Abstract
FT-IR difference spectroscopy can provide detailed informa- tion about structure and conformational changes in proteins.* 1 This approach is greatly enhanced by site-directed isotope labeling (SDIL).2-4 In contrast to earlier methods employing site-directed mutagenesis, SDIL allows vibrational band assign- ments to be made in an essentially unaltered system through the replacement of single amino acid residues with their isotopic analogs.
In this work, we have used FT-IR/SDIL to probe protein conformational changes which occur during the primary photochemical reaction of bacteriorhodopsin (bR), a light-driven proton pump from Halobacterium salinarium. While it is known that this initial step involves an all-trans to 13-cí's isomerization of the retinylidene chromophore,5 ***little is known about the interactions which occur between the retinal and protein during this step or how these interactions eventually facilitate proton transport.6 We have probed the response to chromophore isomerization of three specific tyrosine amide carbonyl groups residing in the retinal binding pocket (Tyr 57, 83, and 8185),7 as well as Tyr 147 outside of this pocket. Our results show that out of the 11 tyrosine residues in bR, only the amide carbonyl group of Tyr 185 is perturbed by chromophore isomerization.