SITE-DIRECTED ISOTOPE LABELING AND FTIR SPECTROSCOPY - ASSIGNMENT OF TYROSINE BANDS IN THE BR-]M DIFFERENCE SPECTRUM OF BACTERIORHODOPSIN

Fourier transform infrared difference spectroscopy has been used exten sively to probe structural changes in bacteriorhodopsin and other reti nal proteins. However, the absence of a general method to assign bands to individual chemical groups in a protein has limited the applicatio n of this technique. While site-directed mutagenesis has been successf ul in special cases for such assignments, in general, this approach in duces perturbations in the structure and function of the protein, ther eby preventing unambiguous band assignments. A new approach has recent ly been reported (Sonar et al., Nature Stnuct. Biol., 1 (1994) 512-517 ) which involves cell-free expression of bacteriorhodopsin and site-di rected isotope labeling (SDIL). We have now used this method to re-exa mine bands assigned in the bR --> M difference spectrum to tyrosine re sidues. Our results show that out of 11 tyrosines in bR, only Tyr 185 is structurally active. This work further demonstrates the power of SD IL and FTIR to probe conformational changes at the level of individual amino acid residues in proteins.