Adaptation of Green Proteorhodopsin to Changes in Membrane Lipid Composition by Rachel Brown A Thesis presented to The University of Guelph In partial fulfilment of requirements for the degree of Master of Science in Biophysics Guelph, Ontario, Canada © Rachel E. Brown, August, 2018 Abstract ADAPTATION OF GREEN PROTEORHODOPSIN TO CHANGES IN MEMBRANE LIPID COMPOSITION Rachel E. Brown Advisors: Dr. V. Ladizhansky University of Guelph, 2018 Dr. L. Brown The protein studied in this thesis is Proteorhodopsin (PR), which is linked to starvation states in marine bacteria. This work examines PR membrane system stability using FTIR. The stable membrane systems, found using FTIR, are further examined using multiple methodologies. The findings show lipid loss over time for PR membrane systems that contained only PC lipids or a DOPC/DOPS mixture. Lipid systems containing DOPE/DOPG were found to be stable and were compared to systems containing DMPC/DMPA in further experiments. These systems mimic the changes involved in the transition from non-starvation to starvation state, suggesting DMPC/ DMPA as a suitable native membrane model for structural study of PR. Photocycle and titration experiments provide evidence of a shift in the pKa of D97 (DMPC/DMPA PR 6.6, DOPE/DOPG PR 7.5). CANCO experiments showed minimal changes in observable residues, however further NMR studies are required for full evaluation. Acknowledgements To my Labmates. I can never repay you all enough for the warmth I have felt being part of this lab-group. To Leonid Brown who believed in my ideas and helped to put me on the path to proving them. To Vladimir Ladizhansky, for your patience in guiding me through the process of learning NMR. I also want to thank Denis Nikitenko, who took the brunt of my stress, and I will be spending quite a while making it up to him. "iii Work Performed Cells were transformed by our lab previously. Growth and purification were performed by the author. Titration experiments were performed by the author. Photocycle experiments were performed by the author with the help of Andrew Harris. BN-PAGE gel was performed by the author with the help of Rachel Munro. NMR experiments were performed by the author with the help of Xiao Peng and Meg Ward: 1D Nitrogen, 1D Carbon, 2D Carbon-Carbon, 2D NCA. CANCO experiments were conducted together with Vladimir Ladizhansky, processing and analysis was completed by the author. "iv Table of Contents Abstract ……….………………..…..………………………………………………………………….…ii! Acknowledgements ..…………..………………………………………………………………….……iii! Work Performed …………………..………………………………………………………………….…iv! Chapter 1: Introduction …………………………………………………………………………….……1! 1.1 Lipid Protein Interactions…………….……………….……………………………….……2! 1.1.1 Membranes.…………………….…………………………….…………….…….2! 1.1.2 Proteins in Membranes………………………..………………………….…..…3! 1.1.3 Hydrophobic Mismatch………………………..………………………………..5! 1.2 Proteorhodopsin…………………………….…………………………………….………..8! 1.3 Marine Bacteria…………….………….…………………………………………………..10! 1.4 Rationale for Our Experiments..………………….….…………………………………..12! Chapter 2: Methods ……………………………………………………………………………………16! 2.1 Production of GPR…………………………….…………………………………….…….17! 2.1.1 Growth…….….…….……….……………………………………….……………17! 2.1.2 Purification……………………….……………………………………………….18! 2.1.3 Reconstitution……………………….……………………………………………19! 2.1.4 Bu$ers…………….……………….…..………………….………………………19! 2.2 Titration Curve…………………………….……………….……….………………………20! 2.3 FTIR…………………………….……..………………….………………………………….21! 2.4 Nuclear Magnetic Resonance Spectroscopy……………….……….…………………22! 2.4.1 NMR Experiment…………….…….….…………………………….…………..22! 2.4.2 Chemical Shift…………………………………………………….……………..23! 2.4.3 Chemical Shift Anisotropy…………………..….………………………………23! 2.4.4 Dipolar Interaction…………..………….…………………………….…………24! 2.4.5 J-coupling…………….…….….…………………………….…………………..24! 2.4.6 Magic Angle Spinning (MAS)…….…….………….…………….……………..24! 2.4.7 Recoupling techniques……………..……….………………….………………25! s 2.4.7.1 Cross-Polarization……………..…….………………………………25! 2.4.7.2 Chemical Shift Correlation Spectroscopy…………………………25! 2.4.8 NMR Experiments……………..……….…………………………….…………26! 2.4.9 NMR Experimental Conditions…..……………..……….…………………….27! Chapter 3: Results ……………………………………………………….………………..……………30! 3.1 Membrane System Stability Experiments……..………………….………………..……31! 3.2 Titration Curve………….…………….…………………………….………………………37! "v 3.3 Laser Spectroscopy Photocycle Experiment………….…………….………………….38! 3.4 Oligomerization State Experiment………………………..…….………………………..41! 3.5 Discussion…………………………….…….…………………..………………………….42! 3.6 NMR…………………………….……………………….…………………………….…….44! 3.6.1 3D Experiments………..…………….…………………………….……………44! 3.6.2 Secondary Structure Investigation……..……………………………………..51! 3.6.3 NMR Discussion…………………….………………………..…….……..…….52! Chapter 4: Conclusions and Future Work …………………….…………………………………..…55! 4.1 Conclusions…….…………………………………………………………………………..56! 4.2 Future Work…………………………………………………………………………………57! "vi List of Tables and Figures Table 1: Synopsis Table……….………..………………………………….……………………………………………………………………36! Figure 1: The surface of the bacteriorhodopsin molecule showing different bound lipids molecules………………………….…4! Figure 2: How lipids may contort to minimize hydrophobic mismatch……………………………………….…………………………6! Figure 3: Photocycle of PR as described by Lindholm et al (2015)…………………………………………….…….………..…………9! Figure 4: Representation of MAS in the SSNMR experiment…….………………………………………………………………………24! Figure 5: Pulse program used in the 3D CANCO experiment….….……………………………………………..………………………26! Figure 6: Baseline-corrected FTIR spectra of GPR Controls.………………………………………………..……………..……………32! Figure 7: Stability of different GPR membrane systems over time using PC headgroups and different length unsaturated acyl tails………………………………………………………………..…………………….…………….………………………32! Figure 8: Stability of different GPR membrane systems using DOPCDOPS (9:1) over several days..………………….…………33! Figure 9: Stability of different GPR membrane systems using DOPE:DOPG (9:1) over time.…………….…..……………………35! Figure 10: Stability of different GPR membrane systems using DOPE:DOPG at different ratios of PE:PG over 13 days.…….36! Figure 11: Titration Curves of GPR in Different Membrane Systems……………….…………………………………………..………37! Figure 12: Absorption Spectra of GPR in in DMPC:DMPA (9:1) (Green) and DOPE:DOPG (7:3) (Purple) at 1:2 P:L at pH 5.5 (A) and pH 9 (B)…………………………………………………………………………………………………………………………..38! Figure 13: Laser spectroscopy of the mixtures of intermediates of the GPR photocycle……………………….…………..……..39! Figure 14: Comparison of laser spectroscopy scans of photocycle intermediates of GPR……….……………….….…………..39! Figure 15: Comparison of laser spectroscopy scans of 600 nm K/O intermediate (A) and 400 nm M intermediate (B) of GPR ……………………….……………………………………………………………………………………….………………………..40! Figure 16: BN-PAGE gel of GPR in different reconstitution states………………….…………………………………………………..41! Figure 17: Examples of minimal chemical shifts between GPR in DMPCDMPA (9:1) at 1:2 P:L (RED) and GPR in DOPEDOPG (7:3) at 1:2 P:L (BLACK).……….……………………………………………………………………………..…………….…..45! Figure 18: Ca chemical shift differences……………………………………………………………………………………………………..45! Figure 19: CO chemical shift differences……………………………………………….….………………………………………………..46! Figure 20: Nitrogen chemical shift differences……………….……….……………………..……………………………………..………47! Figure 21: Examples of large nitrogen chemical shifts between GPR in DMPCDMPA (9:1. (w/w)) at 1:2 (w/w) P:L (RED) and GPR in DOPEDOPG (7:3 (w/w)) at 1:2 (w/w) P:L (BLACK)………….…………………………………..……………..……48! Figure 22: Structure of BPR where homologous residues with chemical shift differences larger than 0.8 ppm are highlighted in red and labeled….……………………………………………………………………….……..………………………………49! Figure 23: Representation of the counter-ion complex in Proteorhodopsin…..…………….…………………..……………………50! Figure 24: Secondary structural analysis using Ca shifts compared to Random coil values from BMRB………………………51% "vii Abbreviations List: Amp = Ampicillin! Chl = chloramphenicol! DDM = n-Dodecyl &-D-maltoside! DMPA = 1,2-dimyristoyl-sn-glycero-3-phosphate ! DMPC = 1,2-dimyristoyl-sn-glycero-3-phosphocholine ! DMPCDMPA = PR in a Mixed-lipid system at a 1:2 P:L ratio containing DMPC and DMPA at a 9:1 w/w ratio ! DOPE = 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine! DOPG = 1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (sodium salt)! DOPEDOPG = PR in a Mixed-lipid system at a 1:2 P:L ratio containing DOPE and DOPG! E. coli = Escherichia coli FTIR = Fourier-transform infrared spectroscopy GPR = green-absorbing proteorhodopsin ! IPTG = Isopropyl-&-D-thiogalactoside! MAS = magic angle spinning! P:L = protein to lipid ratio! PAGE = Polyacrylamide Gel Electrophoresis! PC = a lipid with a phosphatidylcholine headgroup! PR = proteorhodopsin! UCN = uniformly labeled 13C and 15N! w/w = weight per weight% "viii Chapter 1: Introduction "1 1.1 Lipid Protein Interactions! 1.1.1 Membranes! Membranes are used in biology to define the space of the cell. The membrane encases all the substances required for life, and the surface is used to interact with the outside world. Everything on the interior of the space defined by the membrane is directed by proteins transcribed by the genetic material. The membrane defines a living being as being separate from another. Biological membrane systems are made up of phospholipids, integral membrane proteins, glycolipids, sphingolipids, phosphoglycerides, surface membrane proteins, sterols such as cholesterol, and many lipid-soluble components such as vitamin E. Di$erent membranes have di$erent thicknesses, which is dependent on acyl