UC Santa Cruz

The native lipid environment is essential to the functional integrity of membrane proteins. Obtaining detailed information about the dynamics, mechanisms of action and structures of membrane proteins can be challenging, as traditional methods of solubilizing biological membranes can affect the preservation of surrounding lipid molecules. Novel solubilizing agents have been developed to maintain the stability of membrane proteins and improve the application of various biophysical techniques to study them. Unfortunately, in many cases, it is not clear if these solubilizing agents provide a truly natural environment or if they affect the functional properties of membrane proteins. We address this question by comparing the photoactivation kinetics of rhodopsin, a G protein-coupled receptor (GPCR) in the disc membranes of rod cells, in native membrane suspensions, and in the solubilized state. Rhodopsin provides an ideal model system to investigate the functional integrity of a membrane protein because its activation mechanism is initiated with light, and the progression of the reaction intermediates can be monitored with absorption spectroscopy. The kinetics associated with rhodopsin intermediates are well documented at ambient temperature, so we can characterize any deviation to determine the effects due to solubilizing agents. Amphipathic copolymers, detergents, and membrane scaffold proteins were investigated to evaluate the most suitable environment for studies of rhodopsin photokinetics. Styrene maleic acid and diisobutylene maleic acid copolymers significantly slowed the reaction progress of rhodopsin, especially at late stages where significant conformational changes occur. While the highest styrene maleic acid/rhodopsin ratios yielded the most solubilized protein, the rhodopsin produced under these conditions could not reach the active (Meta II) state upon photoactivation and the changes to the protein were not reversible. In contrast, rhodopsin in lauryl maltose neopentyl glycol detergent micelles and membrane scaffold protein nanodiscs present native membrane-like intermediates without perturbing the activation sequence. The results presented in this dissertation may apply to many membrane-bound GPCR proteins.