Equilibrium Composition of Retinal Isomers in Dark-Adapted Bacteriorhodopsin and Effect of High Pressure Probed by Near-Infrared Raman Spectroscopy
Abstract: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.
Document Type: Research Article
Affiliations: 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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