THE EFFECTS OF AN AC FIELD ON THE TRANSPORT OF PEPTIDES AND OTHER MOLECULES THROUGH THE α-HEMOLYSIN PORE A Thesis Submitted to the College of Graduate Studies and Research In Partial Fulfillment of the Requirements for the Degree of Master of Science In the Department of Biochemistry University of Saskatchewan, Saskatoon By Elisabet Jakova © Copyright Elisabet Jakova, June 07, 2016. All Rights Reserved. Permission to Use In presenting this thesis in partial fulfillment of the requirements for a Postgraduate degree from the University of Saskatchewan, I agree that the Libraries of this University may make it freely available for inspection. I further agree that permission for copying of this thesis in any manner, in whole or in part, for scholarly purposes may be granted by the professors who supervised my thesis work, or in their absence, by the Head of the Department or the Dean of the College in which my thesis work was done. It is understood that any copying or publication or use of this thesis or parts thereof for financial gain shall not be allowed without my written permission. It is also understood that due to recognition shall be given to me and to the University of Saskatchewan in any scholarly use which may be made of any material in my thesis. Request for permission to copy or make other uses of material in this thesis in whole or in part should be addressed to: Head of the Department of Biochemistry College of Medicine, University of Saskatchewan Saskatoon, SK Canada S7N 5E5 i ABSTRACT This research is based on superimposing an alternating current (AC) onto a direct current (DC) during the nanopore analysis of various peptides, single-stranded DNA, α-synuclein (AS) protein, and AS-drug complexes. Standard nanopore analysis consists of a constant DC voltage used to electrophoretically drive small molecules towards a pore. The superposition of the AC voltage at constant amplitude and different frequencies causes small molecules to oscillate as they approach the pore vestibule which will lead to alterations of the event parameters, the blockade current and blockade time. The DC voltage was set at 100 mV. It was found that a 200 mV AC voltage imposed over the DC voltage at frequencies from 10 MHz – 1 GHz was optimal to conduct the experiments. Initially, four α-helical peptides with a Fmoc-capping group on the N-terminus with the general formula Fmoc-DDAxKK, were studied since their DC behaviour is well known, e.g. Fmoc-DDA10KK (A10), Fmoc-DDA14KK (A14), Fmoc- DDA18KK (A18) and retro-inverse Fmoc-KKA10DD (RI-A10). These peptides are of different length and consequently different dipole moments, which is hypothesised to alter their mobility in the AC field. It was shown that the ratio of translocations to bumping events could be manipulated by a combination of AC voltage and frequencies. In particular, A10 could be studied without interference from RI-A10. Similarly, a large intrinsically disordered protein of 140 amino acids, AS, which translocates the pore readily in a DC field could be prevented from doing so by application of an AC field of 200 mV at 100 MHz. Additionally, three fragments of AS, C-terminal, N-terminal and the ΔNAC, were studied under the same AC conditions. After an AC field was applied, the C- and N-terminal were prevented from entering the pore due to their charge. Conversely, ΔNAC behaved as expected for a peptide with a negative C- terminus and/or with a large dipole moment, and the ratio of bumping events to translocation events, increased with an increase of the AC frequency. Finally, nanopore analysis with an AC field was useful to probe the conformational states of AS which are induced by drugs. Seven drugs were chosen to be studied in an AC field since their DC blockade histograms and binding properties are known. Five neuroprotective drugs (S-1-aminoindan, 3-methoxytyramine, caffeine, (-)-nicotine, metformin) and two neurotoxic drugs (cocaine and (+)-amphetamine) were studied. Surprisingly, the neuroprotective AS-drug complexes, under the effect of the AC field, showed signs of drug stripping. ii PUBLISHED WORK Articles in refereed journals resulting in the work presented of this Thesis: Jakova, E., and Lee, J.S. (2015). Superposition of an AC field improves the discrimination between peptides in nanopore analysis. Analyst 140, 4813-4819. iii AKNOWLEDGEMENTS First and foremost, I would like to thank my academic mentor and supervisor Professor Jeremy S. Lee for the incredible opportunity to work as a graduate student in his lab. It has been a great three year journey full of excitement and stimulating moments. Thank you, Dr. Lee for your continuous support and precious advice, both academically and personally. Your passion for science, brilliant ideas, and imagination have inspired me to always look deeper and to pursue my goals even if initial outcomes were discouraging. Lastly, I would like to thank you for your encouragement to think creatively and to further improve myself as a scientist. I would also like to thank all my committee members: Dr. Yu Luo, Dr. Oleg Dmitriev, and external member Dr. Peter Howard. I am grateful for their stimulating questions and academic suggestions. Overall, I am thankful for their support. I was lucky enough to take courses from both Dr. Luo and Dr. Dmitriev. I can truly say that through these courses I have learned a great deal about membrane . Furthermore, I would like to thank my old and new lab members of Professor Lee's research team. Having you by my side has been a great pleasure and joy! Finally, I would like to thank the Natural Science and Engineering Research Council of Canada (NSERC) and scholarships from the Department of Biochemistry's Devolved Scholarship Fund to Professor Lee for enabling me to pursue and achieve such results. iv DEDICATION I would like to dedicate this Thesis to my small Canadian family. To my auntie, Pat Atkinson, for being a role model and showing me what a true strong, assertive, experienced and independent woman should be. In addition, for being a good friend and a good listener. Finally, I would like to thank her for teaching me that hard work always pays off and that sacrifices need to be made in order to achieve your goals. To my uncle, Alfons Joseph-Paul Shoshi, for having tremendous patients with me and unconditional support. For teaching me the true meaning of the word perseverance and for having enormous faith in me. Lastly, for helping and being there for me every time and anytime I needed him. v TABLE OF CONTENTS TITLE PAGES ABSTRACT.................................................................................................................................ii ACKNOWLEDGMENTS..........................................................................................................iv LIST OF TABLES......................................................................................................................ix LIST OF FIGURES....................................................................................................................xi LIST OF ABBREVIATIONS...................................................................................................xv 1. INTRODUCTION..............................................................................................................1 1.1 Introduction............................................................................................................1 1.2 Single molecule biosensors.....................................................................................2 1.2.1 Cell membrane transporters.................................................................5 1.3 Nanopore Technology.............................................................................................8 1.3.1 Fundamentals of Nanopore analysis...................................................8 1.4 Structure Alpha-Hemolysin and other pores..........................................................10 1.4.1 Other mutants of Alpha-Hemolysin..................................................16 1.4.2 Solid state pores................................................................................17 1.4.3 Superimposing an AC field on a standard Nanopore analysis..........19 1.5 Peptides and small molecules.................................................................................20 1.5.1 Structure and properties of peptides..................................................21 1.6 Alpha-Synuclein.....................................................................................................22 1.6.1 Structure of Alpha-Synuclein and domains.......................................22 1.6.2 Clinical importance of Alpha-Synuclein............................................24 1.6.2.1 Misfolding that leads to aggregation....................................27 1.6.2.2 Role of other factors in Parkinson's disease........................29 1.7 Protein-Drug complexes........................................................................................30 1.7.1 Different drugs that interact with Alpha-Synuclein...........................30 1.8 Hypothesis.............................................................................................................32 1.9 Objectives..............................................................................................................32 2. MATERIALS AND METHODS.....................................................................................34 vi 2.1 Reagents, Equipment and Software......................................................................34