Resonance Raman microprobe spectroscopy of rhodopsin mutants: effect of substitutions in the third transmembrane helix.
A microprobe system has been developed that can record Raman spectra from as little as 2 microL of solution containing only micrograms of biological pigments. The apparatus consists of a liquid nitrogen (l-N2)-cooled cold stage, an epi-illumination microscope, and a substractive-dispersion, double spectrograph coupled to a l-N2-cooled CCD detector. Experiments were performed on native bovine rhodopsin, rhodopsin expressed in COS cells, and four rhodopsin mutants: Glu134 replaced by Gln (E134Q), Glu122 replaced by Gln (E122Q), and Glu113 replaced by Gln (E113Q) or Ala (E113A). Resonance Raman spectra of photostationary steady-state mixtures of 11-cis-rhodopsin, 9-cis-isorhodopsin, and all-trans-bathorhodopsin at 77 K were recorded. The Raman spectra of E134Q and the wild-type are the same, indicating that Glu134 is not located near the chromophore. Substitution at Glu122 also does not affect the C = NH stretching vibration of the chromophore. The fingerprint and Schiff base regions of the Raman spectra of the 380-nm, pH 7 forms of E113Q and E113A are characteristic of unprotonated retinal Schiff bases. The C = NH modes of the approximately 500-nm, pH 5 forms of E113Q and E113A in H2O (D2O) are found at 1648 (1629) and 1645 (1630) cm-1, respectively. These frequencies indicate that the protonated Schiff base interacts more weakly with its protein counterion in the Glu113 mutants than it does in the native pigment. Furthermore, perturbations of the unique bathorhodopsin hydrogen out-of-plane (HOOP) vibrations in E113Q and E113A indicate that the strength of the protein perturbation near C12 is weakened compared to that in native bathorhodopsin.(ABSTRACT TRUNCATED AT 250 WORDS)
Department of Chemistry, University of California, Berkeley 94720.
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D000409:Alanine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D005971:Glutamates / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D018698:Glutamic Acid; D005973:Glutamine / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007536:Isomerism; D008958:Models, Molecular; D008968:Molecular Conformation; D009154:Mutation; D012243:Rhodopsin / Q000031:analogs & derivatives / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013059:Spectrum Analysis, Raman
D005971:Glutamates; C042882:isorhodopsin; D005973:Glutamine; D018698:Glutamic Acid; C015017:bathorhodopsin; D012243:Rhodopsin; D000409:Alanine