and projects its imidazole ring into the cavity where it rotates freely in order to facilitate its involvement in hydrogen bonding. HisjJo was substituted with Ala, Asn, and Phe which displayed severely reduced ADP-ribosylation activity (> 1000-fold). However, NAD+ glycohydrolase activity remained intact. This result, combined with similar KM values for the wild-type ETA and the~emutants, suggests that NAD+ binding is not affected by substitution rit Hisao. In view of the location of HisMo residue within or close to the proposed NAD+ binding

site. these resuIts suggest that Hisjj* may be a catalytic residue invotved in the transfer of the

ADP-ribose moiety to eEF-2 (Han and Galloway, 1995).

Kessler and Galloway (1992) proposed that the HisQ6 residue is required for ETA

interaction with eEF-2. Previous work indicated that this residue also plays a key role in ADPRT activity (Galloway et al., 1989; Wozniak et al.. 1988) and is located in a major clefl which may be associated with eEF-2 binding (Wozniak et al.. 1988; McGowan. et al., 1990). To study the

ETA site of interaction with eEF-2, an assay usine immobilized eEF-2 to bind ETA was deveioped. The binding assay revealed that ETA, previously incubated in the presence of NADf, bound to the immobilized eEF-2. However. ETA alone. or CRM 66 (a naturally occurring mutant of ETA, H426Y) in the presence or absence of NAD+ does not bind to the imrnobilized eEF-2. These results suggest that incubation with NAD+ alters the conformation of ETA allowing it to bind eEF-2 (Kessler and Galloway, 1992).

In order to understand the relationship behveen NAD+ binding and eEF-2 interaction with ETA, these researchers were interested in detennining whether NADt binding induces a conformational change in ETA. Their approach was to utilize a monoclonal antibody (TC-1) to probe for conforrnationaI change which results in the opening of the major clefi in dornain III and exposure of the HisJ-6 residue. The results indicate that binding of NAD' changes the conformation of ETA and results in the exposure of the His426 residue, to which TC-1 binds.

Furthemore, results from the interaction of ETA and eEF-2 showed that TC-1 binding is blocked when the toxin is bound to the immobilized EF-2. Since TC-1 binds to the His426 residue, these results indicate that eEF-2 binds at or near the His426 site (Kessler and Galloway, 1992).

Kessler and GaIloway (1992) proposed a more accurate mode1 for the ADPRT reaction.

It is proposed that there are two principle sub-sites within domain III; one site associated with

NADf binding and another site for eEF-2 interaction. First, the reaction requires NADC binding at the NAD+ binding site. Second. NAD+ binding alters the conformation of ETA, facilitating the interaction between the ETA- NAD+ complex and eEF-2. The ETA-NAD+ interaction with eEF-2 takes place at the eEF-2 subsite and requires the His426 residue. Subsequent to eEF-2 binding, the ADP-ribose moiety of NAD+ is transferred to eEF-2 which results in the release of nicotinamide and the return of ETA to an NAD+ uncharged state (Kessler and Galloway. 1992).

The recent crystal structure of diphtheria toxin bound to NAD+ (2.3 A resolution) (PDB accession number ITox) revealed a loop region proposed to be involved in binding to eEF-2

(Bell and Eisenberg, 1996). This loop region was found after comparing this diphtheria toxin structure complexed with NAD+ to that of uncomplexed toxin. Since this was the only signifkant structural change in the catalytic domain that was induced by NAD+ binding, it is probable that this loop is involved in eEF-3 binding (Bell and Eisenberg, 1996). The loop consisted of residues 29-46, wliich corresponds to residues 458-465 in ETA. Evidently, the crystal structure of PE24 complexed with nicotinamide and AMP (PDB accession number

I DMA) also revealed a significant conformational difference in the active site region from

Arg458 to Asp463 when compared to the crystal structure of ETA by itself (Li et al.. 1995).

Currently, Our laboratory is attempting to etucidate the role(s) of this loop as well as another loop region using site-directed mutagenesis. 13Priaciples of Fluorescence Spectroscopy

Fluorescence is the light emission process of atoms or molecules excited through the absorption of electromagnetic radiation. Upon absorbing radiation, excited species relax to the ground state. giving up their excess energy as photons. Substances which display significant fluorescence (fluorophores) typical 1y possess delocal ized electrons normally present in conjugated double bonds (Lakowicz. 1983). The absorption and emission of light can be explained in more detail using a modified Jablonski diagram (Fig. 1.5). Following light absorption. a fluorophore is excited to a higher vibrational level of either Si (first electronic state) or S2 (second electronic state) from the ground state (So) at an extremely rapid rate (10-

1%). The absorption of light by a fluorophore is very specific and a particular molecular structure absorbs light of distinct energy or wavelength. Afier reacliing the excited state. the excited molecule rapidly (10-13 to 10-1 1s) relaxes to the lowest vibrational level of Si. This energy relaxation occurs by one of two types of non-radiative means, depending on the amount of excited energy possessed by the fluorophore (i.e., the excited electronic state within which the fluorophore is located). First, the excitation energies of these fluorophores occurring in S l. S.>. etc.. are dissipated by way of interna1 conversion, which involves collisions between excited fluorophores. Second, vibrational relaxation takes place for those fluorophores in the higher vibrational levels of the lowest excited state (SI). The excitation energy in this case is dissipated by energy transfer to the extemal medium (e-g., water). The emission of radiation known as fluorescence, occurs after the fluorophore has reached this lowest vibrational level of the SI state and its transition back to the ground state. Fluorescence decay occurs at a much slower rate

(10-9 S) than non-radiative relaxation processes, which is the reason for fluorescence emission occurring only from the Iowest vibrational level of S 1 (Fig. 1.5). Therefore, fluorescence emission is of lower energy (longer-wavelength)than the initial energy absorbed and may be Vibrational Relaxation

Vibrational Relaxation

FIGURE 1.5 Modified Jablonski diagram showing some of the energy changes that occur during absorption. fluorescence, and non-radiative ret~~ation. attributed to the loss of energy in the various vibrational levels of the excited state.

1.3.1 Tslptophan Absurbance and Fluorescence in Profeins

The three aromatic amino acids. phenylalanine. tyrosine and tryptophan, possess a

system of conjugattd double bonds capable of the absorption of near-UV radiation. Proteins

typically absorb light ma.imally at 280 nm. At this wavelength, tryptophan is usually the

primary contributor to protein absorbance since its molar absorptivity (at neutral pH) is

appro'cimately 4.5 times that of tyrosine. At 295 nrn absorption, the molar absorptivity of

tryptophan is about 85 times that of tyrosine due to the broadness of the tryptophan absorbance

band. As a result, the excitation of a protein at 295 nrn wavelength of light will generally

selectively excite tryptophan only. The m~xirnumabsorption of radiation by tryptophan results

from x+n* transition of the electrons within the indole ring of tryptophan (Lakowicz 1983).

The 10 7c electron cloud of tryptophan is unevenly distributed, which results in a large molecular

dipole moment. This dipole moment is significantly changed during the transition of tryptophan

to its excited state. Thus. the energy of the excited dipole (excited tryptophan) is very

susceptible to fluorescence quenching. in particular to quenching by polar soIvents. Tryptophan

fluorescence is quenched in polar solvent through dipole-dipole interactions where the dipoles of

the solvent molecules rearrange around the excited state dipole of the excited typtophan. As a

result, an altered electron distribution occurs, causing a decrease in the energy of the tryptophan e'ccited state (Lakowicz, 1983).

The ability of tryptophan to be quenched by polar solvents makes it a suitable intrinsic

fluorescent probe sensitive to changes in protein conformation. Generally, solvent exposed

tryptophan residues in proteins have a red-shifted fluorescence wavelength ernission maxima

(A ,,mm), while buried tryptophan residues, shielded from solvent, tend to have a blue-shified

fluorescence A ,,mm. Thus, fluorescence is an excellent tool in monitoring structural changes of proteins as influenced by such factors as pH. ionic strength. substrate binding or protein- protein association.

1.3.2 7-Azafryptoplran as an Inthsic Fluorescence Probe

Tryptophan is one of the most common optical probes of protein structure and function.

However, there is one major limitation to its use in fluorescence. When a protein has more than one tryptophan residue, or when several proteins in a cornplex al1 have tryptophan residues, it becomes difficult to distinguish the contribution of a single residue to the total fluorescence emission (Laws et al.. 1995). The higher the number of tryptophan residues, the lessor is the detailed information that can be obtained about structure, function, and dynamics of the protein or protein-protein cornplex.

Recently. a new technique has been deveioped to allow the study. by tryptophan fluorescence, of a protein's interaction with other proteins that also contain tryptophan residues.

The technique involves the incorporation of spectrally enhanced tryptophan analogues into proteins. which is discussed in more detail in Chapter 2. Schlesinger (1968) was the first to study the absorbance and fluorescence characteristics of a protein incorporated with the tryptophan analogue, 7-azatryptophan (7A W)(Appendix 1 ). Her observations on 7A W- incorporated into alkaline phosphatase revealed many useful properties of 7AW as a fluorescence probe. For example. her results showed that 7AW-incorporated protein is spectroscopically distinguishable with respect to native protein and that this analogue is an extrernely sensitive probe of the local environment. Recent studies have shed more Iight on the properties of this tryptophan anaIogue. Brennan et al ( 1994) studied 7AW in the tripeptide Lys-

7AW-Lys and found that this analogue was far more sensitive to changes in pH over the range of

3-4 than tryptophan. This pH sensitivity was also seen in pH studies on the parent chromophore,

7-azaindole (7Ai)(Négrerie et al., 199 1) and its methyl derivatives (Chen et al., 1993). The additional possibiliry of protonation of the N at position 7 of the 7AI ring (which is not possible

with indole) may explain 7AW's pH sensitivity.

The use of 7AW to monitor protein structure and dynarnics relies on a good

understanding of the photophysics and photochem istry of the parent chromophore, 7A1, and has

been the subject of considerable study. Adler ( 1962) reported the re Iative fluorescence

intensities of various azindoles and noticed a low fluorescence yield of 7AI in water. The

fluorescence of 7AI in water and ethanol was examined in more detail by Avouris et al. ( 1976).

The investigators noticed that the fluorescence spectrum of 7AI in water showed one band, however the emission of 7AI in ethanol consists of two well-separated bands. temed FI

(&,rnax near 400 nm) and F2 (&,max near 530 nm). The possible photophysical pathways of

7AI in protic solvents such as water or alcohols proposed by the researchers is shown in figure

1.6. The tautomerization of 7AI involves a cyclic intermediate formed by hydrogen bonds N 1-H-

OR and N7-HOR benveen the 7AI ring and the alcohol. By comparing ernission spectrum of

7AI. Ni-methyl-7-azaindole, N7-methyl-7H-pyrrolo[3.3-b]pyridine(called N7-methyl tautomer),

Avouris et al. (1976) assigned F 1 to the normal 7AI (n-7AI) and F2 to the tautomer 7AI (t-

7AI)(Figure 1.6).

Petrich and CO-workers(199 1) attributed the single band observed in water to emission from the tautomer or some tautomer-like species. However, Chou et al. (1993) studied the emission of 7AI in watedaprotic solvent mixtures and found that with the addition of a srnaIl amount of water to polar aprotic solvents (ethyl ether and p-dioxane), they observed the growth of a distinct long wavelength band similar to the tautomer band observed in alcohols. These investigators assigned this band to tautomer emission and concluded that the single peak observed in pure water must be from the normal species. Chapman and MaronceIli (1992) found that 7AI undergoes the same tautomerization reaction in water as it does in alcohol. More Normal 7AI Tautomer 7AI

Excited State *a/ N e - *a- H - \ H \ H , - ---. \o',\8++' . -a I F 1 1 R R

Ground State $" \ Y y \ N m-m- H \ H\O""'"" H 8d888%* / I I R R

FIGURE 1.6 Photophysical pathways of azaindole (Avouris et al.. 1976). recentfy, Chen et al. ( 1993), using fluorescence Iifetime measurements. proposed that there are three types of species of 7AI in water that give rise to its fluorescence spectrum (Fig. 1.7). The researchers concluded that 20% of the excited-state 7A1 molecules comprise normal and tautomer species. while the other 80% are blocked species that are unable to tautomerize. It is clear from these solvent experiments that considerations have to be made when studying 7AW fluorescence, which include contributions from rhe following species: normal 7AW, tautomer

7AW and blocked 7AW.

7A W should prove to be a useful bioIogica1 probe not only because of its spectral shifi when incorporated into proteins. but also because of its sensitivity to the local environment.

Although weakly fluorescent in water. the 7A1 ring shows much stronger fluorescence in aprotic solvents. which suggest that 7AW may be very sensitive to its microenvironment in a protein. FIGURE 1.7 Chemical structures of the three types of species of 7AI in water: (A) normal 7AI; (B)tautomer 7AI; (C)btocked 7AI. 1.4 Rationale for studying the interaction between ETA and eEF-2

Residues such as Tyriai, HisJz6 and Hisu* in ETA have been proposed to be involved in

the interaction with eEF-2 (Lukac and Collier. 1988: Kessler and Galloway, 1992: Han and

Galloway, 1995). However, the data presented in the above studies and others are not sufficient

to describe a detailed mechanism of the transfer of the ADP-ribose moiety of NAD+ to the target

protein eEF-2. It is proposed that the properties of these residues rnay contribute to the ETA

interaction with the incoming eEF-2 diphthamide residue, stabilize the transition state

intermediate. and facilitate the transfer of the ADP-ribose moiety to the diphthamide site. For

example. Blanke et al. (1994) proposed that the primary function of DT HisZI, a funciional

homologue of HisJz6. is to fom a hydrogen bond with the nicotinamide carboxyamide of NAD+.

This interaction would facilitate formation of the DT-NAD+ complex. orient the N-glycosidic

bond of NAD+ for nucleophilic attack by the incoming diphthamide residue of eEF-2. and

stabilize the transition state intermediate of this reaction.

In order to further define the interaction of ETA with eEF-2. the approach used in this

thesis was to replace the tryptophan residues of PE24 with 7-azatryptophan. The incorporated

analogue may then be used as an intrinsic fluorescent probe to examine the structure of PE24

upon eEF-2 binding using fluorescence techniques. A study of the toxin-eEF-2 interaction and

the rnolecular events associated with its transferase rnechanism rnay provide the necessary

knowledge to control P. aeruginosa infections among immuno-cornprornised patients. In addition, a greater understanding of the rnechanism by which this toxin and others exert their

ceilular toxicity may assist in the development of dmgs to combat their toxicity. CHAPTER2 - INTRODUCTION TO THE INCORPORATIONOF TRYPTOPHANANALOGUES INTO PROTEINS

2.1 Introduction

Recently. there has been much interest in the technique of incorporating amino acid analogues into proteins. Research groups led by A.G. Szabo (Dept. of Chemistry/Biochemistry,

University of Windsor) and J.B.A. Ross (Dept. of Biochemistry, Mt. Sinai School of Medicine) have focused on replacing tryptophan in proteins with analogues of that amino acid in vivo. capitalizing on the fact that tryptophanyl RNA synthetase will catalyze tRNA acylation with a few tryptophan analogues. The main advantage of the incorporation of tryptophan analogues into proteins is that these analogues have different spectral properties with respect to those of L- tryptophan. Therefore, it is possible to selectively excite these tryptophan analogues in proteins, in the presence of tryptophan residues in other proteins or in the presence of DNA bases. Thus. protein-protein and protein-nucleic acid interactions can be studied by fluorescence spectroscopy. 22 Earty Tryptophan Analogue Experiments

Tryptophan analogues were first used in the 1950's to help elucidate the pathways by which amino acids were incorporated into proteins (Halvorson et al.. 1955). The first report of tryptophan analogues being incorporated into proteins was published by Pardee et ai. in 1956. In this study, cells of a culture of a tryptophan auxotrophic strain of E. coli were washed free of growth media containing tryptophan. The bacteria were then grown in media containing either

7AW. 2-azatryptophan (2AW) or 5-methoxytryptophan (5MW) (Appendix A). It was determined from this study that only media containing 7AW and 2AW were capable of sustaining growth. Sharon and Lipmann (1957) reported the failure of the incorporation of

5M W. 6-methoxytryptophan (6MW) and 5-hydroxytryptophan (5HW) into bovine TrpRS.

These early results lead to the premature conclusion that larger substituents on the 5 carbon of tryptophan prevented incorporation of these analogues. Later. it was determined that if protein synthesis was kept separate from celi growth, the incorporation of tryptophan analogues with substituents at the 5 position was possible. For example. Lark (1969) demonstrated the incorporation of 5MW into proteins only afier ceIl growth occurred. This growth step allowed a large number of cells containing active protein synthetic machinery to be established prior to the addition of analogue. These early experiments demonstrated that tryptophan analogue proteins could be made. however no such modified protein was purified or characterized at this time. 23Analogue-lncorporated Proteins

Schlesinger ( 1968) was the first to report on the purification and characterization of an analogue- substituted prosin. With the replacement of tryptophan residues by 7A W and ZA W into aikaline

phosphatase, Schlesinger observed very 1 ittle effect on enzymatic activity. However. there was a

large etfect on the proteins' absorbance and fluorescence properties. but only recently has the value of this result been appreciated (Ross et al., 1997).

In the 1970's. fluorinated amino acids incorporated into proteins were recognized to offer spectroscopic utitity for NMR (Sykes et al., 1974). Pratt and Ho ( 1979, citing the potential of i9F NMR to study proteins. replaced the trvptophan residues of the E. cofi enzymes lactose pemease. &alactosidase and D-lactate dehydrogenase with either 4-. 5- or 6-fluorotryptophan

(4FW. 5FW and 6FW. respectively) (Appendix A). These researchers found 4FW had little effect on enzyrnatic activity and found the analogue even increased the activity of D-lactate dehydrogenase two-fold. Pratt and Ho concluded that the effects of fluorotryptophan analogues on individual enzymes could not be predicted due to the variability in the results observed.

The first reference to the use of tryptophan analogues to study protein structure and function using fluorescence was made by Hudson and coworkers (t 986). They suggested that azulene and benzo-b-thiophene (Appendix A) amino acid derivatives may be substituted for tryptophan and used as fluorophores. However. the strategies they proposed were never undertaken. Petrich and coworkers have recognized the importance of incorporated 7AW as an ideal in situ probe of protein structure and dynarnics (Negrerie et al.. 1990). Aithough Petrich and coworkers published a series of papers dealing with the photophysics of 7AW (Chapter I ) and 7-azaindole (7AI), they did not conduct any studies dealing with 7AW-incorporated proteins.

The incorporation of tryptophan analogues into proteins for the study of protein structure and function using fluorescence was fint demonstrated independently by Szabo and coworkers (Hogue et al.. 1992) and Ross and coworkers (Ross et al., 1993). Szabo and coworkers

incorporated SHW into a Tyr-57 to tryptophan mutant (Y57W) of the calcium binding protein, oncornodulin. Using a different expression system, Ross and coworkers demonstrated that 5HW

could also be successfully incorporated iiito the bacteriophage /I cl repressor protein. The term

"spectrally enhanced" protein was coined for such analogue-containing proteins. Since then, over a dozen different spectrally enhanced proteins have been made (Table 2.1). Szabo and coworkers have incorporated SHW and 7AW into rat parvalbumin and Bacillus st~btilis tryptophanyl tRNA synthetase (Hogue. 1994: Hogue et al., 1995). Similarly. Ross and coworkers have incorporated 5HW into insulin (Laue et al.. 1993). Also, Heyduk and Heyduk

( 1 993) have incorporated 5HW into CAMPreceptor protein and Soumillion et al. (1 995) replaced the tryptophan residues in phage lambda lysozyme with 7AW. More recently, Wong and Eftink

( 1997) incorporated both 7A W and 5HW into staphylococcal nuclease.

The early experiments demonstrated the potential for incorporating analogues into proteins. However. the unpredictability of the degree of analogue incorporation was likely a factor in the slow growh in popularity of this method (Hogue. 1994). Early studies of analogue incorporation with systems that had natural inducible promoters were successful. for example. alkaline phosphatase (Schlesinger. 1968: Sykes et al., 1974) and aspartate transcarbamylase

(Foote et al. 1980). In these systems. the toxicity of analogues was circumvented by first growing cells in media containing the natural amino acid, while the expression of these proteins were repressed. Afier sufficient ceil growth, analogues were added and expression was induced, which resulted in high levels of incorporation (> 85%)(Ross et al., 1997). The development of highly efficient protein expression systems in the 1980's. made available expression vectors with artifrcial inducible promoters for many proteins. This made it possibIe to achieve eficient incorporation of tryptophan analogues in vivo and allow the study of protein-protein and pmtein- TABLE 2.1 Proteins expressed with tryptophan analoguesa.

Protein # of Promoter Percent Analogue Activity Trps Incorporation 5HW 7AW 4FW Rat oncomoduIin (Y57W) 1 OXYPRO <50 wild type h CI repressor 3 tac 95 wild type CAMPregulatory protein -3 APL 50-90 wild type u subunit RNA polymerase -3 T7 50-90 wiId type 1 1 mutants of W32 1 F 2 T5 >95 wild type (W260.. .. , W270) o subunit RNA polymerase 4 T7 50-60 wild type Cytidine repressor (M 15 1 W) 1 T7 30-50 wild type B iotin repressor 7 tac 85 inactive Soluble human tissue factor 1 tac (20 <30 ? Trp tRNA synthetase 1 tac >95 >95 >95 altered Herpes virus protein VP 16 1 tac 50-95 50-95 wild type W443. W473 MyoD 1 T5 >95 >95 wi Id type Rat parvalbumin F102W 1 T7/pLysE ~50 =50 z50 wild type TBP 1 T7 30-50 ? Staphy lococcal nuclease A 1 temp. >95 >98 wild type sens. h cl repressor h phage lysozyme 4 ? 98 ? a modified from Ross et al., 1997 nucleic acid with these intrinsic probes (Ross et al.. 1997).

2.4 Methods for Incorporation of Tryptophan Analogues into Pro teins

There are a number of methods that can be used to incorporate probes into proteins.

Extrinsic probes have been introduced into proteins by chemical modification and semi-synthetic

methods. Chem ical modification have been used to introduce spin-labels, fluorescent molecules and photoactivatable cross-linking agents ont0 the reactive side chains of amino acids such as

lysine and cysteine (Bmnner. 1993). However. this approach can be complicated by the presence of several reactive side chains in the protein.

Semi-synthetic methods have been used to site-specifically incorporate probes into proteins. In this method, a synthetic peptide containing the probe is ligated to a protein fragment to produce a full-length protein (Offord, 1987). However. this method may be diftlcult to perfbrm because of the need to cleave the protein specifically at the peptide ligation site, and the diftlculties in coupling the protein and peptide termini selectively (Cornish et al.. 1995).

Alternatively. hvo biosynthetic methods of introducing fluorescent probes have been developed. The first method involves a ce1 1-free expression system (in virro transcription/translation using a nonsense suppresser tRNA that is chemically acylated with a non-naturai arnino acid). The second method. which is employed in this study. uses a whole-ce11 expression system in vivo.

2.4.1 Incorpora!ion of Tryptoplran Analogues in vitro

Shultz and coworkers (Noren et al., 1989; Cornish et al., 1995) and Chamberlin (Bain et al., 1989) have used a general hiosynthetic method to incorporate non-natural amino acids into proteins in a site specific manner (Fig. 2.1 j. This method involves the replacement of the codon for the naturally occurring amino acid of interest with a nonsense (stop) codon (usually the amber codon, UAG) by conventional site-directed mutagenesis. The amber codon is not recognized by any of the comrnon tRNAs involved in protein synthesis. However. an amber suppresser tWA can recognize this nonsense codon and can append its amino acid to a growing polypeptide in response to the recognized stop codon. Thus, a non-natural amino acid can be incorporated site-specifically (if it is tolerated by the ribosomal machinery) into the protein of interest (Ellemen et al.. 199 1 : Cornish et al., 1995).

There are a few factors essential to the success of this approach. First, this method rnust be performed in a cell-free, in vitro transcription-translation system. Since there is no general tnethodology that exists for introducing large quantities of the aminoacylated suppresser tRNA into intact dividing cells, proteins are synthesized in virro (Cornish et al.. 1995). This system consists of an E. coli S-30 extract, which contains al1 the proteins and RNAs required for transcription and translation.

Second, the suppresser tRNA must not be recognized by any of the normal tRNA synthetases in the in vitro translation system. If the suppresser tRNA is recognized by any of the aminoacyl-tRNA synthetases, the appended non-natural amino acid could be removed or replaced by one of the common twenty amino acids. The suppresser tRNA derived from yeast tRNAPhc. with specific base changes, has been found to function efficiently with E. cali or rabbit reticulocyte lysate translation systems (Cornish et al., 1995).

Finally, the suppresser tRNA with the appended non-natural amino acid must be chemically synthesized. A method originally developed by Hecht and coworkers (Heckler et al.,

1984) has been used in which the dinucleotide pdCpA is chemically acylated with a Na- protected amino acid. The pdCpA-amino acid is then enzymatically ligated to a tRNA that is missing the terminal dinucleotide pdCpA at the 3'-acceptor stem. Codon for residue of interst Nonsense codon +-'TGG + Oligon ucleotide-directed mutagenesis

Ligase

Chernical Synthesis J 3'C

Mutant Enzyme with non-natural amino acid site-specificaiiy iacorporated i1?AUC Suppressor tRNA- (am ber)

FIGURE 2.1 The strategy for in vitro biosynthetic incorporation of non-natural amino acids into proteins. A, adenosine; C, cytidine; G, guanosine; T, thymidine; U. uridine (adapted from Ellemen et al., 199 1 ). A major limitation of the in virro approach is that protein yields are low. Whether the protein is synthesized using an E. cofi translation system or from a rabbit reticulocyte translational system. protein yield is generaily lower than 100 pg (Comish et al., 1995).

Recently, large quantities of aminoacylated suppresser tRNA and mRNA have been coinjected into single .Yenopus oocytes (Nowak et al.. 1995). Whether this in vivo incorporation in intact cells can eventually increase protein yieid remains to be seen. The in vitro incorporation does hold one major advantage over in vivo incorporation. which is that a non-natural amino acid can be placed at any single site in a protein.

Currently, only a few proteins have been incorporated with tryptophan analogues using the in virro approach. Comish et al. (1994), has reported the site-specific incoïporation of 7AW into bacteriophage T4 lysozyme. More recently, 7AW. 5HW and E-dansyl lysine have been incorponted into Pgalactosidase at a single designated site (Steward et al.. 1997). Both reports demonstrate the possibility of incorporating tryptophan analogues site-specifically into proteins. with minimal structural perturbations.

2.5 Incorporation of Tryptophan Analogues in vivo

Ross et al. (1997) describe two procedures for the in vivo biosynthetic incorporation of tryptophan analogues into recombinant proteins by using plasmids in E. coli host ceiIs. The first rnethod involves a two-step procedure: first, a growth step prior to expression in media containing tryptophan and second. a protein expression step requiring media that does not contain tryptophan. Instead, a tryptophan analogue is added to the media followed by induction

(Heyduk and Heyduk. 1993). The second method involves a single-step process, where cells are cultured overnight, followed by the addition of the analogue and induction. 2.5.1 Two-step method of incorporation

Trp analogues are toxic to bacterial celfs (Ross et al., 1997). The toxic effects of

analogues may be overcome by first growing the tryptophan auxotroph in rich media, which

iillows a large number of cells containing active protein synthetic rnachinery to be established

prior to the addition of the analogue. Thus, cells do not replicate in the presence of these toxic analogues, but instead in the presence of natural tryptophan.

When an appropriate optical density of the culture is reached, the cells are harvested by centrifugation. A washing step may be added at this point. that is, the pellet is resuspended in water and centrifuged again. This washing step reduces tryptophan availability to the ceIl and ensures that less tryptophan is incorporated into the protein of interest. The cells are then resuspended into the expression media (Section 2.5.4) and incubated for 30 min to deplete any residual tryptophan in the culture. The tryptophan analogue is then added to the culture and after

10 min, the culture is induced. The protein rnay then be purified.

2.5.2 Single-step met/.od of incorporation

The single-step method involves exhausting the available tryptophan without replacing the culture medium prior to induction of the cells. It has been suggested that the single-step analogue incorporation procedure is necessary when a T7 RNA polymerase promoter is used in conjunction with the plasmid pLysE (Ross et al., 1997). This requirement is due to the presence of T7 lysozyme in these strains. If a small proportion of cells are damaged. for example during the centrifuge step in the two-step incorporation procedure, this would cause T7 lysozyme to be released. As a result. rapid lysis of the culture may occur (Ross et al., 1997). 2.5.3 E. coiî Tryptophan Auxotroph and Fxpression System

A prerequisite for the in vivo approach requires a plasmid-borne bacterial expression system that has been deveioped for the protein of interest. The plasrnid must be compatible for expression by a tryptophan auxotroph. The strain of E. coii auxotroph setected will depend on the prornoter system of the expression vector. The E. coli tryptophan auxotrophic strains

CY 15077 (W3 1 10 [naAl AfrpEAZ), W3 1 1O TrpA33 and W3 1 10 TrpA88 have been transformed successfully with a variety of expression vectors. Other E. coli strains auxotrophic for tryptophan may be used.

Thus far, only a few different prornoter systems have been used. These include roc, T7,

T5, and temperature- and oxygen-sensitive promoters. Although this method is still in its early stages, it seems that the efficiency of incorporation varies from promoter to promoter. To date, the lac and T5 promoters have produced the highest analogue incorporation (Table 2.1). In contrast, use of the T7 polymerase promoter has generally resulted in poor analogue incorporation (Ross et al., 1997).

2.5.4 Media used for Analogue Incorporation

In the two-step analogue incorporation. the growth of cells prior to expression requires tryptophan-containing media. LB medium, prepared according to Sambrook et al (1989) has been used. Alternatively, M9 minimal medium supplemented with 0.5% glucose, I % casein acid hydrolysate (casamino acids), 0.1% thiamine and 0.25 mM tryptophan may be used (Ross et al.,

1997). Also, Neidhardt's defined medium (Neidhardt et al., 1974) with supplements of the 20 amino acids, thiamine-HCI and biotin have been used (Wong and Eftink, 1997).

During the protein expression step, a growth medium that does not contain tryptophan is needed. M9 minimal medium supplemented with 1% casamino acids may be used (Sambrook et al., 1989). Casamino acids do not contain tryptophan since the condition of acid hydrolysis destroys tryptophan during preparation. in addition, the M9 minimal media needs to be

supplemented with the following sterile solutions per liter of media: 2 mL of IM MgS04, 0.1

mL of IM CaC12, and 1 mL of 100mg/mL thiamine (filter sterilized). Finally. the tryptophan

analogue is added to the minimal media at a final concentration of 0.25 mM for the L-amino acid

form or 0.50 mM for the DL-amino acid fom of the analogue. The analogue may be dissolved

in sterile water prior to addition into media. Altematively, the analogues may be directly added

into the media in dry form (Ross et al.. 1997).

The growth medium for the single-step analogue incorporation is simitar to that used during the protein expression step in the two-step method. However, 2% casamino acids and twice the recommended amount of carbon source (glucose or glycerol) is needed. In addition. a

final L-tryptophan concentration of 0.02 mM is added (Hogue et al.. 1992). The cells may then

be grown overnight in this medium. The above concentration of tryptophan will eventualty Iimit the growth of the cells and this can be checked by measuring the optical density of the culture and then again an hour later. Once confirmation that ceIl growth has leveled off, the tryptophan analogue solution can then be added to the culture as described above (Ross et al., 1997). CHAPTER3 - BIOSYNTHETICINCORPORATION OF 7- AzATRYPTOPHAN INTO P~24

3.1 Introduction

The objective of this study was to develop a method to prepare tryptophan analogue- incorporated toxin (PE24) to study the protein-protein interaction between toxin and eEF-2.

Normally, studies of protein-protein interactions using tryptophan fluorescence spectroscopy would be impaired by the presence of one or more than one t~ptophanresidue in the proteins being investigated. For example. eEF-2 contains seven tryptophan residues and PE24 possesses three tryptophan residues. Sontag et al. ( 1993), showed that excitation of rat liver eEF-2 at either

280 nrn or 295 nm resulted in an emission spectrum with a fluorescence h,,rnax of 333 nm.

Excitation of PE24 at 295 nm resulted in an emission spectrurn with a fluorescence &,ma near

333 nm (335 nm) (Fig. 3.9). This clearly demonstrates that an emission spectntm of a PE24- eEF-2 mixture would be difficult, if not impossible, to resolve. However, by use of an intrinsic fluorescence probe such as 7AW. with an absorption spectrum that would allow its selective excitation in the presence of L-tvptophan. and a fluorescence spectrum that does not significantIy overlap with L-tryptophan. it becornes possible to study protein-protein interactions without the aforementioned spectral interference. in order to better understand the conformational changes in PE24 necessary for docking with its natural substrate, eEF-2. the three naturally occurring tryptophan residues found in PE24 were replaced with 7AW and the resultant protein was characterized by absorption and ernission spectroscopy using tryptophan analogues as intrinsic fluorophores. 3.2 Materiab and Methods

3.2.1 Uverevpression and Purification of Wild-îype PE24

The established procedure for the expression and purification of PE40 was utilized for

PE24 (Beattie and Menill, 1996). For the expression of PE24. the plasmid. pPEA5-399. was used (Fig. 3.18). which is a derivative of the plasmid used for the expression of ETA. pVC45f(+)T (Fig. 3.IA). pPEA5-399 was obtained from the laboratory of Dr. I. Pastan

(Laboratory of Molecular Biology, National Cancer Institute. Bethesda Maryland) and encodes arnino acids 1-4, followed by 400-613 of ETA and preceded by an N-terminal methionine (Fig.

1.1 ). pVCJSf(+)T carries the DNA sequence encoding al1 three domains of ETA and pPEA5-399 and was prepared by genetically deleting dornains Ia, II and Ib of pVC45f(+)T (Li et al.. 1995).

The gene for PE24 in pPEd5-399 is located downstream from a isopropyl PD- th iogalactopyranoside (IPTG) inducible T7 promoter. a ri bosorne binding site. and a sequence encoding an OnpA signal sequence. Afier protein synthesis. the OmpA signal sequence ensures that PE24 is secreted into the periplasm. This sequence is followed by a T7 transcription terminator. which prevents read through. and an fl phage origin of replication which allows the production of single stranded DNA for use in site-directed mutagenesis and sequencing (Fig.

3.1 )(Jinno et al.. 1989: Pastan et al.. 1992). The presence of an ampicillin (Amp) resistance gene allows for selection of cells afier transformation of the plasmid.

pPEd5-399 was transformed into and overexpressed in an E. coli lysogenic ceIl line,

BL21 (XDE3). obtained from Novagen (Madison, WI). One rnicroliter of this plasmid was transformed by heat shock (Brent, 1992) into competent BL2 1 (hDE3) cells and plated ont0 four

2xYT medium plates containing 100 pg/rnL Arnp and incubated overnight at 37 OC. The next day. each plate was scraped into 50 mL of super L-broth (Appendix B). The cells were grown for 1 hr at 37 OCand 10 rnL of this culture was transferred to each of four 1 L cultures. These MAEEA

DNA regions encoding for:

ETA Binding Domain 0)

FIGURE 3.1 The structure of ETA (A) and PE24 (B) expression vectors. The backbone of both vectors consists of a T7 promoter, a ribosome binding site, an OmpA signal sequence, ETA (or PE24) structura1 gene, a transcription term inator (TT), fl filamentous phage origin of rept ication (f(+)),and an am picillin resistance gene (Amp). The translation of PE24 begins with Met and then the first four residues of ETA (Ala-Glu-Glu-Ala) followed by residues 400-6 13 of ETA (Li et ai., 1995). cultures were grown to 0.8-1.0 0D6s0 at 37 OC after which the cells were induced with ImM

IPTG and grown for a further 90 min.

The cells were harvested by centrifuging the 4 L of culture at 1000 x g for IO min. PE24 was then isolated from the periplasm according to the method developed in Our laboratory

(Rasper and MerriIl. 1994). This protocol involves an osmotic shock procedure where each pellet was resuspended in 800 mL (total) of sucrose solution (20% sucrose. 30 rnM Tris-HCI, 1 mM EDTA. pH 7.4) for 10 min. After hornogenization. the suspension was then centrifuged at

1000 .u g for IO min.

The periplasmic fraction containing PE24. now in the supematent. was pooled and loaded ont0 a Q-Sepharose Fast-Flow anion exchange colurnn (Pharmacia-LKB. PQ). previously equilibrated in 20 mM Tris-HCl. pH 7.6 (equilibration buffer). The column was then washed with 500 mL of equilibration buffer and proteins were eluted by a salt gradient. Here, 60 mL of

O. 15 M NaCl in the above buffer \vas used first, followed by 60 rnL of 0.4 M NaCI in the same buffer. Fractions containing PE24 were pooled and dialyzed against equilibration buffer.

The dialyzed sample was concentrated to 5 mt using an Arnicon Centriprep concentrator

(Amicon Inc., Beverly. MA). The sample was then loaded on a 1.25 cm x 3.7 cm HPLC column,

Q-Sepharose HP (Pharmacia-LKB, PQ) and eluted with a linear gradient of NaCl (O - 0.5 M in equilibration buffer. pH 7.4; 1 mL!rnin flow rate) over 120 min. Fractions containing PE74 were pooled. dialyzed and concentrated to 5 mL and the HPLC step repeated at pH 7.2. The purified

PE24 was concentrated to approximately 2 mg/mL, filtered through a 0.2 pm Acrodisc membrane filter (Gelman Science, Rexdale, ON), and the protein concentration was determined by absorbance using ~~280of 2.73 1 x 104 M-km-1. calculated according to the method of Gill and von Hippel (1 989). The protein was dispensed into small aliquots and frozen at -80 OC.

Protein purity was assessed using 12.5% SDS-PAGE and was identified by Western Blot analysis using a polyclonal antibody to whole toxin, ETA (Sigma. St. Louis, MO). 3.2.2 Construction of Tryptcrplzan Auxotroph

The selection of an E. coli auxotrophic host strain wilI depend on the promoter system used (Chapter 2). In our case, it was desirable to express PE24 in the E. cofi Iysogenic cell line,

BL2 1 (kDE3) as already described. However, to Our knowledge. no E. cofi lysogenic ceIl line exists that is auxotrophic for tryptophan. Thus, strain BL21 (lDE3) that is auxotrophic for tryptophan had to be constructed. A generalized transduction mediated by phage P 1 was used in auxotroph construction (Schleif and Wensink 198 1) (Fig 3.2).

3.2.2. t Preparation of Pl Lysogens

The tryptophan auxotroph strain NK7402 (A-.trpB83:: Tn i O. IN(rrnD-rrnE1 i)(E. coli

Genetic Stock Center. Yale University. New Haven, CT) was grown overnight in 2 ml of L-broth

(Appendix B). The next day. 100 pl of 1OOmM CaClr was added to the ovemight culture. After mixing well, 0.1 ml of the ovemight culture was added to 0.1 ml of P 1 lysate (P 1. a gifi from Dr.

J. Wood. Dept. of Microbiology, University of Guelph, carries chloramphenicol (Cam) resistance) for 10 min. The culture was than streaked For single colonies on LB plates containing

Cam (15 pg/mL). Next. a single colony was picked and grown overnight at 30 OC in 5 ml of L- broth containing Cam (25 pg/mL).

3.2.2.2 Preparation of Lysate

The next day, I ml of the overnight culture was added to 9 ml of L-broth containing

1 OmM MgSOj in a 125 ml flask. The cells were grown with shaking at 30 OC to an OD65o of

0.8. At this tirne. the temperature was shifted to 42 OC and the cells grown for 35 min with shaking. Afier 35 min. the temperature was shifted to 37 OC for 60 min. The 42 OC incubation causes loss of repression of P 1 phage DNA, phage DNA replicates and is packaged. The 60 OC temperature with the MgS04 added earlier, causes ceIl lysis. FIGURE 3.2 Schematic for the construction of the tryptophan auxotroph, BL21 (hDE3)/NK7402. by Pl transduction. (i) Pl phage (Camr) was added to a culture of the tryptophan auxotroph, NK7402 (contains the Tetr transposable eIement, TnlO) and incubated overnight; (ii) a single colony was grown to an ODasoof 0.8, after which the temperature was shifted to 42 OC; (iii) the shifi in temperature causes loss of repression of Pl phage DNA and progeny phages begins to replicate; in addition, bacterial DNA breaks apart and is packaged into some phages to become transducing phages; (iv) ce11 lysis occurs and P 1 lysate is collected; (v) Pl Iysate was used to infect the recipient strain BL21 (hDE3); (vi) transductants were streaked for Tetr and checked for tryptophan auxotrophy. 3.2.2.3 Tra~tsducfionwifh PI Lysates

The recipient strain, BL2 1 (IDEj), was grown for transduction overnight at 37 OC in 5

ml Li3 broth. The cells were then centrifuged (10 min, 10,000 rpm) and the supernatant was

decimted. After resuspending the ce11 pellet in 5 ml of MC medium. the cells were incubated at

37 OC for 15 min. Next. 100 pL of the suspended cells were added to each of 5 test tubes. To

the first tube, 100 PL of the Pl Lysate (prepared above) was added. Ten-fold dilutions of the

lysate were added to the second, third and fourth tubes, respectively. Nothing was added to the

fifth tube, which served as a control. Finally, in a sixth tube. 100 PL of the lysate was added (no

cells) as another control. After incubating al1 tubes at room temperature for 20 min. phage

adsorption was stopped by the addition of 200 pL of 1M sodium citrate (pH 7.0) to each tube.

LB broth (400 pL) was added to each tube and incubated for 1 hr at 30 OC to allow for the

expression of the Tet phenotype of the auxotroph. Next. 3 mL of pre-warmed top agar (45 - 50

OC)was added to each tube and plated ont0 LB plates containing Tet (1 O kg/rnL). Afler the agar

gelled, the plates were incubated overnight at 37 OC. The colonies (transductants) were streaked

ont0 Tetr plates to get rid of any background expression. The transductants were then streaked

ont0 MOPS media (Appendix B) (Sarnbrook et al., 1989) in plates containing (1% L-tryptophan)

and plates lacking tryptophan, to test for tryptophan auxotrophy. Transductants (herein called

BL2 1 (hDE3)MK7402) were rendered competent by the CaCI->rnethod (Silhavy et al., 1984).

3.2.3 Mini-prep Testfor Overexpression of 7A W-incorporated PE24

To determine if our expression system was suitable for analogue incorporation and to see

if we could obtain a useful yield of protein, a mini-prep test for overexpression was fint

conducted. One rnicroliter of the plasmid. pPEA5-399, was transformed into competent BL21

(hDE3)/NK7402 cells and grown on a ZxYT medium plate containing 100 pg/mL Amp and 10

pg/mL Tet and incubated overnight at 37 OC. Cells from the transformation were scraped into 5 mL of super L-broth containing the above antibiotics. Afier growing the cells at 37 OC to a high cell density ( about 2 h), 1 mL of cell-containing growth medium was transferred to a 25 mL culture of the sarne medium as above.

The culture was grown at 37 OC until an OD650 of 0.5 was reached. The cells were then

harvested by centrifugation (1 000 x g for 10 min) and the supernatant was decanted.

The cell pellet was then resuspended in M9 media (Appendix B) supptemented with

0.5% glucose. 1 % casamino acids and 0.1 % thiamine. and grown for a further 20 min. The 7A W

(DL-forrn) (Sigma) was added to the minimal medium at a final concentration of 0.50 mM and the cells induced with 1 mM IPTG. At the same tirne. 500 PL of the cell-containing media was placed in a 1.5 mL eppendorf tube and centrifuged. The supernatant was removed and the ce11 pellets placed on ice. Subsequently. 500 pL of the cell culture was collected every hour for 3 hours and centrifuged. Each ceIl pellet was then resuspended in Laemmli buffer (Laemmli,

1970) and placed in boiling water for 1 min to lyse the cells. Each tube was then placed in a sonicator for 1 min and the ceII contents Ioaded ont0 a 12.5 % SDS-PAGE gel.

3.2.4 Expression and Purification of 7AW-incorporatecl PE24

The overexpression and purification of PE24 containing 7AW was essentially as described for wild-type PE24 and in the mini-expression test. However, after the first HPLC step with the Q-Sepharose HP column, the pooled and concentrated (2mL) sample was applied to a 2.5 cm x 60 cm HPLC Superose 12 gel filtration column (Pharmacia-LKB. PQ). previously equilibrated in 20 mM Tris-HCI. 50 mM NaCl, pH 7.6. The proteins were eluted with this equilibration buffer at lmt/rnin flow rate for 180 min and the purified protein was concentrated to approxirnately 2 mg/mL. filtered and the protein concentration determined by absorbance. 3.2.5 AD PR T Assay

Samples were assayed for ADP-ribosyltransferase activity by using a newly developed fluorometric ADPRT assay based on the work of Klebl and Pette ( 1996). The reaction medium for assessrnent of ADPRT activity contained 20 mM Tris-HCI, 50 pg/mL BSA, pH 7.4. O - 600 pM IJW-etheno NAD (ENAD; Sigma) and 14 pM eEF-2 in a total volume of 70 PL in a 3mm quartz cuvette. Afier 5 min of equilibration of the above mixture, the reaction was started by the addition of 23.8 nM of 7AW-PE24 or 1.4 nM of wiId-type PE24 and the progress of the ADPRT reaction was rnonitored by excitation at 305 nm and fluorescence emission at 4 10 nm. for 10 min. The EADP-ribosy 1-eEF-2 produced was quantified by cal ibration with CAMP (Sigma), which exhibits the same fluorescence quantum yield as EADP-ribose.

Absotbance spectra were obtained at room temperature with a Perkin-Elmer À6 double beam. scanning absorption spectrometer (Perkin-Elmer. Norwalk, CT) interfaced to a persona1 cornputer. Al1 absorbance spectra of proteins and amino acid mode1 compounds were scanned from 250 to 360 nm (2 nm slit width) in Tris-HCI buffer. pH 7.6. Fluorescence emission spectra were recorded on 3 cornputer-controlled PT1 Alphascan-2 spectrofluorometer (Photon

Technology Inc., South Brunswick, NJ). Fluorescence emission spectra of wild-type PES4 and

7AW-containing PE24 were scanned from 3 IO to 450 nm (excitation at 295 nm, 4 nm excitation and emission bandpass) and measured in 20 mM Tris-HCI buffer. 50 mM NaCl pH 7.6 at 20 OC.

Wavelength dependent bias of the optical and detection systems was corrected and appropriate blanks were su btracted. 3.2.7 Anuiysis of Analogue Incorporation Using Absorbunce Spectra

To assess the degree of analogue incorporation. the absorption spectrum of 7AW- containing PE24 was analyzed by sumrning the expected contributions of the model cornpounds

N-acetyltryptophanamide (NATrpA), N-acetyltyrosinarnide (NATyrA) and f-boc(a-amino)-7AW tryptophan (a gifi from Dr. J.B.A. Ross, Dept. of Biochemistry, Mount Sinai School of Medicine.

City University of New York. NY). This was perfonned by first obtaining basis spectn for these model amino acids in 6 M guanidine hydrochloride (Gn-HCI) in 20 rnM Tris-HCI, 50 mMNaCl buffer (pH 7.6). The absorbance spectrum of 6 M Gn-HCI denatured 7AW-PE24 was then fitted. between 270-350 nrn. with the bais set spectra using the fitting program, LNCS (linear combination of basis spectra, obtained frorn Dr. J.B.A. Ross) as described by Waman et al.

(1993). To avoid overlap with the absorption of phenylalanine residues. we did not fit the protein spectrum at wavelengths shorter than 270 nrn.

In the LINCS analysis program, individual absorbance spectra were fit to the relationship

AT(À) = ZU~~(À) where AT(h) is the absorbance of the protein at each wavelength A, Ai(h)are the absorbances of the basis spectra and u; is the relative molar arnount of each basis spectnim in the protein spectrum (Ross et al.. 1992). The relative molar amounts of each basis spectrurn (relative scaling factor) were determined from analysis of the absorbance spectra of hvo polypeptides, adrenocorticotropin and glucagon, each containing one tryptophan and two tyrosine residues

(Appendix C) (Wmrnan et al.. 1993). The quality of the fit was evaluated by visual inspection and from the residual plots of the fitted data sets. 33 Results and Discussion

3.3.1 PI Transduction

PI is widely used by bacterial geneticists to incorporate bacterial genes into the phage genome and to transduce recipient strains. When the packaged host DNA is injected into the recipient bacteria, BLZl (hDE3). the DNA become stably integrated into the recipient cell's chromosome by homologous recombination (Schleif and Wensink 198 1 ). Specifically, the double-stranded segment of the recipient's chromosome is replaced by the homologous double- stranded segment from the transducing fragment. This event requires hvo crossovers, one at each end of the replaced segment. The tryptophan auxotroph, BL21 (lDE3)/NK7402, was demonstrated to have tryptophan auxotrophy (data not shown). This tryptophan auxotroph showed good growth in media supplemented with tryptophan. but no growth on tryptophan deficient plates (data not shown).

3.3.2 Expression of 7A W-incorporatedPE24

The tryptophan auxotroph was tested for IPTG-inducible expression of 7AW- incorporated PE2J (7AW-PE24) and the results are shown in Figure 3.3. The expression of

7AW- PE24 was seen one hour afier induction (lane 7) with expression leveling off afier three hours of induction (lane 9). Close inspection shows bands corresponding to the molecular weight of PE24 prior to induction (lane 6) which would suggest that this promoter is not completely silent in the absence of inducer (Le., basal expression of protein was evident prior to induction). This implies that a fraction of PE24 protein pool incorporated L-tryptophan instead of the analogue. during growth. The extent of "gene leakage" was characterized later by spectroscopy. FIGURE 3.3 Mini-prep test for protein expression of wild-type PE24 (Lanes 1-4) and 7AW- PE24 (Lanes 6-9). (Lanes 1 and 6) cells prior to induction, (Lanes 2-4 and 7-9) represent 1, 2 and 3 hours afier induction, respectively. (Lane 5) molecular weight standards: (Daltons, top to bottom) phosphorylase b (97.400), bovine semm albumin (66,200), ovalbumin (42,6991, carbonic anhydrase (3 1,000), soybean trypsin inhibitor (21,500) and lysozyme (14,400). The arrow indicates the position of the PE24 protein. SDS-PAGE gel was stained with Coornasssie brilliant blue stain. In chapter 2. it was mentioned that the single-step analogue incorporation method is used

when a T7 RNA polymerase promoter is employed in protein expression. This is to prevent any

T7 lysozyme from being released during the centrifugation step in the two-step

incorporation procedure. which may result in rapid lysis of the culture. However. since we have discovered that the optimal optical density of our culture prior to induction was 0.5 and that an ovemight growth of culture (necessary in the single-step method) would have caused poor expression of our protein. the two-step analogue incorporation procedure was utilized.

3.3.3 Pur~jïcationof 7A W-incorporated PE24

Purification of 7AW-PE24 followed the procedure described in sections 3.2. I and 3.2.4.

Afier extraction of proteins from the periplasm, the periplasmic extract was separated using Q-

Sepharose Fast-Flow anion-eschange resin eluted by two NaCl elution steps (O. 15 and 0.4 M). A typical chrornatographic profile of this step is seen in Figure 3.4A as detemined by UV absorption at 280 nm. Two peaks were seen, with the majority of the analogue-containing protein eluting in the second peak. Fractions 50-54 were found to contain 7AW-PE24 as determined by SDS-PAGE. Afier pooling, dialyzing and concentrating the fractions, the protein solution was purified by HPLC anion-exchange chromatography (Fig. 3.48). SDS-PAGE revealed that fractions between 4 1-44 and 7 1 - 106 min contained 7AW-PE24 and these fractions were pooled and dialyzed. Figure 3.5 shows the HPLC gel filtration step following anion- exchange chrornatography and revealed that fractions eluting between 90-95 min contained the purified 7AW-PE24 (Fig. 3.5). The isolated protein was subjected to 12.5% SDS-PAGE and determined to be at least 90% pure (Fig. 3.6).

Wiid-type PE24 was purified according to section 3.2.1. The yields of both wild-type

PE24 and 7AW-PE24 were noticeably different. Typical yields for both wild-type and 7AW- containing PE24 were 7- 1 O mg and 0.7-1 mg, respectively, starting from a 4L culture of medium. I 1 I I 1 O 20 40 60 80 Fraction No.

Time (min)

FIGURE 3.4 Chromatographie purification of 7AW-PE24. (A) Q-Sepharose Fast-Flow anion- exchange elution profite of the separation of 7AW-incorporated PE24 from the periplasmic extract. Contaminating proteins were eluted with 0.15 hl NaCl into 40, 1.3 mL fractions followed by elution of PE24 and other contaminating proteins with 0.4 M NaCl into 40, 1.5 mL fractions. (B) HPLC elution profile of the purification of 7AW-incorporated PE24 using a Q- Sepharose HP anion exchange column. A linear NaCl gradient (O - 0.5 M, pH 7.4. 1 mL/min flow rate for 175 min) was applied to elute the protein. FIGURE 3.5 Superose 12 gel fikation elution profile for the purification of 7AW-PE24. Gel filtration was performed in a 20 mM Tris-HC1, 50 mM NaCI, pH 7.6 buffer at 1 mL/min flow rate for 180 min. FIGURE 3.6 SDS-PAGE of purified 7AW-PE24 (arrow). (Lane 1) molecular weight standards: (Daltons. top to bottom) phosphorylase b (97,4001, bovine serum albumin (66.200), ovalbumin (42.699), carbonic anhydrase (3 1,000). soybean trypsin inhibitor (21.500) and lysozyme (14,400); (Lane 2) purified 7AW-PE24 (15 ug). SDS-PAGE gel was stained with Coomasssie brilliant blue stain. Thus, we observe a ten-fold reduction in protein yield when 7AW is incorporated into PE24.

This is inconsistent with the findings from other incorporation studies. For example, Hogue

( 1994) only observed a hvo-fold decrease in analogue-containing protein recovered using a tac

promoter. Wong and Eflink (1997). using a temperature sensitive A cl repressor. received

comparable yields in their analogue-substituted protein with respect to wild-type protein.

The T7 polymerase promoter expression system used in the incorporation of 7AW into

PE14 may account for the reduction in protein yield observed in this study. The T7 promoter

requires newly synthesized proteins for translation, for example. T7 RNA polymerase (Studier et al., 1990). Expression of proteins is induced by providing a source of T7 RNA polymerase in the

host cell. which in our case, is the tryptophan analogue, BL2I (hDE3)/NK7402. This auxotroph, originally derived from BL21 (hDE3), contain a chromosomal copy of the T7 RNA polymerase gene under the control of the inducible lacUV5 promoter. Addition of IPTG to a growing culture

induces the polymerase. which in turn tranccribes the target DNA in the plasmid (Studier et al.,

1990). If an essential tryptophan residue of T7 RNA polymerase or any of the other proteins required for translation is replaced by an analogue. function and therefore expression may be compromised (Ross et al.. 1997). Thus far, the T7 polymerase promoter has not proven to be efficient for analogue incorporation (Table 3.1).

A new assay for diphthamide-specific ADPRT activity determination was developed in

Our laboratory, based on a existing assay for NAD' glycohydroIase (Perkin and Anderson, 1978) and an extension of an arginine-specific ADPRT assay (Klebl and Pette. 1996). ADPRT activity was measured fluorometrically using the NAD+ analogue, ENAD, as a substrate. Upon addition of eEF-2 to a mixture of PE24 and ENAD. the toxin catalyzes the cleavage of the bond between nicotinamide and EADP-ribose, which results in a 10-fold increase in fluorescence intensity (Perkin and Anderson. 1978). Thus. it was possible to detemine the ADPRT activity by the gain

in fluorescence after addition of eEF-2 to the assay. The EADP-ribose produced was quantified

by calibration with aAMP. which exhibits the same molar fluorescence intensity as aADP-ribose.

The Michaelis-Menten saturation curve for the ADP-ribosylation of wheat genn eEF-2

by 7AW-PE24 is shown in Figure 3.7. The kinetic parameters for wild-type PE24 and 7AW-

PE24 are shown in Table 3.1. Wild-tvpe PE24 showed a turnover number (k,,) just over 3000

min-1 with a KM value of almost 210 pM and a kCat/Kb1close to 1.5 .u 106 M-1 min-1. The analogue incorporated PE24 resulted in a reduction in the k,,, value (less than 2% of the catalytic ability of the wild-type protein). However, the Khi value was affected to a lesser extent (2-fold decrease). The catalytic efficiency was substantially reduced (4% of wiid-type value) by substitution of the tryptophan residues with 7AW. due largely to the kCatvalue (Table 3.1).

The reduction in the turnover number of 7AW-PE24 is not surprising since studies conducted on the active-site tryptophan residues in PE24 (Trp-il? TrpJ66 and Trpjj8) found

TrpJ66 was important in the conformational stability of the protein (Li et al., 1995; Beattie and

Merrill, 1996). Beattie and Merrill ( 19%) observed a 20-fold decrease on the catalytic rate of

PE40 upon replacement of Trp-166 with phenylalanine. Minimal effects were seen for both the mutants, W417F and W558F. However, the effect was cumulative for the double mutant,

W466F/W558F, where the k,, decreased by 200-fold. One might expect the double mutant,

W466F/W417F, would also be catalytically defective, although this mutation was not prepared for the study. The cumulative effect of the conservative replacement of the 3 tryptophan residues with 7AW was much lower (60-fold decrease) than the 200-fold decrease observed by

Beattie and Merrill (1996). Additional studies (not presented in this thesis) on the binding of

7AW-PE24 to its nucleotide substrate (NAD+) found that the association of enzyme with substrate increased modestly IO-fold (dissociation constant (K,) decreased) with respect to the wild-type protein value (Dr. Rod Merrill. personal communication). These two results strongly Mean O trial 1

FlGURE 3.7 Michaelis-Menten saturation curve for the ADP-ribosylation of wheat germ eEF-2 (14 PM) by 7AW-PE24 (23.8 nM). -- -

Table 3.1 : Kinetics of ADP-ribosyl Transferase Activity for PE24 and 7A W-PE24a

kat KM kat/ KM Protein (min-') (&-NADf)(PM) (1 06 M-1 min-[)

Minetic parameters were obtained by analysis of Lineweaver-Burk and Hanes-Woolf plots of initial rates of reaction as a function of &-NADf concentration and where eEF-2 was in excess. suggest the low turnover number for 7AW-PE24 may be the result of increased binding afinity for NAD' rather than structural perturbation of the enzyme. However. structural integrity experiments on 7AW-PE24 were not performed.

3.3.5 Absorbance and Fluorescence of 7AW-PE24

The absorption and fluorescence spectra of wild-type and 7AW-incorporated PE24 are shown in Figures 3.7 and 3.8 under native protein conditions. The absorbance of wild-type PE24 is dominated by three tryptophan residues. producing a A,, at 278 nrn (Fig. 3.8). The incorporation of 7AW produced a protein absorbance spectrum red-shifted (A,, at 280 nm) with respect to wild-type protein. The spectrum of the analogue-containing protein also acquires the characteristic red-extended absorbance tail of 7AW (Hogue. 1994). Figure 3.9 shows the steady-state fluorescence emission spectra of both PE24 and 7AW-PE24 with 300 nm excitation.

The spectra were peak-normalized ro depict the maximum emission waveIength (h,,max) differences between the hvo proteins. A difference in the &,ma of 19 nrn (red-shift) for the analogue-containing protein was observed.

The absorption and fluorescence spectra of both proteins were also measured under denaturing conditions (Figs. 3.10 and 3.1 1). Both figures show spectra of wild-type PE23 and

7AW-PE24 obtained in the presence of 6 M Gn-HCI, which eliminates essentially al1 protein structure-induced effects on extinction coefficients. This loss of protein structure can be seen in the red-shift in the Lemmm for both wild-type PE24 and 7AW-PE24 when going from native

(Fig. 3.9) to denaturing conditions (Fig. 3.1 1). This red-shift is usually indicative of a increase in the exposure of the protein's tryptophan residues to the surrounding solvent.

The spectral enhancement of 7AW-PE24 with respect to wild-type FE24 is illustrated by the characteristic red-shift seen in both the absorption spectra (Fig. 3.8 and 3.10) and emission Wavelength (nrn)

FIGURE 3.8 Peak normalized absorbance spectra of PE24 and 7AW-PE24, showing a red shifi in absorbance for the analogue-incorporated protein compared to wild-type protein. The intrinsic protein fluorescence of the 7AW-PE24-eEF-2 protein-protein complex can be studied by exciting, for example. at 3 10 nm. where absorption of light by the tryptophan residues in eEF-? is negligible. The structure of L-tryptophan (blue) and 7AW (red) are also shown. (20 rnM Tris- HCI, 50 mM NaCI, pH 7.6, room temperature). Wavelength (nm)

FIGURE 3.9 Normalized steady state fluorescence emission spectra of PE24 and 7AW-FE24 excited at 300 nm (4 nm slits). A red shift of 19 nm is observed for 7AW-PE24. (20 mM Tris- HCl, 50 mM NaCI, pH 7.6, room temperature). Wavelength (nrn)

FIGURE 3.10 Peak normaIized absorbance spectra of PE24 and 7AW-PE24, both denatured in 6 M Gn-HCI to essentially eliminate ail protein structure-induced effects on extinction coefficients of tryptophan and 7AW. A red shift in A,, of 6 nm is observed. (20 mM Tris-HCI, 6M Gn-HCI. 50 mM NaCI, pH 7.6, room temperature). Wavelength (nm)

FIGURE 3.1 1 Normalized steady state fluorescence emission spectra of PE24 and 7AW-PE24, both denatured in 6 M Gn-HCI and excited at 300 nm (4 nm slits). (20 mM Tris-HCI, 6M Gn- HCI, 50 mM NaCI. pH 7.6, room temperature). spectra (Fig. 3.9 and 3. i 1). In either case. excitation at approximately 3 10 nm should provide selective excitation of the 7AW chromophore. even in the presence of tryptophan residues (in eEF-2). since at this wavelength. the EM for L-tryptophan is near zero (Fig. 3.8). This would enable the study of the interactions behveen PE-24 and eEF-2.

3.3.6 Efficiency of 7AW Incorporation into PE24

Figure 3.12 shows wild-type PE24 (A) and 7AW-PE24 (B) absorption spectra of the proteins denatured in 6 M Gn-HCI. It also shows the spectra of NATrpA. NATyrA and t-boc(a- amino)-7AW under the same conditions. The wild-type PE24 spectra was initially fitted to the two basis spectra, NATrpA and NATyrA (Fig. 3.12A) and the scaled buis method (section

3.2.6) was used to assess the degree of incorporation of 7AW into PE24. The LINCS anaIysis gave a NATyrAI NATrpA ratio of 2.70 which is consistent with the 85 (2.67) molar ratio expected from the amino acid composition of PE24. The 7A W-PE24 spectrum gave a best fit when al1 three basis spectra were used in the LINC analysis (Fig. 3.12B). According to the anal ysis. approximately 60% of the incorporated tryptophan residues are 7AW. Thus. the other

40% consists of L-tryptophan-containing PE24. The tryptophan-containing portion of PE24 can be visualized in the fluorescence emission spectrum of 7AW-PE24 where a small tryptophan hump can be seen at approximately 345 nm (Fig. 3.9).

The relatively low incorporation may be attributed to several factors. The T7 expression system used for the 7AW incorporation into PE24 is susceptible to gene leakage. Figure 3.3 shows that prior to induction and addition of analogue- there is basal expression of PE24 (lane 6).

Thus. a portion of PE24 that is being expressed contains tryptophan, and likely contributes to the low efficiency of incorporation. However, this basal expression cannot account for al1 40% of the tryptophan-containing PE24, since there is only approximately 5% leakage inherent to this promoter (Studier et al., 1990). Thus, there are likely other factors involved. First. there is the wtPE23Fit ...... NATrpA

260 280 300 320 340 360 Waveiength (nm)

PE247.4W PE247AW Fit Boc7AW

A . . . . . NATrpA

.... N ATy rA

Wavelength (nm)

FIGURE 3.12 LINCS analysis of protein absorption spectra. Spectra are arbitrarily scaled and do not reflect relative extinctions. (A) Absorbance spectrum of wild-type FE24 fitted to the NATrpA and NATyrA basis spectra. (B) absorbance spectrum of 7AW-PE24 fitted to the r- boc(a-amino)-7AW. NATrpA and NATyrA basis spectra. Spectra of mode1 amino acids and proteins were measured in 6 M Gn-HCI in 20 mM Tris-HCI, 50 mM NaCI buffer (pH 7.6). presence of residual L-tryptophan from growth media that were not rernoved after centrifugation.

Any tryptophan that is available after induction will compete effectively with the tryptophan analogue for incorporation since these analogues are not natural substrates. L-tryptophan can also become available from protein turnover with cells afier induction. Second, the expressed protein itself may not tolerate uniform replacement of its tryptophan residues. For exmple, the eficiency of incorporation of analogues into the soluble extracellular domain of human tissue factor (sTF), was 20 and 30% for 5HW and 7AW. respectively. even though expression was through a tac promoter, which has proven efficient for analogue incorporation in other proteins

(Table Z.I)(Ross et al.. 1997). In addition, mutation of these tryptophan residues to either tyrosine or phenylalanine resulted in low yields of expression (Hasselbacher et al., 1995). Third, as mentioned earlier, the function of newly synthesized proteins required for translation using the

T7 promoter may be compromised if an essential tryptophan residue is replaced in these proteins.

Thus. the low efficiency of 7AW incorporation into PE24 may be explained by the interplay of the several different factors described above.

Attempts were made to improve the efficiency of incorporation by addressing each of the factors described above. Even in the presence of IPTG. there is sorne expression of T7 RNA polymerase from the hcW5 prornoter (Studier et al.. 1990). To minimize Ieakage, the plasmid pLysS was transformed into the tryptophan auxotroph along with the vector for PE24. pLysS provides a small arnount of T7 lysozyme, which is a natural inhibitor of T7 RNA polymerase, but does not prevent induction of high levels of target proteins. We observed reduction in basal activity using pLysS when expressing PE24 in minimal media containing tryptophan. However, expression of protein with pLysS in minimal media containing analogue was compromised and we did not observe any PE24 expression.

Residual tryptophan present from growth media after the centrifugation step can be reduced by resuspending the cell pellet in M9 minimal media (L-tryptophan and analogue free). However. due to the presence of T7 lysozyme. one must be careful when resuspending the cells.

As described above, T7 lysozyme is a natural inhibitor of T7 RNA polymerase. However, this enzyme is also able to cleave bonds in the peptidogiycan layer of the E. coli ceIl wall and can cause cell lysis under mild conditions. If a srnall fraction of these cells are damaged during the centrifugation and resuspension steps, T7 lysozyme will escape and cause rapid lysis of the entire culture. Thus, the "washing" step to reduce tryptophan availability was not performed.

Instead, after resuspension in minimal media. the cells were allowed to çrow for 30-40 min prior to the addition of analogue and induction to deplete the residual tryptophan residues.

Perhaps the main reason for low analogue incorporation is the T7 promoter used. for reasons already discussed. In additional work not presented in this thesis. anempts were made to express PE24 using a tac promoter which has proven effective for analogue incorporation (Ross et al., 1997). The commercially available. intein-mediated purification with an afinity chitin- binding tag (IMPACT)(New England Biolabs) system of purification was utilized. This system involved making a recombinant protein where the gene encoding PE24 was inserted into the multiple cloning site of a pCYB vector to create a fusion between the C-terminus of the PE24 gene and the N-terminus of the gene encoding for the protein splicing element. intein. DNA encoding a chitin binding domain (CBD) was added to the C-terminus of the intein to allow affinity purification of the 3-part fusion. The expression of the fusion gene was under the control of a tac promoter and is inducible by IPTG.

In theory, when crude extracts of ceIls from an E. coli expression system are passed over a chitin column, the target protein-intein-CBD fusion protein binds to the chitin beads while al1 other contaminants are washed away through the colurnn. The target protein is cleaved away from the intein-CBD fusion by cleavage induced at 4 OC by addition of a reducing agent such as dithiothreitol. The target protein is released while the intein-CBD fusion remains bound to the column. Unfortunately, we obsewed poor expression of the PE24-intein-CBD fusion as well as proteolytic cleavage. Despite experimenting with different growth and induction conditions to optimize the expression of the fusion. as well as trying different protease deftcient hosts to reduce proteolysis, we were unsuccessful at obtaining useful amounts of PE24 using this new expression system. CHAPTER4 - SUMMARY,CONCLUSIONS AND FUTURESTUDIES

This study has demonstrated that a previously unavailable lysogenic strain of tryptophan

auxotroph can be constructed using a Pl transduction. It was shown that this trvptophan

auxotroph was able to express 7AW-incorporated PE24. however a 1 O-fold reduction in protein yield was observed. The absorbance and emission spectra of the analogue-incorporated protein were consistent with those of other spectrally enhanced proteins and therefore the product appears to be applicable for the study of toxin-eEF-2 interaction using intrinsic fluorescence.

However, the utility of 7AW-PE24 depends on the extent of incorporation. which was found to be approximately 60%. In order for this method to be successful. a higher incorporation rate is desirable, which was could not be achieved in this study using the T7 polymerase promoter.

Based on the experirnents of other investigators. the best results to date for analogue incorporation have been obtained by using a lac promoter (Ross et al.. 1997).

This study has laid the groundwork for the incorporation of trvptophan analogues into

ETA. The conditions for overexpression and purification for analogue-containing protein have been established. Since this method shows good promise for the study of toxin-eEF-2 protein- protein interaction, further experiments should be performed to optim ize the incorporation of analogues. Although attempts in expressing 7AW-PE24 using the IMPACT method have failed, there are other expression and purification methods available. Currentty, the Merrill lab is attempting to express and puri& PE24 expressed by a tac promoter with a His tag N-terrninally attached to the protein.

If the extent of incorporation proves to be high using a tac promoter, other analogues such as 5HW and 4FW may be incorporated since each analogue has different and important attributes that may aid the study of protein structure and function. For example, Bronskill and

Wong (1988) demonstrated that 4FW in water was essentially nonfluorescent at room ternperature. Thus. JFW incorporation could be used as a general technique to eliminate tryptophan fluorescence, allowing the study of other fluorophores such as the tryptophan

residues in eEF-2 upon binding to toxin. However, the utility of 4FW to remove tryptophan

fluorescence will depend on the full incorporation ( 100%) of this analogue. The B. subtilis strain

used by Bronskill and Wong (1988) was --de-adapted away fiom tryptophan towards 4FW and rnay be a more appropriate rnethod for preparing 4FW-incorporated proteins.

It is important to determine the extent to which tryptophan analogues perturb the structure, stability and function of the protein. Since 5HW and 7AW have additional polar hydroxy and ring nitrogens. respectively. not found in the indole ring, and because tryptophan residues are usually buried in nonpolar regions of a protein, the expectation is that they may cause some destabilization within proteins. However, in rnany cases. these analogue-containing proteins retain wild-type function. structure and stability (Ross et al., 1997; Wong and Efiink.

1997). Techniques that can be used to study the structural integrity of analogue-incorporated proteins include circular dichroism. limited proteolysis and fluorescence denaturation experiments.

The ADP-transfer reaction catalyzed by PE24 has been proposed to include formation of a binary complex of NADt and toxin, binding of eEF-2 to the binary complex, and transfer of the

ADP-ribose moiety of NAD+ to diphthamide. If the structure and function of the toxin-eEF-2 cornptex is to be studied using analogues, the ADP-ribosyl transferase activity must be blocked, i.e. hydrolysis of NADf rnust be prevented. Thus, an analogue of NAD+ rnust be used in which the N-glycosidic bond to ADP-ribose and nicotinarnide does not get hydrolyzed. The NAD+ analogue, prnethylene-thiazole-4-carboxamide adenine dinucleotide (PTAD). shows good promise as a substrate, since PTAD has been crystallized in the presence of PE24 and shows very slow hydrolysis (Li et al.. 1996). hlthough intrinsic tryptophan analogue fluorescence is an excellent tool for studying protein structure and function, most proteins contain many tryptophan residues. Therefore, changes in the tryptophan anaIogue flriorescence of one protein upon interaction with another protein only display global events. The use of mutant proteins, possessing single-tryptophan residues, allows the probing of site-specific regions using fluorescence spectroscopy. Currently, the Menill lab possesses single tryptophan mutants of PE24. each of which may be reptaced by a single analogue. This would aIIow the identification of specific regions involved in interacting with eEF-2. Adler, T.K. 1962. Fluorescence properties of mono- and poly-azaindoles. Anal. Chem. 34:685- 689.

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Wozniak, D.J., L.Y. Hsu, D.R. Galloway. 1988. His-426 of the Pseudomonas aeruginosa exotoxin A is required for ADP-ribosylation of elongation factor II. Proc. Natl. Acad. Sci. USA85:8880-8884. APPENDIXA - STRUCTURESOF TRYPTOPHAN ANALOGUES. GBroth, per liter 3 g casamino acids 5 g yeast extract 5 g NaCl

Adjust the pH to 7.0 with NaOH. Sterilize by autoclaving for 20 minutes at 15 Iblsq. in. on liquid cycle.

Super L-Broth, per Iiter 3 g peptone 5 g yeast extract IO g NaCl 0.4 g MgSOj 0.5% glucose

Adjust the pH to 7.0 with NaOH. Sterilize by autoclaving for 20 minutes at 15 lblsq? on liquid cycle. Afier autoclaving, just before innoculation add 0.5% glucose.

M9 Minimal Medium. per liter 6 g Na2HPO4 3 g KH2P04 1 g NH&I 0.5 g NaCl 3 mg CaClz

Add ingredients to water and heat with stirring until dissolved. Pour into bonles with loosened caps and autoclave 15 minutes at 15 Ib/sq?. Cool media to <50 OC before adding nutritional supplements and antibiotics. Waxman et al. (1993), generated relative extinction coefficients frorn analysis of the absorbance spectra of adrenocorticotropin (ACTH) and glucagon, each containing one tryptophan and hvo tyrosine residues. These researchers used the LINCS fitting procedure to detemine scaling coefficients a and b that produced the best fit of the model

to the absorbance spectra of the peptides ACTH and glucagon. Here. the A" is the measured pe~ih absorbance of the polypeptide at the specified wavelength and is the concentration of the known peptide. The quantities aEi.- and b,$? are thus the apparent extinctions for tryptophan T~P )Ir and tyrosine residues in the peptides. Although it is not possible to simultaneously determine al1 three parameten cPPIide,a and b. the quantity a/b, and thus a,+;7r/bdjycan be determined.

Therefore, if the rnolar extinction coefficient of one model compound is known. then the extinction coefficient of the other model compound can be calculated. For example. assurning that the absoIute molar extinction coeficient at 280 nm of NATrpA in 6 M Gn-HCL is 5690 cm-' M-1, a best fit /bZ8O of 5.42 is obtained, making the apparent extinction coefficient for Trp 7p NATyrA at 280 nm, l OS0 cm-[ M-1 (Waxrnan et al.. 1993). IMAGE NALUATION TEST TARGET (QA-3)

APPLIED -4 IMAGE. Inc -= 1 ô53 East Marn Street --. - Rochester. NY 14609 USA ------Phone: ilW48XKUM ------Fa: 7t 61288-5989

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