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Tight Asp-85--Thr-89 association during the pump switch of bacteriorhodopsin.

ABSTRACT: Unidirectional proton transport in bacteriorhodopsin is enforced by the switching machinery of the active site. Threonine 89 is located in this region, with its O--H group forming a hydrogen bond with Asp-85, the acceptor for proton transfer from the Schiff base of the retinal chromophore. Previous IR spectroscopy of [3-(18)O]threonine-labeled bacteriorhodopsin showed that the hydrogen bond of the O--D group of Thr-89 in D(2)O is strengthened in the K photocycle intermediate. Here, we show that the strength and orientation of this hydrogen bond remains unchanged in the L intermediate and through the M intermediate. Furthermore, a strong interaction between Asp-85 and the O--H (O--D) group of Thr-89 in M is indicated by a shift in the C==O stretching vibration of the former because of (18)O substitution in the latter. Thus, the strong hydrogen bond between Asp-85 and Thr-89 in K persists through M, contrary to structural models based on x-ray crystallography of the photocycle intermediates. We propose that, upon photoisomerization of the chromophore, Thr-89 forms a tight, persistent complex with one of the side-chain oxygens of Asp-85 and is thereby precluded from participating in the switching process. On the other hand, the loss of hydrogen bonding at the other oxygen of Asp-85 in M may be related to the switching event.

SUBMITTER: Kandori H

PROVIDER: S-EPMC29298 | biostudies-literature | 2001 Feb

REPOSITORIES: biostudies-literature

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Tight Asp-85--Thr-89 association during the pump switch of bacteriorhodopsin.

Kandori H H Yamazaki Y Y Shichida Y Y Raap J J Lugtenburg J J Belenky M M Herzfeld J J

Proceedings of the National Academy of Sciences of the United States of America 20010201 4

Unidirectional proton transport in bacteriorhodopsin is enforced by the switching machinery of the active site. Threonine 89 is located in this region, with its O--H group forming a hydrogen bond with Asp-85, the acceptor for proton transfer from the Schiff base of the retinal chromophore. Previous IR spectroscopy of [3-(18)O]threonine-labeled bacteriorhodopsin showed that the hydrogen bond of the O--D group of Thr-89 in D(2)O is strengthened in the K photocycle intermediate. Here, we show that ...[more]

PMID: 11171992

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Project description:Photosensitive proteins embedded in the cell membrane (about 5 nm thickness) act as photoactivated proton pumps, ion gates, enzymes, or more generally, as initiators of stimuli for the cell activity. They are composed of a protein backbone and a covalently bound cofactor (e.g. the retinal chromophore in bacteriorhodopsin (BR), channelrhodopsin, and other opsins). The light-induced conformational changes of both the cofactor and the protein are at the basis of the physiological functions of photosensitive proteins. Despite the dramatic development of microscopy techniques, investigating conformational changes of proteins at the membrane monolayer level is still a big challenge. Techniques based on atomic force microscopy (AFM) can detect electric currents through protein monolayers and even molecular binding forces in single-protein molecules but not the conformational changes. For the latter, Fourier-transform infrared spectroscopy (FTIR) using difference-spectroscopy mode is typically employed, but it is performed on macroscopic liquid suspensions or thick films containing large amounts of purified photosensitive proteins. In this work, we develop AFM-assisted, tip-enhanced infrared difference-nanospectroscopy to investigate light-induced conformational changes of the bacteriorhodopsin mutant D96N in single submicrometric native purple membrane patches. We obtain a significant improvement compared with the signal-to-noise ratio of standard IR nanospectroscopy techniques by exploiting the field enhancement in the plasmonic nanogap that forms between a gold-coated AFM probe tip and an ultraflat gold surface, as further supported by electromagnetic and thermal simulations. IR difference-spectra in the 1450-1800 cm-1 range are recorded from individual patches as thin as 10 nm, with a diameter of less than 500 nm, well beyond the diffraction limit for FTIR microspectroscopy. We find clear spectroscopic evidence of a branching of the photocycle for BR molecules in direct contact with the gold surfaces, with equal amounts of proteins either following the standard proton-pump photocycle or being trapped in an intermediate state not directly contributing to light-induced proton transport. Our results are particularly relevant for BR-based optoelectronic and energy-harvesting devices, where BR molecular monolayers are put in contact with metal surfaces, and, more generally, for AFM-based IR spectroscopy studies of conformational changes of proteins embedded in intrinsically heterogeneous native cell membranes.

Dataset Information

Tight Asp-85--Thr-89 association during the pump switch of bacteriorhodopsin.

Publications

Tight Asp-85--Thr-89 association during the pump switch of bacteriorhodopsin.

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