title: Suppressed or recovered intensities analysis in site-directed (13)C NMR: assessment of low-frequency fluctuations in bacteriorhodopsin and D85N mutants revisited.
abstract: The first proton transfer of bacteriorhodopsin (bR) occurs from the protonated Schiff base to the anionic Asp 85 at the central part of the protein in the L to M states. Low-frequency dynamics accompanied by this process can be revealed by suppressed or recovered intensities (SRI) analysis of site-directed (13)C solid-state NMR spectra of 2D crystalline preparations. First of all, we examined a relationship of fluctuation frequencies available from [1-(13)C]Val- and [3-(13)C]Ala-labeled preparations, by taking the effective correlation time of internal methyl rotations into account. We analyzed the SRI data of [1-(13)C]Val-labeled wild-type bR and D85N mutants, as a function of temperature and pH, respectively, based on so-far assigned peaks including newly assigned or revised ones. Global conformational change of the protein backbone, caused by neutralization of the anionic D85 by D85N, can be visualized by characteristic displacement of peaks due to the conformation-dependent (13)C chemical shifts. Concomitant dynamics changes if any, with fluctuation frequencies in the order of 10(4) Hz, were evaluated by the decreased peak intensities in the B-C and D-E loops of D85N mutant. The resulting fluctuation frequencies, owing to subsequent, accelerated dynamics changes in the M-like state by deprotonation of the Schiff base at alkaline pH, were successfully evaluated based on the SRI plots as a function of pH, which were varied depending upon the extent of interference of induced fluctuation frequency with frequency of magic angle spinning or escape from such interference. Distinguishing fluctuation frequencies between the higher and lower than 10(4) Hz is now possible, instead of a simple description of the data around 10(4) Hz available from one-point data analysis previously reported.
authors.affiliation: Department of Life Science, Himeji Institute of Technology, University of Hyogo, Harima Science Garden City, Kamigori, Hyogo 678-1297, Japan.
authors.forename: Hazime
authors.initials: H
authors.lastname: Saitô
authors.forename: Atsushi
authors.initials: A
authors.lastname: Kira
authors.forename: Tadashi
authors.initials: T
authors.lastname: Arakawa
authors.forename: Michikazu
authors.initials: M
authors.lastname: Tanio
authors.forename: Satoru
authors.initials: S
authors.lastname: Tuzi
authors.forename: Akira
authors.initials: A
authors.lastname: Naito
mesh_terms: D019943:Amino Acid Substitution; D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000235:genetics; D002247:Carbon Isotopes / Q000737:chemistry; D019617:Halobacterium salinarum / Q000737:chemistry* / Q000235:genetics; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D017433:Protein Structure, Secondary / Q000235:genetics
chemical_list: D002247:Carbon Isotopes; D001436:Bacteriorhodopsins