Interpretation of Amide I Difference Bands Observed during Protein Reactions Using Site-Directed Isotopically Labeled Bacteriorhodopsin as a Model System

Reaction-induced infrared difference spectra show characteristic amide I spectral changes, which indicate conformational changes of the protein backbone but which cannot be interpreted at a molecular level. To obtain some insights into their causes, we used bacteriorhodopsin as a model system and investigated its BR f N transition during which the largest amide I changes are observed. For the molecular interpretation, we labeled a single peptide CdO group at specific positions of the backbone with 13C and monitored the resulting isotope effects. This has been achieved by replacing specific amino acids with a cysteine. Because wild-type bacteriorhodopsin does not contain this amino acid, (1-13C)cysteine can be incorporated into the mutants for site-directed isotopic labeling. Although the isotope-induced spectral changes are very small, we observed clear isotope effects for the middle to extracellular part of helices B, C, and F, indicating that the backbone of these parts of the protein is distorted during the reaction, whereas no label effects could be identified for the E-F loop and for the cytosolic regions of helices E and F. The results are discussed within the framework of recent experimental and theoretical studies of the amide I band, and they are correlated to the structural changes observed by other methods.