BIOPHYSICAL MEASUREMENTS OF MEMBRANE STRUCTURE

我们的长期目标是确定脂质结构之间的关系

Our long-term goal is to identify relationships between lipid structure and functional properties of membranes, with a particular emphasis upon understanding the enzymatic regulation of energy transducing integral membrane proteins. An important aspect of this work is the further development of new spectroscopic probes to measure lipid structural properties, and the use of complimentary spectroscopic techniques (i.e., fluorescence resonance energy transfer, fluorescence anisotropy, and spin-label EPR methods) to eliminate ambiguities regarding the interpretation of experimental data. As a.first step aimed at understanding the fundamental physical forces that modulate membrane structure, we propose to utilize model membranes composed of well defined single or binary mixtures of lipids. These simple systems will allow us to systematically investigate the underlying molecular relationships that influence the macroscopic properties of the membrane. We will Investigate possible relationships between i) the conformation of individual phospholipid acyl chains, ii) the rotational dynamics at defined positions on the phospholipid hydrocarbon chain, iii) structural changes regarding the conformation of phospholipid headgroups, and iv) the phase transition temperature(s) of the bilayer, which provide a simple physical measure regarding the stabilizing interactions between individual membrane lipids. A second theme, and ultimate goal of the project, is to apply these methods in order to gain insight into fundamental regulatory mechanisms whereby changes in lipid structural properties modulate the function of membrane proteins. We will primarily utilize the well characterized Ca-ATPase found in sarcoplasmic reticulum membranes obtained from skeletal muscle that, in addition to its important role in regulating intracellular calcium levels, serves as a model system in which to directly assess fundamental mechanisms whereby active transport proteins efficiently couple ATP hydrolysis to the physical movement of ions against a concentration gradient. This will involve measurements of both the lipid and protein structural properties, subsequent to the incorporation of the Ca-ATPase into defined lipid environments. In addition to the measurements of lipid structure already mentioned, we will measure the protein's conformation and dynamics taking advantage of i) intrinsic fluorophores (i.e., tryptophans) located on the transmembrane helices of the Ca-ATPase that provide a direct measure regarding changes in the conformation of the calcium ion channel, and ii) site-directed extrinsic chromophores covalently bound to the cytoplasmic portion of the Ca-ATPase in order to measure both Intramolecular conformational changes of the ATPase associated with the enzymatic coupling between spatially distant nucleotide and calcium sites, as well as oligomeric interactions between ATPase polypeptide chains. This comprehensive approach will allow us to identify the functionally relevant interplay between membrane lipid and protein structure. In addition, an understanding of the physical mechanism regulating this relatively simple ion transport protein should provide fundamental insights into how the functional properties of other membrane transport and channel proteins are regulated.