Michael F Brown
Publications
Rhodopsin is an important example of a G protein-coupled receptor (GPCR) in which 11-cis-retinal is the ligand and acts as an inverse agonist. Photolysis of rhodopsin leads to formation of the activated meta II state from its precursor meta I. Various mechanisms have been proposed to explain how the membrane composition affects the meta I-meta II conformational equilibrium in the visual process. For rod disk membranes and recombinant membranes containing rhodopsin, the lipid properties have been discussed in terms of elastic deformation of the bilayer. Here we have investigated the relation of nonlamellar-forming lipids, such as dioleoylphosphatidylethanolamine (DOPE), together with dioleoylphosphatidylcholine (DOPC), to the photochemistry of membrane-bound rhodopsin. We conducted flash photolysis experiments for bovine rhodopsin recombined with DOPE/DOPC mixtures (0:100 to 75:25) as a function of pH to explore the dependence of the photochemical activity on the monolayer curvature free energy of the membrane. It is well-known that DOPC forms bilayers, whereas DOPE has a propensity to adopt the nonlamellar, reverse hexagonal (H(II)) phase. In the case of neutral DOPE/DOPC recombinants, calculations of the membrane surface pH confirmed that an increase in DOPE favored the meta II state. Moreover, doubling the PE headgroup content versus the native rod membranes substituted for the polyunsaturated, docosahexaenoic acyl chains (22:6 omega 3), suggesting rhodopsin function is associated with a balance of forces within the bilayer. The data are interpreted by applying a flexible surface model, in which the meta II state is stabilized by lipids tending to form the H(II) phase, with a negative spontaneous curvature. A simple theory, based on principles of surface chemistry, for coupling the energetics of membrane proteins to material properties of the bilayer lipids is described. For rhodopsin, the free energy balance of the receptor and the lipids is altered by photoisomerization of retinal and involves curvature stress/strain of the membrane (frustration). A new biophysical principle is introduced: matching of the spontaneous curvature of the lipid bilayer to the mean curvature of the lipid/water interface adjacent to the protein, which balances the lipid/protein solvation energy. In this manner, the thermodynamic driving force for the meta I-meta II conformational change of rhodopsin is tightly controlled by mixtures of nonlamellar-forming lipids having distinctive material properties.
Lipid bilayers represent a fascinating class of biomaterials whose properties are altered by changes in pressure or temperature. Functions of cellular membranes can be affected by nonspecific lipid-protein interactions that depend on bilayer material properties. Here we address the changes in lipid bilayer structure induced by external pressure. Solid-state ²H NMR spectroscopy of phospholipid bilayers under osmotic stress allows structural fluctuations and deformation of membranes to be investigated. We highlight the results from NMR experiments utilizing pressure-based force techniques that control membrane structure and tension. Our ²H NMR results using both dehydration pressure (low water activity) and osmotic pressure (poly(ethylene glycol) as osmolyte) show that the segmental order parameters (S(CD)) of DMPC approach very large values of ≈ 0.35 in the liquid-crystalline state. The two stresses are thermodynamically equivalent, because the change in chemical potential when transferring water from the interlamellar space to the bulk water phase corresponds to the induced pressure. This theoretical equivalence is experimentally revealed by considering the solid-state ²H NMR spectrometer as a virtual osmometer. Moreover, we extend this approach to include the correspondence between osmotic pressure and hydrostatic pressure. Our results establish the magnitude of the pressures that lead to significant bilayer deformation including changes in area per lipid and volumetric bilayer thickness. We find that appreciable bilayer structural changes occur with osmotic pressures in the range of 10-100 atm or lower. This research demonstrates the applicability of solid-state ²H NMR spectroscopy together with bilayer stress techniques for investigating the mechanism of pressure sensitivity of membrane proteins.