Time-resolved microspectroscopy on a single crystal of bacteriorhodopsin reveals lattice-induced differences in the photocycle kinetics.

TitleTime-resolved microspectroscopy on a single crystal of bacteriorhodopsin reveals lattice-induced differences in the photocycle kinetics.
Publication TypeJournal Article
Year of Publication2006
AuthorsEfremov, R. G., Gordeliy V. I., Heberle J., and B├╝ldt G.
JournalBiophys J
Volume91
Issue4
Pagination1441-51
Date Published2006 Aug 15
ISSN0006-3495
KeywordsBacteriorhodopsins, Crystallography, Dose-Response Relationship, Radiation, Kinetics, Light, Photobiology, Photochemistry, Radiation Dosage, Spectroscopy, Fourier Transform Infrared, Time Factors
Abstract

The determination of the intermediate state structures of the bacteriorhodopsin photocycle has lead to an unprecedented level of understanding of the catalytic process exerted by a membrane protein. However, the crystallographic structures of the intermediate states are only relevant if the working cycle is not impaired by the crystal lattice. Therefore, we applied visible and Fourier transform infrared spectroscopy (FTIR) microspectroscopy with microsecond time resolution to compare the photoreaction of a single bacteriorhodopsin crystal to that of bacteriorhodopsin residing in the native purple membrane. The analysis of the FTIR difference spectra of the resolved intermediate states reveals great similarity in structural changes taking place in the crystal and in PM. However, the kinetics of the photocycle are significantly altered in the three-dimensional crystal as compared to PM. Strikingly, the L state decay is accelerated in the crystal, whereas the M decay is delayed. The physical origin of this deviation and the implications for trapping of intermediate states are discussed. As a methodological advance, time-resolved step-scan FTIR spectroscopy on a single protein crystal is demonstrated for the first time which may be used in the future to gauge the functionality of other crystallized proteins with the molecular resolution of vibrational spectroscopy.

DOI10.1529/biophysj.106.083345
Alternate JournalBiophys. J.
PubMed ID16731567
PubMed Central IDPMC1518640
Research group: