title: Structural insight into proteorhodopsin oligomers.
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.
authors: Department of Chemistry and Biochemistry, University of California, Santa Barbara, Santa Barbara, California, USA.
authors: Katherine M
authors: KM
authors: Stone
authors: Jeda
authors: J
authors: Voska
authors: Maia
authors: M
authors: Kinnebrew
authors: Anna
authors: A
authors: Pavlova
authors: Matthias J N
authors: MJ
authors: Junk
authors: Songi
authors: S
authors: Han
mesh_terms: D002850:Chromatography, Gel; D002853:Chromatography, Liquid; D003545:Cysteine / Q000235:genetics; D003902:Detergents / Q000737:chemistry; D004578:Electron Spin Resonance Spectroscopy; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry; D055503:Protein Multimerization; D020836:Protein Structure, Quaternary; D021122:Protein Subunits / Q000737:chemistry; D012031:Refractometry; D012243:Rhodopsin / Q000737:chemistry*; D025604:Rhodopsins, Microbial; D012542:Scattering, Radiation; D012995:Solubility; D013113:Spin Labels; D013696:Temperature
chemical_list: D003902:Detergents; D050505:Mutant Proteins; D021122:Protein Subunits; D025604:Rhodopsins, Microbial; D013113:Spin Labels; C413548:proteorhodopsin; D012243:Rhodopsin; D003545:Cysteine