Amphibian Peptides: Their Structures and Bioactivity A thesis submitted for the Degree of Doctor of Philosophy by Kate Louise Wegener B. Sc. (Hons.) from the Department of Chemistry The University of Adelaide December, 2001 -Preface- GONTENTS Acknowledgements vii Statement of OriginalitY ix Abstract x Part l. lsolation and ldentification of Novel Amphibian Peptides 1 Ghapter I lntroduction 2 1.1 AmPhibian Skin Secretions 2 1.2 Peptides from Australian Frogs 5 1.2a NeuroPePtides 5 1.2b Antibiotics 6 1.2c Anticancer Agents 6 1.2d Pheromones 7 1.2e Other Functions 7 1.3 PePtideBiosYnthesis I 1.4 MethodologY 10 1.4a Collection of Frog Secretions 10 1.4b HPLC AnalYsis 11 1.4c Mass SPectrometry 11 1.4d Q-TOF 2 Hybrid Quadrupole Time of Flight Mass Spectrometer 12 1.5 PePtide Sequencing 15 1.5a Mass SPectrometry 15 1.5b Automated Edman Sequencing 16 1.5c EnzYme Digestion 16 1.5d C-terminalEndGroupDetermination 16 1.6 BioactivitYTesting 18 Chapter 2 Peptides l¡om Litoria dahlii 19 2.1 lntroduction 19 2.2 Results 22 2.2a General 22 2.2b Dahlein 1.2 24 2.2c Dahlein 4.1 27 2.2d Dahlein 5.6 31 2.2e BioactivitYTesting 36 2.3 Discussion 39 2.3a PePtides Írom Litoria dahlii 39 2.3b Structure and Bioactivity of the Dahleins 40 2.4 ExPerimental 46 -Preface- 2.4a Secretion Collection and Preparation 46 2.4b HPLC Separation of Glandular Secretion 46 2.4c Mass Spectrometry AnalYsis 47 2.4d Determination of the Peptide Terminal Group 47 2.4e Lys-C Digestion 47 2.4r Automated Edman Sequencing 48 2.49 Preparation of Synthetic Peptides 48 2.4h Antibacterial Testing 48 2.4i Anticancer Testing 48 2.4j Neuronal Nitric Oxide Synthase lnhibition Testing 49 part ll. Amphibian Antimicrobial Peptides - Structural and Mechanistic Studies 50 Chapter 3 lntroduction 51 3.1 AntibacterialPePtides 51 3.2 Mechanisms of Action of Antibacterial Peptides 55 3.2a GeneralMechanism 55 3.2b The Barrel-Stave Model 55 3.2c The Toroidal Model 59 3.2d The CarPet Model 62 3.3 Specificity of Action 65 3.4 Structural Features of cr-Helical Antibacterial Peptides 68 3.4a C-TerminalAmideGrouP 68 3.4b Helicity 69 3.4c Charge State 70 3.4d HYdroPhobicitY 71 3.4e AmPhiPathicitY 72 3.4f HYdroPhilic angle 73 3.4g Summary 74 3.5 Hinged a-Helices 75 Chapter 4 Protein Structure Determination 77 4.1 General 77 4.2 Circular Dichroism 78 4.3 Nuclear Magnetic Resonance 82 4.3a One-dimensionalSpectroscopy 84 4.3b Two-dimensionalSpectroscopy 85 4.3c CorrelatedSpectroscopy(COSY) 85 4.3d TotalCorrelation Spectroscopy(TOCSY) 87 4.3e HeteronuclearGorrelation Spectroscopy 88 4.3Í Nuclear Overhauser Effect Spectroscopy (NOESY) 89 -Preface- 4.4 SequentialAssignments 91 4.4a ProtonAssignments 91 4.4b CarbonAssignments 92 4.5 Secondary Structure ldentification Using NMR Spectroscopy 94 4.5a Secondary Shifts 94 4.5b NOEConnectivities 96 4.5c CouplingConstants 98 4.5d Amide Exchange Measurements 100 4.5e Summary 101 4.6 StructureCalculations 102 4.6a DistanceRestraints 102 4.6b Ambiguous NOEs 106 4.6c Stereo-specificAssignments 107 4.6d DihedralAngleRestraints 108 4.6e Restrained Molecular Dynamics and Simulated Annealing 109 4.6f The Potential Energy Function 111 4.6g Quality of Structures 113 4.7 Models for a Membrane Environment 116 4.7a 2,2,2-T¡illuoroethanol 117 4.7b Micelles 118 Chapter 5 Orientation Studies 120 5.1 General 120 5.2 Solid-State NMR SPectroscopy 123 5.2a Orientation of PePtides 124 5.2b PePtide-LiPidlnteractions 126 5.2c Head-grouPs - PhosPhorus NMR 127 5.2d AcylChains - Deuterium NMR 129 5.2e MechanicallyAligned Bilayers 133 5.2f MagneticallyAlignedBicelles 134 5.3 Oriented Circular Dichroism 137 Ghapter 6 Short Antibacterial Peptides 140 6.1 lntroduction 140 6.2 Citropin 1.1 145 ' 6.2a Circular Dichroism Studies 145 6.2b NMR Studies - Assignments 146 6.2c Secondary Shifts 149 6.2d Amide SecondarY Shifts 151 6.2e NOEConnectivities 151 6.21 CouPling Constants 152 IV -Preface- 6.2g Structure Calculations 152 6.3 Aurein 1.2 156 6.3a NMR Studies - TrifluoroethanolTitration 156 6.3b Assignments 157 6.3c Secondary Shifts 1ô0 6.3d Amide SecondarY Shifts 161 6.3e NOEConnectivities 162 6.3f Coupling Constants 163 6.3g StructureCalculations 163 6.4 Orientation Studies 167 6.4a Oriented Circular Dichroism - Unoriented Vesicles 167 6.4b Oriented LiPid BilaYers 168 6.4c Solid-state NMR - Unoriented Multilayers 171 6.4d MechanicallyOrientedMultilayers 173 6.5 Discussion 181 6.6 Conclusion 185 Chapter 7 The Gaerin PePtides 186 7.1 lntroduction 186 7.2 Structure of Caerin 1.1 in DPC Micelles 192 7.2a NMR Studies - Assignments 192 7.2b Secondary Shifts 195 7.2c Amide SecondarY Shifts 196 7.2d NOEConnectivities 197 7.2e Amide Exchange 198 7.2f StructureCalculations 199 7.3 Caerin 1.4 204 7.3a Circular Dichroism Studies 204 7.3b NMR Studies - Assignments 204 7.3c Secondary Shifts 210 7.3d Amide SecondarY Shifts 211 7.3e NOEConnectivities 212 7.31 Amide Exchange 213 7.39 StructureCalculations 214 7.4 Discussion 221 7.5 Conclusion 225 Chapter 8 Experimental 226 8.1 Materials 226 8.2 CD Spectroscopy - Trifluoroethanol Titrations 226 8.3 Solution NMR SPectroscopy 226 v -Preface- 8.3a SamPlePreParation 226 8.3b NMR SPectroscopy 227 8.3c Structural Restraints 229 8.3d StructureCalculations 229 8.4 Oriented Circular Dichroism 230 8.4a SamplePreParation 230 8.4b CD Measurement 231 8.5 Solid-state NMR SPectroscopy 231 8.5a Sample Preparation - Unoriented Bilayers 231 8.5b Mechanically,Oriented Bilayers 232 8.5c NMR Spectroscopy - Unoriented Bilayers 232 8.5d MechanicallyOrientedBilayers 232 8.5e Peptide Powders 233 Summary and Future Directions 234 References 237 Appendix - HSQC and HMBC SPectra 269 Publications 275 VI -Preface- Acknowledgements I would firstly like to thank my supervisor, Professor John H. Bowie, for his advice and guidance and for giving me the opportunity to work on such an interesting and wide- ranging project. I would also like to acknowledge the Australian Post-graduate Research Award that enabled me to undertake the research presented in this thesis. Thanks also to Associate Professor John Carver, from the University of Wollongong, for his help and advice regarding NMR techniques and Dr. Robyn Lindner for her assistance with the acquisition of CD spectra. Many thanks as well to Associate Professor Frances Separovic and Tracy Lam, from the University of Melbourne, for teaching me aspects of solid-state NMR spectroscopy and Dr. Maurits de Planque for his help with oriented CD. Special thanks must go to Dr. Grant Booker, from the Department of Molecular Biosciences, University of Adelaide, for allowing me the use of his computers and for being so generous with his time and advice - it was very much appreciated. Thanks also to Dr. Terr)' Mulhem, now of the University of Melboume, for teaching me the computational aspects of peptide structure determination. In addition, I'd like to acknowledge Associate Professor Mike J. Tyler and Ben Smith, from the Department of Environmental Biology, University of Adelaide, for providing the amphibian secretions and Associate Professor John C. Wallace, from the Department of Molecular Biosciences, University of Adelaide, for providing the automated Edman sequencing data. Thanks also to Dr. Lyndon E. Llewellyn, from the Australian Institute of Marine Science, for conducting the nNOS inhibition assays and the National Cancer Institute (V/ashington) for performing the anticancer tests. Much thanks to the academic, research and technical staff at the University of Adelaide for their help and advice - particularly Dr. Brian Chia, Phil Clements and Dr. Simon Pyke for assistance with NMR spectroscopy, Dr. Paul V/abnitz and Andrew McAnoy for mass spectrometry advice and Craig Brinkworth for his help with the Q-TOF mass spectrometer and Edman sequencing. Thanks also to the members of the Bowie research group, especially Craig Brinkworth and Charm Boontheung for the comic relief they provide in the lab! vil -Preface- Thanks to Sam Lienert for being so generous with his lap-top computer and my brother Tim for his patient help with computer scripts and for attempting to answer millions of incidental questions - and both of them for being such accommodating house-mates. Thanks also to Becky Lienert and Adrian Harris for looking after me so well when I was in Melbourne. Special thanks as well to my 'Ph. D. buddy' Duncan Grove for the helpful chats (did I win the bet?) and for understanding and to my U.S. mate Anita Kishore for her waffn friendship. Thanks also to the Lawrie family (particularly Mrs Adams) for supporting me throughout my Ph.D., taking an interest in my work and for keeping me well-fed! Also, to all my family and friends, thank-you for putting up with me and for constantly asking 'how are the frogs going?'. Oh, and thanks for all the frogs Liesel. I would especially like to thank my Mum and Dad for all the support they've given me throughout my life. It has been greatly appreciated and this wouldn't have been possible without it. And finally, I'd like to thank Mark Lawrie, firstly for his technical assistance with the 3D lipid figures and computer scripts, but mostly for his extraordinary patience and the amazing support and encouragement he's given me over the last few years. vill -Preface- Abstract Amphibians secrete a cocktail of chemicals onto their skin in response to stressful stimuli, such as attack by predators. The compounds secreted include bioactive peptides, which can protect against infection, relieve pain and regulate aspects of the animal's physiology. Such peptides are potential therapeutic agents and can also be used to investigate the relationships between different amphibian species.