Project Summary: Lipid intrinsic curvature is a fundamental physical feature that regulates membrane interactions. The goal is to elucidate intrinsic curvature induced effects on the growth of membrane curvature and packing heterogeneity upon mixing lipids of dissimilar curvature and the consequent distribution of cholesterol and the neurotoxic 25-35 segment of Aβ peptides in bilayers, using a novel steady state fluorescence method that utilizes the computed second harmonic (SH) of the zeroth order spectra to better resolve spectral lines that are not obvious in the zeroth harmonic. The polarity sensitive fluorophore Laurdan will be employed. Laurdan emission in bilayers is a composite of a line, referred to as blue, from the denser packed region at 432 nm and a red line from the less packed region starting at 440 nm in single component gel phases evolving to 490 nm upon melting or mixing with a higher curvature lipid. This path of structure evolution was a new finding. The present hypothesis is: Any added molecule of a dissimilar curvature enters the less packed region of the bilayer. A higher curvature molecule increases stress which is then relieved by growth in flat domains. Laurdan’s preference for flat regions together with growth in such domains contribute to an increase in Laurdan presence in flat domains. Fluorescence spectra register an increase in the area, Ab, of the blue line from flat domains and decrease in the red line area, Ar, along with a red-shift of the red peak and no shift in the blue peak. A lower curvature molecule decreases stress and the membrane tends toward a homogeneous curvature distribution resulting in an Ab decrease, Ar increase, blue shift of the red peak and no shift of the blue peak. Enhancement of heterogeneity in regional curvature due to dissimilarity of intrinsic curvature of mixing lipids and preferential distribution of cholesterol or Aβ peptide that manifest distinct difference in behaviors of areas and peak positions are predicted. Laurdan fluorescence spectra will be measured without and with cholesterol or Aβ (25-35) in mixed bilayers of (i and ii) 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) with (i) 1-palmitoyl-2-oleoyl-sn- glycero-3-phosphocholine (POPC), and (ii) 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol (POPG) and (iii) brain lipids. SH of spectra will be computed to determine the number of elementary emissions. Spectra will be fitted to this number of log-normal functions according to developed methodology to resolve spectral lines. Area and peak position changes with mixture composition will be examined to determine packing heterogeneity in the bilayers without the transmembrane molecules and the type of distribution (uniform or non-uniform) of these molecules. Computing the SH of fluorescence spectra by modulating the wavenumber in analogy to field modulation in ESR spectroscopy is an innovative approach to bring superior definition to features missed in broad featureless in measured zeroth harmonics. The proposed research will significantly enhance current knowledge by elucidating the key role of intrinsic curvature in the mechanism by which the added molecule enters, accumulates preferentially, inhomogenously rigidifies the bilayer and changes its shape.

Project Narrative: This project will elucidate the key role of lipid intrinsic curvature in the mechanism of development of packing heterogeneity and preferential accumulation of cholesterol and the neurotoxic Aβ peptide in biological membranes.