Protein-Protein and Protein-Lipid Interactions Tune Proteorhodopsin Function by Altering Water Dynamics

Abstract

Currently, the role of water and the membrane assembly in tuning the function of seven-helical transmembrane (7TM) proteins is not well-understood. Here, we focus on the light activation and functional properties of a prototypical example, the Proteorhodopsin (PR) proton pump from marine bacteria, observing how the protein and surrounding hydration water rearrange upon activation. This is made possible by the application of the powerful residue-specific magnetic resonance methods of electron paramagnetic resonance (EPR), which measures protein segment mobility, and Overhauser dynamic nuclear polarization (ODNP), as recently developed for probing local water diffusivity within 10 Å of a nitroxide spin-label. We investigate further how these dynamics are affected by the surrounding environment, encompassing both protein-protein and protein-lipid interactions. With these techniques together with optical absorption spectroscopy, we find that water dynamics (both ps scale translational motion and ns scale “bound” water) at the membrane protein surface is dramatically affected by the lipid bilayer or surfactant micelle environment. Furthermore, hydration is correlated to functional changes such that water could modulate the timescale of conformational motion. Specifically, the slowdown of translational water motion at the membrane surface, coupled to a lack of bound waters, may facilitate proton uptake by PR. The association of PR with other PR molecules within the membrane, or oligomerization, has similar functional consequences in addition to effects on the protonation properties of key residues for ion transport (pKa of D97). The implication of our study is that PR-PR association alters the hydrogen bond network within the channel, possibly mediated by an altered interaction with the surfactant and surface hydration water upon oligomerization. This result, combined with the homology of PR with sensory receptors, elicits the intriguing possibility that PR has a functional flexibility mediated by oligomerization.