Abstract
Oligomerization has important functional implications for many membrane proteins. However, obtaining structural insight into oligomeric assemblies is challenging, as they are large and resist crystallization. We focus on proteorhodopsin (PR), a protein with seven transmembrane α-helices that was found to assemble to hexamers in densely packed lipid membrane, or detergent-solubilized environments. Yet, the structural organization and the subunit interface of these PR oligomers were unknown. We used site-directed spin-labeling together with electron spin-resonance lineshape and Overhauser dynamic nuclear polarization analysis to construct a model for the specific orientation of PR subunits within the hexameric complex. We found intersubunit distances to average 16 Å between neighboring 55 residues and that residues 177 are >20 Å apart from each other. These distance constraints show that PR has a defined and radial orientation within a hexamer, with the 55-site of the A-B loop facing the hexamer core and the 177-site of the E-F loop facing the hexamer exterior. Dynamic nuclear polarization measurements of the local solvent dynamics complement the electron spin-resonance-based distance analysis, by resolving whether protein surfaces at positions 55, 58, and 177 are exposed to solvent, or covered by protein-protein or protein-detergent contacts.