9406983 Engelman We conceptualize membrane protein stability in terms of a two-stage model: many transbilayer helices can be regarded as independently stable folding units which interact specifically in a side-to-side fashion to generate tertiary and quaternary structures. This contention is supported by experiments and suggests that a detailed understanding of membrane helix interactions will lead to greater understanding of membrane protein structure and to more powerful prediction methods. During the past grant period, we established the independent stability of many (but not all) of the helices of bacteriorhodopsin (BR), evolved a model system permitting detailed computational and experimental studies of the dimerization of glycophorin A (GpA) transmembrane helices, studied the possible roles of polypeptide links and retinal in bacteriorhodopsin stability, and discovered that one bacteriorhodopsin helix is capable of reversible spontaneous insertion across lipid bilayers. These studies have now permitted a more refined investigation to be designed: 1. Spontaneous insertion will be studied using the reversible transbilayer insertion of BR helix C as a starting point. Which sequence features permit or abolish such insertion? Can a pH difference translocate the peptide across a lipid bilayer? Can an experimental delta delta G be assigned to each amino acid? 2. Folding intermediates and alternative domain structures will be studied using BR as a model. Is the helix F - helix G hairpin a stable domain? Do helices A or B interact separately with the rest of the molecule? 3. Structural studies exploiting NMR will be used to define the detailed structure of the glycophorin dimer. Is it as predicted from mutagenesis and computation? 4. Measurement of the energy of helix-helix interactions will be made using sequence variation in the glycophorin helix. These will be used to test computational approaches and to seek chemical principles in the interactio ns. 5. Effects of the lipid environment on the stability of helix- helix interactions will be studied. Possible insights into the relative contributions of lipid-lipid, lipid-helix and helix-helix interactions will be elucidated. %%% Many important biological events happen within membranes. The responses of cells to their environment, the conversion of energy from one form to another, the regulation of the living state of a cell, and many other vital processes involve the interactions of molecules inside the membrane structure. Our work to this date and our planned experiments are aimed at understanding how specific interactions occur and describing these in chemical terms. Since so many important events happen in this environment, and since it has been shown that the specificity of different binding events can be very high, it may well be that new pharmacological agents can be found that will act within membranes to modulate cell activity. Indeed, it may prove to be the case that some drugs already discovered act in this way, but we do not yet understand their mode of action. A higher level of understanding will permit the design of search methods to find new drugs and, perhaps, the de novo design or modification of drugs for specific effects. ***
