of Work: Our research efforts encompassed two general areas: (1) The modulatory effects of bilayer lipids on the structural reorganizations of integral membrane proteins, and (2) the instrumental development and applications of vibrational Raman and infrared spectroscopic imaging techniques. (1A) Our interest in characterizing the effects of fluctuating lipid microdomains within biomembranes has recently focused on cluster formation within bilayer matrices comprised of lipid mono- or polyunsaturated sn-2 chain and saturated sn-1 chain assemblies. The lateral compressibility properties of these lipid microaggregates are effective in exerting a modulatory influence on induced conformational changes occurring within integral membrane proteins. In studying spectroscopically specific lipid bilayers, appropriate acyl chain deuteration allows the vibrational dynamics of each chain moiety to be monitored separately. We have continued the utilization of both Raman and infrared spectroscopic techniques, in conjunction with freeze-quenching methodologies, toward examining the bilayer series comprised of 1-eicosanoyl(d39)-2-eicosenoyl-sn-glycero-3-phosphocholine [C(20-d39):C(20:1 delta j)PC, with j=5, 8, 11, and 13]. The acyl chain packing characteristics and microdomain formation of 1-palmitoyl-2-oleoyl-sn-glycerol-3-phosphocholine (POPC-d31) bilayers were also investigated, Established order/disorder parameters pertinent to each lipid class and to each chain system were determined. Polycrystalline samples were examined using Raman spectroscopic techniques, while infrared spectroscopic procedures were applied to the aqueous bilayer dispersions. Computational techniques provided estimates of chain cluster sizes, which vary between 3-19 saturated acyl chains for these various series of phospholipids. For example, the largest sn-1 lipid chain domain, originating specifically from van der Waals interactions between the chains of neighboring molecules, occurs for the C(20-d39):C(20:1 delta 5)PC species. Using these spectroscopic approaches, we have also examined in detail the microheterogeneity of a variety of bilayer assemblies comprised of highly unsaturated and completely saturated lipid assemblies. (1B) Membrane complexes of sphingolipid and cholesterol, or rafts, have been recently proposed as having roles involving cell trafficking, signal transduction and a variety of membrane related activities. In order to assess the structural properties and domain behavior of these putative complexes, infrared spectroscopic techniques were used to investigate the interactions of binary and ternary mixtures comprised of non-hydroxy fatty acid galactocerebroside (GalCer) and cholesterol in the presence and absence of membrane bilayer matrices of dipalmitoylphosphatidylcholine. In the lipid gel phase, cholesterol acts preferentially with the interfacial region of GalCer. Additionally, cholesterol induces the longer GalCer chains to pack into orthorhombic subcell clusters supporting the existence of domains in the sphingolipid and cholesterol-rich ordered phase in the plasma membrane. (2) Emphasis has been placed on enhancing our mid-infrared spectroscopic chemical imaging microscopy techniques by combining step-scan interferometry with state-of-the-art infrared senstive two-dimensional focal plane array detectors. The integration of high performance digital imaging with noninvasive, high resolution optical spectroscopy allows a visualization of the spatial distribution of distinct chemical species in a variety of host environments. The power of the technique is also manifest in the simultaneous acquisition of an infrared spectrum for each spatial location. This approach has also been extended to the visible spectral region in which reflectance spectra are obtained using a CCD detector and appropriate liquid crystal tunable filters for wavelength discrimination. This particular reflectance unit has been used clinically in assessing blood flow in the skin of burn victims. As examples of the utility of the technique in diagnostic pathology, promising results were obtained from prostate tissue sections and sections from a particular skin lesion (Birt-Hogg-Dube syndrome). The infrared absorbance and Raman emission images, along with their associated vibrational spectra, provided qualitative descriptions of the biochemical differences between unstained tissue from diseased and control animals. Statistical analyses of the individual spectra reflecting the various cell types comprising the tissue provided concise quantitative descriptions of the observed biochemical variations.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Intramural Research (Z01)
Project #
1Z01DK029001-27
Application #
6289744
Study Section
Special Emphasis Panel (LCP)
Project Start
Project End
Budget Start
Budget End
Support Year
27
Fiscal Year
1999
Total Cost
Indirect Cost
City
State
Country
United States
Zip Code
Bhargava, Rohit; Fernandez, Daniel C; Hewitt, Stephen M et al. (2006) High throughput assessment of cells and tissues: Bayesian classification of spectral metrics from infrared vibrational spectroscopic imaging data. Biochim Biophys Acta 1758:830-45
Schlucker, S; Liang, C; Strehle, K R et al. (2006) Conformational differences in protein disulfide linkages between normal hair and hair from subjects with trichothiodystrophy: a quantitative analysis by Raman microspectroscopy. Biopolymers 82:615-22
Levin, Ira W; Bhargava, Rohit (2005) Fourier transform infrared vibrational spectroscopic imaging: integrating microscopy and molecular recognition. Annu Rev Phys Chem 56:429-74
Fernandez, Daniel C; Bhargava, Rohit; Hewitt, Stephen M et al. (2005) Infrared spectroscopic imaging for histopathologic recognition. Nat Biotechnol 23:469-74
Huffman, Scott W; Schlucker, Sebastian; Levin, Ira W (2004) Reorganizational dynamics of multilamellar lipid bilayer assemblies using continuously scanning Fourier transform infrared spectroscopic imaging. Chem Phys Lipids 130:167-74
Bhargava, Rohit; Levin, Ira W (2004) Gram-Schmidt orthogonalization for rapid reconstructions of Fourier transform infrared spectroscopic imaging data. Appl Spectrosc 58:995-1000
Zuzak, Karel J; Gladwin, Mark T; Cannon 3rd, Richard O et al. (2003) Imaging hemoglobin oxygen saturation in sickle cell disease patients using noninvasive visible reflectance hyperspectral techniques: effects of nitric oxide. Am J Physiol Heart Circ Physiol 285:H1183-9
Schlucker, Sebastian; Schaeberle, Michael D; Huffman, Scott W et al. (2003) Raman microspectroscopy: a comparison of point, line, and wide-field imaging methodologies. Anal Chem 75:4312-8
Hendler, Richard W; Barnett, Steven M; Dracheva, Swetlana et al. (2003) Purple membrane lipid control of bacteriorhodopsin conformational flexibility and photocycle activity. Eur J Biochem 270:1920-5
Bhargava, Rohit; Levin, Ira W (2003) Time-resolved Fourier transform infrared spectroscopic imaging. Appl Spectrosc 57:357-66

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