The primary goals of this project are 1-to continue to develop coupled plasmon-waveguide resonance (CPWR) spectroscopy as an important new tool for studying proteolipid membranes, and 2-to obtain new insights into a key question in membrane biophysics: how do the physicochemical properties of the lipid bilayer influence the insertion and assembly of transmembrane peptide helices into functional aggregates? The following new capabilities will be developed for CPWR spectroscopy. a-The use of varying wavelengths to separately monitor lipid and protein components of membranes. This will be accomplished by chromophore labelling of lipids, by using the optical absorption coefficient in data analysis, and by extending plasmon generation and detection into the near UV region. b- Design of plasmon resonators that are able to simultaneously monitor CPWR spectra and electrical current flow across a proteolipid membrane. The influence of membrane physicochemical properties will be investigated using structural variations in a small synthetic hydrophobic helical peptide, and the ion-channel forming protein colicin El. These will be reconstituted into solid-supported lipid bilayers, in which the lipid composition can be readily varied. CPWR spectroscopy will be used to monitor three parameters: a- binding isotherms and peptideiprotein insertion into a preformed lipid bilayer; b- changes in refractive index and optical extinction coefficient anisotropies upon peptide/protein binding and insertion; c- in the case of colicin, the ability to form a functional ion channel, as determined by simultaneous measurements of the kinetics of CPWR spectral changes and membrane electrical conductivity subsequent to establishment of a transmembrane potential to initiate insertion and channel formation. The significance and role of the incorporation of nonbilayer-forming lipids; hydrophobic matching between lipids and peptide/proteins; lipid packing density; electrical surface charge will be studied. These properties will be varied by changes in phospholipid head group identity; fatty acid chain length; lipid packing density; and degree of unsaturation of fatty acid chains. These studies have broad implications for membrane-based processes important in many disease states.
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