Fourier transform infrared difference spectroscopy of rhodopsin mutants: light activation of rhodopsin causes hydrogen-bonding change in residue aspartic acid-83 during meta II formation.
Fourier transform infrared (FTIR) difference spectroscopy and site-directed mutagenesis have been used to investigate structural changes which occur during rhodopsin photoactivation at the level of individual amino acid residues. The rhodopsin-->bathorhodopsin FTIR difference spectra of the mutants Asp-83-->Asn (D83N) and Glu-134-->Asp (E134D) incorporated into membranes are similar to that of native rhodopsin in the photoreceptor membrane, demonstrating that the retinal chromophores of these mutants undergo a normal 11-cis to all-trans photoisomerization. Two bands assigned to the C = O stretching mode of Asp and/or Glu carboxylic acid groups are absent in the D83N rhodopsin-->metarhodopsin II FTIR difference spectrum. Corresponding changes are not observed in the carboxylate C = O stretching region. The most straightforward explanation is that the carboxylic acid group of Asp-83 remains protonated in rhodopsin and its bleaching intermediates but undergoes an increase in its hydrogen bonding during the metarhodopsin I-->metarhodopsin II transition. The mutant E134D produced a normal rhodopsin-->bathorhodopsin and rhodopsin-->metarhodopsin II difference spectrum, but a fraction of misfolded protein was observed, supporting earlier evidence that Glu-134 plays a role in proper protein insertion and/or folding in the membrane.
Rath
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Department of Physics, Boston University, Massachusetts 02215.
DeCaluwé
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Bovee-Geurts
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DeGrip
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Rothschild
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D000818:Animals; D001224:Aspartic Acid / Q000737:chemistry*; D002417:Cattle; D006860:Hydrogen Bonding; D008027:Light; D016297:Mutagenesis, Site-Directed; D010777:Photochemistry; D017433:Protein Structure, Secondary; D012243:Rhodopsin / Q000737:chemistry* / Q000235:genetics / Q000528:radiation effects; D017550:Spectroscopy, Fourier Transform Infrared
D001224:Aspartic Acid; D012243:Rhodopsin