Studies of Proteorhodopsin to Investigate Transmembrane Protein Function and Dynamics

Transmembrane proteins represent a vital class of biomolecules at the cell surface that accomplish many critical tasks, including sensing and transport. In spite of their physiological importance, they historically have eluded traditional biophysical techniques. As transmembrane proteins are largely hydrophobic, to fold properly and carry out their functions they require a complex physical and chemical environment, encompassing water and amphiphilic lipid surfactant molecules as well as similarly embedded or water-soluble proteins.;Here, we seek to understand the fundamental features underlying the function of these proteins by the study of Proteorhodopsin (PR), a light-driven proton pump from marine bacteria. PR represents a recently-discovered mode by which abundant oceanic micro-organisms can harvest solar energy, and its structure is of the class of seven-helical transmembrane (7TM) proteins, which includes many physiologically important receptors for higher organisms. PR is therefore an interesting biological system from a variety of perspectives, with intriguing ecological, technological, and basic science facets that have implications towards a general understanding of how membrane proteins work. The theme of the work presented in this dissertation is to dissect the complex "environmental" interactions occurring in the cell membrane (i.e. protein-protein, protein-surfactant) through the use of spectroscopic techniques that capture molecular details of motion, or dynamics, and protein function, which for the light-absorbing PR is accessible through optical absorption measurements.;There is a particular focus on the unique measurement of water diffusion at the surface of the protein, available through recent developments of the Overhauser dynamic nuclear polarization (ODNP) technique, which is introduced here as a method to monitor conformational changes of PR during the photoactivation process distinctly from the point of view of hydration water. ODNP proves successful for both mapping an alpha-helical secondary structure element within an intracellular loop of PR by quantification of residue-site-specific hydration and also is coupled to electron paramagnetic resonance (EPR) measurements of protein side-chain dynamics to obtain a comprehensive snapshot of conformational change.;With this glimpse into the mechanical aspect of membrane protein function, we obtain a signature of molecular motion that could then be applied to observe conformational dynamics upon tuning protein-protein interactions. The in-depth studies of PR function that follow from the separation of oligomeric forms of PR from the monomeric protein reveal that the surface protein-protein contacts conferred by oligomerization have critical functional consequences. These are observable both by an altered timescale of conformational motion as well as from a change in optical absorption behavior propagating from inside the protein, and have a stronger influence than using different surfactant membrane-mimetic micelle environments for PR. The modulation of PR function through protein-protein interactions is also explored within the context of a more native-like lipid bilayer, as it is found that oligomers can be trapped and characterized by chemical crosslinking. Upon further probing the effect of surfactant on PR dynamics and function, we identify the micelle vs. bilayer environment to have altered hydration properties. These changes in surface water dynamics are coupled with functional changes for PR, and implicate a role for water in supporting transmembrane protein function.;Altogether, the PR studies presented here connect transmembrane protein function to two critical types of interactions that are often overlooked relative to the influence of the expansive lipid bilayer, or are difficult to capture experimentally: protein-water and protein-protein (oligomeric) interactions. This work demonstrates that hydration water can serve as a probe of transmembrane protein function as it participates in the process of conformational change and is affected by altering the surfactant environment in ways that are known to be functionally significant. Similarly, oligomerization emerges as a significant modulator of trasmembrane protein function that should be considered in forthcoming investigations in this particularly exciting area of membrane protein research.