MSc Thesis Presentation: Adaptation of Green Proteorhodopsin to Changes in Membrane Lipid Composition

MSc Thesis Presentation

Candidate: Rachel Brown

Abstract

Proteorhodopsin (PR) is a commonly studied membrane protein that pumps protons from the cytoplasm to the periplasm. It is implicated in energy generation using light. Previous studies suggested minimal functional changes in PR when reconstituted into different membrane systems [1]. This work examines PR membrane system stability using FTIR. The stable membrane systems, found using FTIR, are further examined using NMR, Laser Spectroscopy, and UV-Vis spectroscopy. The findings show lipid loss over time for PR membrane systems at 1:2 (w/w) protein to lipid ratios that contained only PC lipids, as well as for DOPC/DOPS mixture. Lipid membrane systems that contained DOPE/DOPG showed no lipid loss over time. DOPE/DOPG at a 7:3 (w/w) ratio of PE to PG was compared to DMPC/DMPA at a 9:1 (w/w) ratio of PC to PA, both of which showed stability over time and were chosen for further experiments. These systems mimic the changes involved in the transition from non-starvation state to starvation state, which is a known phenomena in marine bacteria. Photocycle and Titration experiments provide evidence of a shift in the pKa of D97, where DMPC/ DMPA has PR in the basic (functional) form at a lower pH compared to DOPE/DOPG. MAS SSNMR experiments were conducted to explore the structural changes involved in the pKa shift between the two systems. CANCO experiments show minimal changes in observable residues. Possible locations for further experiments include the Counterion complex, which includes the residues W34, H75, D227, Y200, and D97. Oligomerization could be a possible cause for the change in this complex, as H75 is involved both in the counter-ion complex and is an oligomeric contact.

[1] Lindholm, L., Ariöz, C., Jawurek, M., Liebau, J., Mäler, L., Wieslander, Å., et al. (2015). Effect of lipid bilayer properties on the photocycle of green proteorhodopsin. Biochemica Et Bioshyica Acta, 1847(8), 698–708. http://doi.org/10.1016/j.bbabio.2015.04.011

Examination Committee:

• Dr. Joanne O'Meara, Chair
• Dr. Leonid Brown, Advisor
• Dr. Steffen Graether
• Dr. John Dutcher