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Biophysical Studies of the First Nucleotide Binding Domain of SUR2A

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Biophysical Studies of the First Nucleotide Binding Domain of SUR2A Biophysical Studies of the First Nucleotide Binding Domain of SUR2A by Elvin Dominic de Araujo A thesis submitted in conformity with the requirements for the degree of Master of Science Department of Chemistry University of Toronto © Copyright by Elvin de Araujo, 2011 Biophysical Studies of the First Nucleotide Binding Domain of SUR2A Elvin Dominic de Araujo Master of Science, 2011 Department of Chemistry University of Toronto Abstract ATP-sensitive potassium (KATP) channels have crucial roles in several biological processes. KATP channels possess four regulatory sulfonylurea receptors. The SUR proteins are members of the ubiquitous ATP-binding cassette (ABC) superfamily. However, unlike most ABC proteins, SURs do not transport substrates but function strictly as regulators of KATP channel activity. Currently, studies into the molecular basis by which various mutations in SUR2A cause disease are highly limited. This is primarily a consequence of poor solubility of isolated SUR2A NBDs, as is typical for many eukaryotic NBDs. By employing structure-based sequence alignments and biophysical studies, we determined domain boundaries for SUR2A NBD1 that enabled, for the first time, NMR studies of NBD1. Our biophysical studies demonstrate that the isolated SUR2A NBD1 is folded and exhibits differential dynamics upon ATP binding activity. Additional studies are now possible to examine the effects of disease-causing mutations on structure, dynamics, and interactions of NBD1. ii Acknowledgments I would like to thank my supervisor, Prof. Voula Kanelis for her valuable support and enduring guidance throughout my Masters program. Her enthusiasm for science is truly motivating. Prof. Kanelis is not only a dedicated scientist but also helped further my development as a researcher. I would also like to thank Prof. R. Scott Prosser for providing me with both insightful suggestions and technical support. As an avid biophysicist, his eagerness has helped instill my interest in biophysics. Working in his lab during my undergraduate years, encouraged me to pursue research. Prof. Ulrich J. Krull has taught me valuable lessons about analytical chemistry, widening my scope in my scientific approach. Past and present colleagues, Serisha Moodley, Lynn K. Ikeda, Dennis Guo, Marijana Staglijar and Dr. Jorge Pédro Lopez-Alonso have been instrumental both in creating an enjoyable and stimulating environment to work in the lab. Thanks to Prof. George S. Espie, Prof. Virginijus Barzda and Prof. Jumi Shin for allowing me to use their scientific equipment. iii Table of Contents 1. Introduction 1.1. Background of ABC Transporters 1 1.2. Structural Architecture of ABC Transporters 2 1.3. Nucleotide Binding Domains 4 1.4. Sulfonylurea Receptors 7 1.5. KATP channels 9 1.6. Inwardly Rectifying Potassium Channels 10 1.7. Physiological Importance of KATP Channels 13 1.8. Regulation of KATP Channels Activity 16 1.9. Cardiac Disease-Causing Mutations in SUR2A 17 1.10. Biophysical Methods for Investigation of SUR2A 19 1.10.1 Nuclear Magnetic Resonance Spectroscopy 19 1.10.2 Circular Dichroism Spectroscopy 22 1.10.3 Fluorescence Spectroscopy 23 2. Materials and Methods 26 2.1 Structure-Based Sequence Alignment of NBDs 26 2.2 Homology modeling of SUR2A NBD1 26 2.3 Generation of Recombinant NBDs 27 2.4 Expression and Purification of SUR2A NBD1 28 2.5 Phosphorylation of Wild Type and Mutant SUR2A NBD1 30 2.6 NMR Spectroscopy 31 2.7 Circular Dichroism Spectroscopy 31 2.8 Fluorescence Spectroscopy 32 3. Results 33 3.1 Structure-Based Sequence Alignment 33 3.2 Generations of Constructs with Variable Domains Boundaries for NBD1 35 3.3 Expression and Purification of SUR2A NBD1 37 3.4 Biophysical Characterization of SUR2A NBD1 40 iv 3.4.1 NMR Studies of 15N labeled S615-L933 40 3.4.2 NBD1 SUR2A S615-L933 functionally binds ATP 44 3.4.3 NMR Studies of T618-L933 47 3.4.4 Phosphorylation of S615-L933 NBD1 SUR2A 53 3.4.5 Role of R934-K972 in NBD1 SUR2A 56 3.4.6 Secondary Structural Analysis of NBD1 SUR2A 61 3.4.7 Fluorescence Spectroscopy of NBD1 SUR2A 64 3.5 Biophysical studies of SUR2A NBD1 Val734Ile 68 4. Discussions and Conclusions 4.1 Identification of SUR2A NBD1 Domain Boundaries 72 4.2 Characterization of ATP binding in SUR2A NBD1 74 4.3 Effects of Phosphorylation on NBD1 of SUR2A 77 4.4 Role of the ED Domain in NBD1 78 4.5 Characterization of SUR2A NBD1 Val734Ile 79 4.6 Conclusions and Future Directions 81 5. Appendix 82 6. References 87 v List of Tables Table 1: Subfamilies of human ABC transporters 2 Table 2: Subunit assembly of KATP channels in different tissues 14 Table 3: Gyromagnetic ratios of some NMR active nuclei 20 Table 4: Primers used in the PCR amplification of SUR2A NDBs 28 Table 5: Total protein yields of various NBD1 SUR2A constructs 37 Table 6: Number of amide proton crosspeaks in NBD1-S615-L933 42 Table 7: Number of amide proton crosspeaks in NBD1-S615-L933 (± ATP) 46 Table 8: Number of amide proton crosspeaks in NBD1-T618-L933 48 Table 9: Number of amide proton crosspeaks in phosphorylated NBD1-S615 -L933 55 Table 10: Number of amide proton crosspeaks in NBD1-S615-K972 at 35oC 59 Table 11: Melting temperatures determined by fluorescence 66 Table 12: Tryptophan solvent exposure determined by fluorescence quenching 67 Table 13: Tryptophan solvent exposure of Val734Ile SUR2A NBD1 determined by fluorescence Val734Ile 71 vi List of Figures Figure 1. General structure of an ABC transporter 3 Figure 2. General structure of the nucleotide binding domain of ABC transporters 5 Figure 3. Dimerization of NBDs upon ATP binding 6 Figure 4. Model for regulation of KATP channels by the SURs 8 Figure 5. Schematic representation of the KATP channel 9 Figure 6. Model of the KATP channel 10 Figure 7. Classification of potassium channels 11 Figure 8. Representation of dimeric Kir subunits 12 Figure 9. Role of KATP channels in various tissues 15 Figure 10. Mutation and phosphorylation sites in SUR2A NBDs 17 Figure 11. Circular dichroism spectra of common secondary structure features 22 Figure 12. Possible excitation and relaxation pathways of a chromophore with light 24 Figure 13. Structure-based sequence alignment of SUR2A NBD1 with other ABCC transporters 34 Figure 14. Protein profile of pre- and post- induction for proteins of various domain boundaries 36 Figure 15. Localization of expressed protein to soluble or insoluble fractions 36 Figure 16. Purification of NBD1 S615-L933 38 Figure 17. Time course of Ulp1 activity on 6xHisSUMO-NBD1 S615-L933 38 Figure 18. Fractions following gel-filtration of cleaved 6xHisSUMO-NBD1 S615-L933 39 Figure 19. 2D 15N-1H TROSY-HSQC spectra of NBD1-SUR2A S615-L933 at 30oC 40 Figure 20. 2D 15N -1H TROSY-HSQC spectra of NBD1-SUR2A S615-L933 at various temperatures 43 Figure 21. 2D 15N -1H TROSY-HSQC spectra of NBD1-SUR2A S615-L933 with varying levels of ATP 45 vii Figure 22. Overlay of 2D 15N -1H TROSY-HSQC spectra of NBD1-SUR2A S615-L933 with and without ATP 46 Figure 23. Homology model of NBD1 of SUR2A 47 Figure 24a. Purification of T618-L933 47 Figure 24b. 2D 15N -1H TROSY-HSQC spectra of NBD1-SUR2A S615-L933 at varying temperatures 48 Figure 25. Overlay of 2D 15N -1H TROSY-HSQC spectra of NBD1-SUR2A S615-L933 with T618-L933 51 Figure 26. Overlays of 2D 15N -1H TROSY-HSQC spectra of NBD1-SUR2A T618-L933 52 Figure 27. 2D 15N -1H TROSY-HSQC spectra of Phosphorylated NBD1-SUR2A S615-L933 54 Figure 28. 2D 15N -1H TROSY-HSQC spectra of Phosphorylated NBD1-SUR2A S615-L933 without ATP at 30oC 55 Figure 29. Purification of NBD1 S615-K972 56 Figure 30. Fractions following gel-filtration of cleaved 6xHisSUMO-NBD1 S615-L933 56 Figure 31. 2D 15N -1H TROSY-HSQC spectra of NBD1-SUR2A S615-K972 58 Figure 32. 2D 15N -1H TROSY-HSQC spectra of NBD1-SUR2A S615-K972 at 35oC 59 Figure 33. 2D 15N -1H TROSY-HSQC Phosphorylated of NBD1-SUR2A S615-L972 at 30oC 60 Figure 34. Circular dichroism spectrum of S615-L933 61 Figure 35. Guanidine-HCl melt of NBD1 SUR2A S615-L933 62 Figure 36. CD spectra of phosphorylated S615-L933 (A) and S615-K972 (B) 63 Figure 37. Fluorescence temperature melting curve of S615-L933 64 Figure 38. Fluorescence temperature melting curve of S615-L933 under varying Conditions 65 Figure 39. Quenching of fluorescence of S615-L933 by potassium iodide 66 Figure 40. Modified Stern-Volmer plots of fluorescence quenching of S615-L933 67 viii Figure 41. 2D 15N -1H TROSY-HSQC of Mutant Val734Ile of NBD1-SUR2A S615-L933 69 Figure 42. CD spectra of Mutant Ile734Val in S615-L933 70 Figure 43. Fluorescence melt curves of Mutant Ile734Val in S615-L933 in the presence/absence of ATP 70 Figure 44. Modified Stern-Volmer plots of fluorescence quenching with Val734Ile3 of S615-L933 71 Figure 45. Altered protein dynamics with ATP binding 75 Supplementary Figure 1: Structure-based sequence alignment of NBD1 with other ABCC transporters 82 ix List of Appendices Supplementary Figures 89 x List of Abbreviations ABC ATP-binding cassette ABCR rod outer segment ABC transporter ADP Adenosine diphosphate ATP Adenosine triphosphate Bo External magnetic field CD Circular dichroism CFTR Cystic fibrosis transmembrane conductance regulator DNA Deoxyribonucleic acid DSS 4,4-dimethyl-4-silapentane-1-sulfonic acid DTT Dithiothreitol fa Fraction of fluorophore population accessible to quenching Go Ground-state energy h Planck’s constant, 6.626068 × 10-34 m2 kg/s HSQC Heteronuclear single quantum coherence HUGO Human Genome Organization ICD Intracellular domain IPTG Isopropyl-β-D-thio-galactoside K Quenching constant K+ Potassium ion KATP channel ATP-sensitive potassium channel
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