Abstract
A step-scan Fourier-transform interferometer was employed to collect the time-evolved difference infrared spectra of bacteriorhodopsin upon photoexcitation. Without a kinetics model being established or target analysis to extract the difference spectra of the intermediates in the conventional photocycle, correlation analysis of the infrared features was used to examine the evolution of the interactive strengths between the retinal Schiff base and its proton donor and acceptor in different periods of the photocycle. Following the evolution of the correlation, those correlative characteristics satisfactorily coincided with the transitions of M N, N O, and O bR. We therefore have illustrated the trajectory of the driving forces on the localized molecular moieties during the photocycle: proton acceptor Asp85 (1762 and 1755 cm–1) → proton donor Asp96 (1400 cm–1) → C–C stretch of 13-cis retinal (1186 cm–1) → C14=C15 stretch of all-trans retinal (1506 cm–1) → C10–C11 stretch of all-trans retinal (1168 cm–1). The correlation analysis successfully provided the dynamic and local conformational alterations as the bR photocycle evolved, without the need to establish a kinetics model, introduce discrete intermediates, or use deconvolution algebra. Including more bands in the correlation analysis provides more thorough information of the structural alteration of the photocycle. The combination of the time-resolved infrared spectroscopic method and the correlation analysis could be a promising method of illustrating the photochemistry of photosynthetic proteins at the molecular level.