The Effect of Detergent on the Oligomerization of a 7-Transmembrane Protein

Abstract

Membrane proteins serve a broad range of functions necessary for life; in fact, they are coded for by 30% of genes in the human genome. Unfortunately, the complex native membrane is irreproducible for many spectroscopic methods, and the necessary use of solubilizing detergents for biochemical and biophysical characterization can alter protein stability and function. We explore the effects of such environments on Proteorhodopsin (PR), a proton-pumping membrane protein from marine bacteria. PR has significant structural and dynamical commonalities to mammalian seven-transmembrane 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 forms are thought to play a significant role in function. Using fast protein liquid chromatography and optical absorption experiments we show that the hexameric state of PR in the nonionic dodecylmaltoside (DDM) detergent has a lower pKa value for the primary proton-acceptor residue than both the monomeric and dimeric protein, suggesting that the hexamer is more optimized for proton transport. We have also observed this phenomenon in the zwitterionic detergent dodecylphosphocholine (DPC). Next, we studied the environmental effects on PR's oligomeric state, using crosslinking followed by SDS-PAGE, and found hexamers in DDM, DPC, and E. coli membranes expressing PR. Additionally, we examine structure and dynamics using continuous wave Electron Paramagnetic Resonance (EPR). Site-directed spin-labeling EPR elucidates site-specific dynamics and detects short distances (<2 nm), making it useful for studying these complex environments. We thereby determine that a change in detergent type can change side-chain dynamics. Our work lends insight into the study of more complex mammalian proteins by demonstrating the effect of detergent on function, dynamics, and protein-protein complexes.