Abstract
The plasma membrane is the crucial interface between the cell and its exterior, packed with embedded proteins experiencing simultaneous protein–protein and protein–membrane interactions. A prominent example of cell membrane complexity is the assembly of transmembrane proteins into oligomeric structures, with potential functional consequences that are not well understood. From the study of proteorhodopsin (PR), a prototypical seven-transmembrane light-driven bacterial proton pump, we find evidence that the inter-protein interaction modulated by self-association yields functional changes observable from the protein interior. We also demonstrate that the oligomer is likely a physiologically relevant form of PR, as crosslinking of recombinantly expressed PR reveals an oligomeric population within the Escherichia coli membrane (putatively hexameric). Upon chromatographic isolation of oligomeric and monomeric PR in surfactant micelles, the oligomer exhibits distinctly different optical absorption properties from monomeric PR, as reflected in a prominent decrease in the pKa of the primary proton acceptor residue (D97) and slowing of the light-driven conformational change. These functional effects are predominantly determined by specific PR–PR contacts over nonspecific surfactant interactions. Interestingly, varying the surfactant type alters the population of oligomeric states and the proximity of proteins within an oligomer, as determined by sparse electron paramagnetic resonance distance measurements. Nevertheless, the dynamic surfactant environment retains the key function-tuning property exerted by oligomeric contacts. A potentially general design principle for transmembrane protein function emerges from this work, one that hinges on specific oligomeric contacts that can be modulated by protein expression or membrane composition.
Highlights
The cell membrane is a hydrophobic barrier that is vital to life, studded with proteins that fulfill the important role of relaying substances and information from the cell’s environment to its interior and vice versa
The association of multiple transmembrane helices within the membrane has been hypothesized to be a general mode of membrane protein assembly[2] and the analogous formation of oligomeric structures has been experimentally found in archaea, bacteria, and eukaryotes.3; 4; 5 Some valuable molecular-level guidelines for the formation of transmembrane protein-protein contacts have emerged from prior structural studies.6; 7; 8
PR displays the well-documented tendency of transmembrane proteins to migrate at rates faster than expected for their true size during SDS-PAGE, recently established to be variable due to detergent-binding and acrylamide concentration.21; 22 The resulting discrepancy in apparent molecular weight is caused by interactions of the protein with the anionic SDS surfactant, which can mediate the dissolution of oligomeric contacts.[23]
Summary
The cell membrane is a hydrophobic barrier that is vital to life, studded with proteins that fulfill the important role of relaying substances and information from the cell’s environment to its interior and vice versa. Since transmembrane proteins occupy a significant portion of the cell surface (from 20–80%1), there is a need to understand whether there is a direct functional effect of their assembly into dimers or higher-order oligomers. The association of multiple transmembrane helices within the membrane has been hypothesized to be a general mode of membrane protein assembly[2] and the analogous formation of oligomeric structures has been experimentally found in archaea, bacteria, and eukaryotes.3; 4; 5 Some valuable molecular-level guidelines for the formation of transmembrane protein-protein contacts have emerged from prior structural studies.6; 7; 8. Any dominant and direct functional effect of transmembrane protein oligomerization can be difficult to separate from the other coexisting, and potentially correlated, environmental factors, especially if the oligomeric contacts are weak
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