During the past year our research efforts encompassed two areas; (A) The modulatory effects of bilayer lipids on the structural reorganizations of integral membrane proteins, and (B) the continued development and applications of vibrational infrared spectroscopic imaging techniques. (A) Infrared spectroscopic measurements demonstrate the critical role of the purple membrane lipid environment of Halobacterium halobium in mediating conformational changes in bacteriorhodopsin (BR), an integral membrane protein. The strategy consists of assessing the flexibility of BR by monitoring the temperature dependence, pressure dependence and retinal-apoprotein coupling of vibrational spectral features characteristic of the conformational reorganizations and dynamical properties of both the transmembrane helices and surrounding lipids. Photocycle activity was correlated to helix mobility in reconstituted membranes, in addition to probing aII to a1 helical conversions. Raman and infrared spectroscopic measurements were performed on membrane bilayer systems comprised of lipid systems containing a saturated and unsaturated chain. The double bond of the saturated chain occupies one of the possible multiple chain positions. The properties, dimensions, and behavior of the bilayer's microheterogeneity, or fluctuating microdomain structures, were elucidated. (B) Our mid-infrared spectroscopic chemical imaging techniques, combining step-scan interferometry with an infrared microscope, were substantially refined and increased in spectroscopic capabilities by incorporating a variety of state-of-the-art infrared sensitive two-dimensional focal plane array detectors. These imaging systems, exhibiting instrumental multiplex/multichannel advantages, operate from 4000-400cm-1 and provide high fidelity, chemically specific images at nearly diffraction limited conditions. Applications consisted of imaging the effects of antineoplastic drugs on rat cerebellar tissue and examining intact brain sections from transgenic mice to elucidate the pathology of lipid distribution induced by the neurodegenerative Niemann-Pick C disease.
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