Maia Kinnebrew

OLIGOMERIZATION AND ITS EFFECT ON FUNCTION IN 7-TRANSMEMBRANE PROTEINS

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, different structural forms that 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. Timeresolved electron paramagnetic resonance (EPR) and optical absorption experiments show that the hexameric state of PR has much slower photocycle dynamics than the monomeric state. Varying surfactant environment appears to have less of an effect on kinetics and function than does

Summer 2013

Oligomerization And Its Effect On The Function Of A 7-transmembrane Proton Transporting Protein 

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, different structural forms that 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. Varying surfactant environment appears to have less of an effect on kinetics and function than does oligomerization—an important result towards investigating similar effects in mammalian membrane proteins.