Biosynthetic Incorporation of 7-Azatryptophan Into The
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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 @ Foo-Lim Yeh. 1997 NationaI Library Bibliothèque nationale 1*1 ofCanada du Canada Acquisitions and Acquisitions et Bibliographic SeMces services bibliographiques 395 Wellington Street 395, rue Weilington Ottawa ON K1A ON4 UttawaON K1A ON4 Canada Canada The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant à la National Library of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or sell reproduire, prêter, distribuer ou copies of this thesis in microfom, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/film, de reproduction sur papier ou sur format électronique. The author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantial extracts kom it Ni la thèse ni des extraits substantiels may be printed or otherwise de ceile-ci ne doivent être Unprimés reproduced without the author's ou autrement reproduits sans son permission. autorisation. ABSTRACT 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,