Summary. 
A quantitative understanding of the structure and function of biological membranes, including the physical chemistry of the membrane-water interface, lipid-protein interactions, and bilayer dynamics, lies at the heart of the general field of membrane biophysics.  Members of the Sakmar Laboratory apply principles of membrane biophysics to study how heptahelical receptors function in biological membrane bilayers.  In particular, they use advanced analytical tools to probe bilayer structure and the interplay between membrane and GPCRs.  Some of the key questions being addressed include the mechanism of receptor oligomerization in bilayers.  Is receptor oligomerization a self-assembly process driven by the physical properties of the bilayer, or is it some intrinsic property of the proteins themselves?  Can proteins assemble into specific heteromeric structures, or are the higher-order structures random?  What is the functional consequence of GPCR oligomerization?  Also, what is the precise complex that forms in the membrane that defines the “signalosome” – the minimum unit required to transmit the signal of ligand binding outside of the cell to signal output inside of the cell?  Membrane biophysics is a demanding interdisciplinary field that is well suited for the Sakmar Laboratory team environment. 

Membrane Curvature and Hydrophobic Forces Drive Receptor Assembly.  Members of the Sakmar Laboratory, working in collaboration with Prof. Michael F. Brown, University of Arizona, demonstrated how association or oligomerization of rhodopsin occurs by long-range lipid-protein interactions due to geometrical curvature forces in model bilayers.  In membrane biophysics studies of GPCR oligomerization in cellular membranes, as well as proteins in lipid rafts, fluorescence energy transfer (FRET) has emerged as a pivotal tool.  FRET studies of fluorescent protein-tagged GPCRs in native cellular membranes have provided evidence for significant receptor association in living cells.  Alternatively, for glycosylphosphatidylinositol (GPI)-linked proteins, FRET microscopy indicates a random (ideal) distribution – a finding that is seemingly incompatible with protein clustering in lipid rafts.  What is lacking, however, are investigations of well-defined membrane systems where lipid-protein interactions can be distinguished from protein-protein interactions, for example as seen in the crystal structure of rhodopsin. 
Using FRET (fluorescence resonance energy transfer), rhodopsin function was measured simultaneously with its degree of association with other receptors. 
Rhodopsin oligomerization was promoted by a reduction in membrane thickness (hydrophobic mismatch) and also by an increase in the protein/lipid molar ratio, showing the importance of interactions extending well beyond a single annulus of protein boundary lipids.  The non-random mixing of rhodopsin was correlated with its photoactivation, suggesting a link to visual phototransduction.  Both association and photoactivation of rhodopsin were strongly influenced by the protein packing density within the membrane as well as hydrophobic matching.  Crowding of receptors decreased the efficiency of signal transduction in the model membrane bilayers.  A more dispersed membrane receptor environment optimized the active protein conformational.  This important initial work provides a conceptual framework for understanding how the tightly regulated lipid compositions of cellular membranes can influence the functions of membrane proteins.

Thermodynamics of Ligand Binding to Rhodopsin in Bilayers.  Many GPCRs, including visual pigments such as rhodopsin, bind to intensely hydrophobic ligands.  Most classical pharmacology models of ligand-receptor interaction are over-simplifications that neglect the effect of the bilayer and assume equilibrium, or pseudo-equilibrium, states that are not likely to exist in actual biological reactions.  Members of the Sakmar Laboratory had employed interdisciplinary methods of membrane biophysics to attempt to understand how hydrophobic ligands bind to their receptors in membranes?  The initial assumption was that these ligands must partition from the aqueous phase into the hydrophobic core of the bilayer and then enter the receptor’s ligand-binding pocket through a flexible pore in the receptor.  Furthermore, it was assumed that the pore might have features of a selectivity filter that might determine the kinetic rate constants for the binding reaction, but not the binding energy for the reaction.   
Using rhodopsin as a model system, a combination of mutagenesis experiments and molecular dynamics (MD) simulations suggested an intramembranous pathway and a ligand gating mechanism that involved a conserved motif between transmembrane (TM) helices 5 and 6.  Based on an all-atom rhodopsin model in a phospholipid bilayer membrane, Thomas Huber, a member of the Sakmar Laboratory, performed multi-nanosecond free energy calculations of ligand binding.  The free energy landscape was calculated as a potential of mean force (PMF) along the binding pathway from reversible MD simulations using umbrella potentials and the weighted histogram analysis method (WHAM).