Oligomerization and its Effect on Function in 7-Transmembrane Proteins

Abstract

Proteorhodopsin (PR) is a solar-powered membrane protein–a proton pump from marine bacteria that has significant structural and dynamical commonalities to seven-transmembrane (7TM) mammalian proteins, including the G-protein Coupled Receptors (GPCRs). Furthermore, PR associates with itself in the membrane to form oligomers similarly to GPCRs, and these different structural forms are thought to play a large role in function. Unfortunately, studying the structure and function of membrane proteins in oligomeric assemblies is very challenging at the molecular level due to the oligomers' large size, disordered nature, and ability to resist crystallization–making PR a facile model system for capturing elusive details of dynamics and function. Past work has revealed PR's hexameric interface in dodecylmaltoside (DDM) micelles, and here we focus on understanding the effect of oligomerization on function. We seek to probe oligomeric interfaces as we vary surfactant environment and oligomeric state. using fast protein liquid chromatography (FPLC) and optical absorption experiments we show that the hexameric state of PR in DDM surfactant has a lower pKa value for the proton accepting residue than both the monomeric and dimeric protein, suggesting that the hexamer is more optimized for proton transport. Time-resolved electron paramagnetic resonance (EPR) and optical absorption experiments show that the hexameric state of PR has much slower photocycle dynamics than the monomeric state. Our work shows that varying surfactant environment appears to have less of an effect on kinetics and function than does oligomerization. Protein-protein interactions therefore have a central role in tuning protein function, a result that lends insight into the functional mechanisms of more complex mammalian membrane proteins.