title: Time-resolved ultraviolet resonance Raman studies of protein structure: application to bacteriorhodopsin.
abstract: Time-resolved ultraviolet resonance Raman spectra of bacteriorhodopsin are used to study protein structural changes on the nanosecond and millisecond time scales. Excitation at 240 nm is used to selectively enhance vibrational scattering from tyrosine so that changes in its hydrogen bonding and protonation state can be examined. Both nanosecond and millisecond UV Raman difference spectra indicate that none of the tyrosine residues change ionization state during the BR----K and BR----M transitions. However, intensity changes are observed at 1172 and 1615 cm-1 in the BR----M UV Raman difference spectra. The 1615-cm-1 feature shifts down 25 cm-1 in tyrosine-d4-labeled BR, consistent with its assignment as a tyrosine vibration. The intensity changes in the BR----M UV Raman difference spectra most likely reflect an increase in resonance enhancement that occurs when one or more tyrosine residues interact more strongly with a hydrogen-bond acceptor in M412. The frequency of the v7a feature (1172 cm-1) in the BR----M UV Raman difference spectra supports this interpretation. The proximity of Tyr-185 and Asp-212 in the retinal binding pocket suggests that deprotonation of the Schiff base in M412 causes Tyr-185 to stabilize ionized Asp-212 by forming a stronger hydrogen bond.
authors.affiliation: Department of Chemistry, University of California, Berkeley 94720.
authors.forename: J B
authors.initials: JB
authors.lastname: Ames
authors.forename: M
authors.initials: M
authors.lastname: Ros
authors.forename: J
authors.initials: J
authors.lastname: Raap
authors.forename: J
authors.initials: J
authors.lastname: Lugtenburg
authors.forename: R A
authors.initials: RA
authors.lastname: Mathies
mesh_terms: D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000378:metabolism; D006217:Halobacterium; D006860:Hydrogen Bonding; D006863:Hydrogen-Ion Concentration; D010777:Photochemistry; D013056:Spectrophotometry, Ultraviolet; D013059:Spectrum Analysis, Raman / Q000379:methods*; D013997:Time Factors; D014443:Tyrosine / Q000737:chemistry
chemical_list: D014443:Tyrosine; D001436:Bacteriorhodopsins