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.