Time-resolved microspectroscopy on a single crystal of
bacteriorhodopsin reveals lattice induced differences in
the photocycle kinetics
Ruslan Efremov 1, Valentin Ivanovich Gordeliy 1, Joachim Heberle 2* and Georg Bueldt 1
1 Forschungszentrum Juelich
2 University of Bielefeld
* To whom correspondence should be addressed. E-mail: joachim.heberle{at}uni-bielefeld.de.
Submitted on February 15, 2006
Revised on March 22, 2006
Accepted on 16 May 2006
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Abstract |
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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 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 the structural changes taking place in the crystal and in PM. However, the kinetics of the photocycle are significantly altered in the 3D crystal as compared to PM. Strikingly, the L state decay is accelerated in the crystal while 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.
Key Words:
FTIR, X-ray, cytochrome c oxidase, proton translocation, retinal protein, vibrational spectroscopy
