Chromophore and Protein Reactions of Bacteriorhodopsin Studied by Sub-Microsecond Time-Resolved Step-Scan FTIR Spectroscopy

Abstract

Time-resolved IR difference spectroscopy is an ideal tool to study molecular processes in photobiological systems, since it is sensitive to both chromophore and protein alterations. Recently, a new powerful method, time-resolved FT-IR absorption spectroscopy using a step-scan interferometer, has been described [1]. The time-resolution has now been increased to ca. 200 ns, and the accuracy for the slower processes has been increased by the use of a quasi-logarithmic transient-recorder. In this report, this method is applied to the study of the BR-gt K (KL) transition and to the molecular discrimination of the M and N intermediates. In Fig. 1, the time-resoved (500 ns after the flash) and the static (temperature 80 K) BR-gt K difference spectra are compared. The overall agreement is surprisingly good. It is noteworthy that the same spectral features in the amide-I range are observed in both spectra, indicating comparable protein structural changes at 80 K vs. 500 ns after the flash at room temperature. However, the small band at 1555 cm⁻¹, present in the low-temperature spectrum, is missing in the time-resolved studies. The main difference, however, can be seen in the HOOP spectral range below 1000 cm⁻¹: whereas in the low-temperature spectrum a HOOP mode at 955 cm⁻¹ (11,12-HOOP) dominates, in the room-temperature spectrum a mode at 980 cm⁻¹, which is absent in the former spectrum, exhibits the largest intensity.