BIOPHYSICAL CHARACTERIZATION OF CHEMICALLY UNFOLDED STATES OF THE MEMBRANE PROTEIN RHODOPSIN

Membrane proteins function as important communication channels of the cell and its environment that aid in regulating the overall homeostasis of organisms. Understanding the pathways by which these proteins adopt their three-dimensional structures can provide us with key insights into their functions. Failure of a membrane protein to fold into its native structure can lead to disruption of their functions and cause diseases. Through an understanding of the folding mechanisms of membrane proteins it may be possible to identify avenues for the treatment of such diseases. Towards these goals, this thesis describes the biophysical characterization of denatured states of rhodopsin, a model system selected to study helical membrane protein folding. The first contribution of this thesis was to establish approaches that can be used to identify suitable conditions for studying membrane protein folding in vitro. This required screening different denaturing conditions to obtain maximum unfolding without causing aggregation of rhodopsin. 30% SDS and 3% SDS + 8 M urea were found to be the most suitable denaturing conditions. Next, structural features of largely unfolded states of rhodopsin under optimized denaturing conditions were systematically characterized focussing on three levels of structural resolution: global, local and site-specific. Global tertiary structure changes upon SDS denaturation were observed to correlate with SDS micellar structure changes and also hinted at formation of compact intermediate states. Local structural dynamics, probed by NMR spectroscopy, showed that the cytoplasmic domain is more flexible than extracellular and transmembrane domains taken together in spite of an overall increase in flexibility with denaturation. Mobility studies probing site-specific changes by EPR spectroscopy, showed that specific extracellular residues retain more rigidity than cytoplasmic residues in denatured states. These results indicate that the former domain is invol