NONPOLAR CONTRIBUTIONS TO CONFORMATIONAL SPECIFICITY IN ASSEMBLES OF DESIGNED SHORT HELICAL PEPTIDES Chandra Lynn Boon A thesis subrnitted in conformity with the requirernents for the degree of Master of Science Graduate Department of Medicai Biophysics University of Toronto O Copyright by Chandra Lynn Boon 2000 National Library Bibliothèque nationale l*l of Canada du Canada Acquisitions and Acquisitions et Bibliographie Sewices services bibliographiques 395 WelIington Street 395. rue Wellington OttawaON KtAON4 ûttawaON K1AON4 Canada Canada The author has granted a non- L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant à la National Li"brary 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 fome de microfiche/= 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 fiom it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author' s ou autrement reproduits sans son permission. autorisation. Nonpolar Contributions to Conformational Specifkity in Assemblies of Designed Short Helical Peptides Chandra Lynn Boon Master of Science 2000 Graduate Department of Medical Biophysics University of Toronto Abstract A series of designed short helical peptides was used to study the effect of nonpolar interactions on conformational specificity. A consensus sequence was designed to contain a heptad repeat (abcdefg), yielding short helices (17 residues), with minimal interhelical polar interactions. Heptad positions a and d were occupied by al1 possible combinations of the hydrophobie residues Leu, Ile, or Val, and positions e and g were occupied by Ala, yielding a series of nine peptides. An experimental methodology was developed to characterize the nine peptides and their pairwise mixtures; the results indicated that a vast arny of structural states were formed possessing different degrees of conformational specificity. In other work, the control of specificity has been linked to polar interactions. This work demonstrated that subtie changes in the configuration of nonpolar interactions can have a significant effect on the conformational specificity of oiigomenc short helices. Acknowledgements 1 am very grateful to my supervisor, Dr. Avijit Chakrabartty, for taking the chance with me and for teaching me how to pipette! 1 have really enjoyed "brainstorming" sessions with him and 1 find his enthusiasm for thinking and learning very contagious. 1 look fonvard to many interesting discussions in the future (even though these discussions tend io draw out lab meetings!). T would like to thank the members of my supervisory cornmittee: Dr. David Rose, Dr. Gil Pnve, and Dr. Dwayne Barber for their interest, guidance, and challenging questions. 1 want to thank Kevin Galley and Thomas Goldthorpe from Research Information Systems for al1 their patience and assistance with my thesis and my defense presentation. Past and present members of the Chakrabartty lab have been a great help. Thank you to Xiao Fei Qi for his help with the NMR data. 1 want to thank Paul Gorman for his friendship and support, and for being so much fun! 1 owe infinite thanks to Sandy Go for her patience while teaching me ALL of my lab techniques (1'11 never forget the pasteur pipette incident!). 1 also appreciate her friendship, sense of humour, and her talent for organizing surprise parties! 1 am very gnteful to Cynthia Qum for being a good friend and for al1 her support, especially during the wnting process. We still have to go for bubble tea! Thank you to JoAnne McLaunn for her support and guidance early in my Master's and for her friendship. During the course of my Master's, 1 experienced some difficulties that I could never have overcome without the help of my fnenâs. 1 would like to thank (in no particular order) Tim Davison, Brenda Rutherford, Linda Mark, Rey Interior, Tarek Harb, Kevin Laiiberte, Jason Maydan, Barbara Guinn, Seishi Shimizu, Shireen Khi, Sandy Batten, George Jones, Jim and Nancy Kost, and Jason Hinek for being so supportive and encouraging. 1 feel extremely forninate to have such amazing fnends! 1 would also like to Say a special thank you to Elyssa Elton and Judi Bechard. E!yssa, 1 can't properly express how much your friendship has meant to me. Your kindness and suppon helped so much. You are a fabulous friend and 1 have great respect for you. Judi, what can 1 say? Thank you for being such a true friend, dl these yean! Your encouragement and your belief in me were a great help, especially right before my defence. Lastly, 1would like to thank my family. 1want to thank my parents, Henry and Mary, for their love and support through al1 these years of school. 1 am so grateful to them for allowing me to explore my own interests and for always encounging me to pursue whatever 1 wanted. 1 also want to thank my sister, Khrista, for that 230 am phone conversation two weeks before my defence - thank you for being there for me. TABLE OF CONTENTS LIST OF TABLES ............................ ............ .......................................... ........... ...................... v LIST OF FIGURES ....,...................... ..................t.....l..... ..t..... ........ .................. v .. LEXICON .." ... t.w.C....we..~"....~U........................................................œ.......H...... ................. .VU CHAPTER 1: INTRODUCTION ...w...w.HH.........mHc.t,..-m." .......-... .............................................. ...........-. 1 THE CL-HELIX ...................................................................................................................................................... -3 ISOLATED HELR FORMATION........................................................................................................................... 3 DESIGNOF CX-HELICALBUNDLES .................................................................................................................... 10 Asm~cr........................................................................................................................................................ 19 INTRODUCTION ................................................................................................................................................ 20 TERIALS AND METHODS........................................................................................................................... 23 Peptide Synthesis and Purification.............................................................................................................. 23 Peptide Concentration Determination ............................................................................................................... 33 Circulrir Dichroism Spectmscopy...................................................................................................................... 24 Fluorescence Specuoscopy................................................................................................................................ 24 SolubiIity Measurements ................................................................................................................................... 3 Light Scattering.................................................................................................................................................. 3 Sedimentation Equilibrium Ultracentrifugation ............................................................................................... 36 Assessrnent of Pimile1 venus Antiparallei Alignment of Heiices in Helical Bundles Through Disuifide Cross-linking ...................................................................................................................................................... 77 Amide Proton Exchmge Rate Mesisurernents ................................................................................................... 28 RESULTS ........................................................................................................................................................... 29 Peptide Design .................................................................................................................................................... 29 Determining Presence of SpeciîÏc Oligomen in Peptide Mixtures ................................................................. 33 OIigornenzrition Strrte ........................................................................................................................................ 39 Stability of the adeg Oiigornen ......................................................................................................................... -Ml Assessrnent of Pimilel versus Antipdlel iüignment of Helices in rideg Oligomers .................................. 44 Amide Proton Exchange Kinetics of vlaa ........................................................................................................ 51 Discuss~o~..................................................................................................................................................... 55 CONCLUSIONS.................................................................................................................................................