CONFORMATIONAL-CHANGES IN THE CORE STRUCTURE OF BACTERIORHODOPSIN

Bacteriorhodopsin (bR) is the light-driven proton pump found in the pu rple membrane of Halobacterium salinarium, In this work, structural ch anges occurring during the bR photocycle in the core structure of bR, which is normally inaccessible to hydrogen/deuterium (H/D) exchange, h ave been probed. FTIR difference bands due to vibrations of peptide gr oups in the core region of bR have been assigned by reconstituting and regenerating delipidated bR in the presence of D2O. Exposure of bR to D2O even after long periods causes only a partial shift of the amide II band due to peptide NH --> ND exchange only of peripheral peptide s tructure. However, the amide II band completely downshifts when recons titution/regeneration of bR is performed in the presence of D2O, indic ating that almost the entire core backbone structure of bR undergoes H /D exchange. Peripheral regions can then be reexchanged in H2O, leavin g the core backbone region deuterated. Low-temperature FTIR difference spectra on these core-deuterated samples reveal that peptide groups i n the core region respond to retinal isomerization as early as the K i ntermediate. By formation of the M intermediate, infrared differences in the amide I region are dominated by much larger structural changes occurring in the core structure. In the amide II region, difference ba nds appear upon K formation and increase upon M formation which are si milar to those observed upon the cooling of bacteriorhodopsin. This wo rk shows that retinal isomerization induces conformational changes in the bacteriorhodopsin core structure during the early photocycle which may involve an increase in the strength of intramolecular ct-helical hydrogen bonds.