The combination of near-infrared Raman spectroscopy and variations in external parameters offers new opportunities for site-specific studies of proteins. Using excitation at 840 nm, we have measured the near-infrared Raman spectrum of dark-adapted bacteriorhodopsin at ambient and high
pressure. The C=C ethylenic stretching region shows two resolved bands at 1526 and 1534 cm-1, corresponding to the all-trans and 13-cis isomers. From deconvolution of these bands we find an isomeric ratio between 13-cis and all-trans retinal equal to
1 at ambient pressure. The Raman spectrum gives direct spectroscopic evidence that the 13-cis component is favored at high pressure, implying that it has a smaller volume. The pressure dependence of the isomeric ratio yields a molar volume of -6.6 mL/mol, which suggests ionization of
one or two residues or the formation of three hydrogen bonds.
Department of Physics and Center for Research and Education in Optics and Lasers, University of Central Florida, Orlando, Florida 32816-2385
Publication date: January 1, 1995
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