Membrane protein function is regulated by changes in the host membrane's lipid composition. This regulation occurs through mechanisms ranging from specific (chemical) interactions between proteins and individual lipid molecules, to non-specific (physical) interactions between proteins and the bilayer behaving as a structure with collective physical properties (e.g., thickness, intrinsic monolayer curvature and elastic moduli). The proposed studies focus on the physical regulation of membrane protein function with special emphasis on how drugs, drug candidates and other molecules that are used to manipulate cell function, alter lipid bilayer properties, as sensed by a bilayer-spanning channel. Such physical regulation of function occurs because hydrophobic interactions between a membrane protein and its host bilayer, couples the protein conformational preference to the bilayer physical properties. This coupling occurs because bilayer-spanning proteins perturb their surrounding bilayer, which incurs an energetic cost, the bilayer deformation energy (?Gdef), where ?Gdef varies with changes in the host lipid bilayer physical properties. Conformational changes that involve the protein/bilayer interface will alter the packing and dynamics of the adjacent lipids, such that the difference in bilayer deformation energy between two protein conformations (I and II) becomes the bilayer contribution (?GbilayerI->II = ?GdefII - ?GdefI) to th free energy difference (?GtotalI-II) for the conformational transition. Membrane protein function varies with changes in lipid bilayer properties that alter ?GbilayerI-II. The experiments will combine fluorescence quench single-channel electrophysiology measurements to explore how changes in lipid bilayer properties alter the energetics and kinetics of gramicidin (gA) channel formation, where the gA channels are probes for changes in membrane properties. The focus will be how libraries of drugs, drug candidates, and other biologically active molecules alter lipid bilayer properties, have "membrane effects", as sensed by bilayer-spanning channels. This is important because many biologically active molecules are amphiphiles, meaning that they will alter lipid bilayer properties at some concentration and thus become promiscuous modifiers of membrane protein function. The screen for bilayer modifying potency will be done using fluorescence quench measurements to determine how different molecules alter the gA monomer dimer equilibrium, which allows for quantification of the compound-induced change in ?GbilayerM->D, the bilayer contribution to the free energy of dimerization. This provides for quantitative information about the bilayer-modifying potency of diverse molecules and the concentration range where a compound is "safe," where it has little membrane effect. Selected molecules will be studied using single channel methods, to determine which bilayer properties that have been altered. These experiments provide a foundation for the development of novel screens of drug candidates, as well as systematic approaches to understand, and eventually avoid (or exploit), the membrane effects caused by biologically active molecules.
Many drugs, drug candidates and other molecules that are used to manipulate biological function have physico-chemical properties that will cause them to adsorb to lipid bilayers and thereby alter lipid bilayer properties. This is important because the lipid bilayer is a common feature among all integral membrane proteins, meaning that drug-induced changes in lipid bilayer properties may cause a drug to alter the function of a large number of off-target membrane proteins in addition to the function of their desired target(s). The proposed studies are aimed at understanding the bilayer-modifying effects of clinically and biologically important molecules, as experienced by a bilayer-spanning channel. This important for public health because the experimental paradigm that is used in these studies will allow for the identification of molecules that are likely to have unacceptable off-target effects, and at wha concentrations, which will be important for future drug development because it will provide a mechanistic basis for the development of novel screens of drug candidates.
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