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.

Characterization of an Unconventional Rhodopsin from the Freshwater Actinobacterium Rhodoluna lacicola

“…However, because the cells do not synthesize retinal, an alternative role for the actinorhodopsin apoprotein cannot be ruled out. Many microbial rhodopsins oligomerize in the membrane and form large aggregates (55)(56)(57)(58)(59)(60). Without a cofactor, actinorhodopsin might still aggregate in the membrane, providing structural stability against membrane stresses.…”

The light‐driven sodium ion pump: A new player in rhodopsin research

“…3A); in contrast, it is suggested that KR2 forms pentamers (Fig. 3B), and PRs assemble into pentamers or hexamers [32,[38][39][40] (Fig. 3C).…”

Determining the Oligomeric Structure of Proteorhodopsin by Gd3+-Based Pulsed Dipolar Spectroscopy of Multiple Distances

“…G-PR likely acts as a light-driven proton pump that enables bacteria to harvest solar energy in a useable form (DeLong and Bé jà , 2010), but it displays many unique properties that suggest differences in mechanism or function from the well-studied bacteriorhodopsin proton pump (Hempelmann et al, 2011;Lö rinczi et al, 2009;Schä fer et al, 2009). Prior studies have demonstrated that G-PR assembles into hexamers in both 2D crystalline lipids (Klyszejko et al, 2008) and detergent micelle environments (Hoffmann et al, 2010;Stone et al, 2013). Although the functional implications of this assembly are still being investigated, few precise details of the G-PR oligomer structure are currently available.…”

“…A recent crystal structure (Ran et al, 2013) reveals a doughnutshaped hexameric assembly for blue-absorbing proteorhodopsin (B-PR), a protein that is homologous to G-PR but differs in its maximum absorption wavelength and photocycle timescale (Hillebrecht et al, 2006;Xi et al, 2008). However, in G-PR hexamers, there has been only a single measurement of a short interprotein distance, which identified a radial orientation of the protein within the hexamer (Stone et al, 2013).…”