Adenosine A2a receptors form distinct oligomers in protein detergent complexes
N.S. Schonenbach, M.D. Rieth, S. Han, and M.A. O’Malley, FEBS Lett 590, 3295 (2016). DOI:10.1002/1873-3468.12367
The human adenosine A2a receptor (A2aR) tunes its function by forming homo-oligomers and hetero-oligomers with other GPCRs, but the biophysical characterization of these oligomeric species is limited. Here, we show that upon reconstitution into an optimized mixed micelle system, and purification via an antagonist affinity column, full length A2aR exists as a distribution of oligomers. We isolated the dimer population from the other oligomers via size exclusion chromatography and showed that it is stable upon dilution, thus supporting the hypotheses that the A2aR dimer has a defined structure and function. This study presents a crucial enabling step to a detailed biophysical characterization of A2aR homodimers.
Functional Consequences of the Oligomeric Assembly of Proteorhodopsin
S. Hussain, M. Kinnebrew, N.S. Schonenbach, E. Aye, and S. Han, J Mol Biol 427, 1278 (2015). DOI: 10.1016/j.jmb.2015.01.004
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), we find evidence that the inter-protein interaction modulated by selfassociation 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 E. 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 as well as the proximity of proteins within an oligomer, as determined by sparse electron paramagnetic resonance (EPR) 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 tuning emerges from this work, one that hinges on specific oligomeric contacts that can be modulated by protein expression or membrane composition.
Transmembrane Protein Activation Refined by Site-Specific Hydration Dynamics
S. Hussain, J.M. Franck, and S. Han, Angewandte Chemie International Edition 52, 1953 (2013). DOI: 10.1002/anie.201206147
Transmembrane proteins are the gatekeepers of the cell—channels that allow ions and molecules to enter or exit, or receptors that respond to their surroundings to change conditions inside the cell. The conformational dynamics of membrane protein activation are key to understanding the details of their function, and yet such information has proven to be difficult to obtain because of the experimental challenges involved in obtaining dynamics information for large hydrophobic protein complexes. Various magnetic resonance techniques, including magic‐angle‐spinning solid‐state NMR (ssNMR) spectroscopy, NMR relaxation measurements, and electron paramagnetic resonance (EPR) spectroscopy, are at the frontier for capturing elusive details of membrane protein structure and dynamics. Here, we demonstrate the use of a novel combination of methods to present a dynamics‐based picture that relates the structure of a membrane protein segment to the functional movement it supports. This is achieved by observing the surrounding hydration water, which rearranges simultaneously with protein conformational changes incurred upon activation.