Protein conformational changes in the bacteriorhodopsin photocycle 1 1Edited by B. Honig

Subramaniam, Sriram; Lindahl, Martin; Bullough, Per A.; Faruqi, A.R.; Tittor, Jörg; Oesterhelt, Dieter; Brown, Leonid S.; Lanyi, Janos Κ.; Henderson, Richard A.

doi:10.1006/jmbi.1999.2589

Cited by 239 publications

(190 citation statements)

References 61 publications

Supporting

Mentioning

171

Contrasting

Unclassified

Order By: Relevance

“…Consistent with observations from experiments that in the first half of the reaction cycle there are no significant protein conformational changes [31], our computations indicate changes upon proton transfer that are largely restricted to the active site [22,30,32]. Nevertheless, flexibility of the protein is essential for proton transfer [30,32,33].…”

Section: Introductionsupporting

confidence: 89%

Mechanism of a proton pump analyzed with computer simulations

2009

View full text Add to dashboard Cite

Understanding the mechanism of proton pumping requires a detailed description of the energetics and sequence of events associated with the proton transfers, and of how proton transfer couples to conformational rearrangements of the protein. Here, we discuss our recent advances in using computer simulations to understand how bacteriorhodopsin pumps protons. We emphasize the importance of accurately describing the retinal geometry and the location of water molecules.

show abstract

Section: Introductionsupporting

confidence: 89%

Mechanism of a proton pump analyzed with computer simulations

2009

View full text Add to dashboard Cite

show abstract

“…Replacement of hydrophobic residues located close to the cytoplasmic proton channel, F219, F171, and T46, prolongs the lifetime of the N intermediate. Moreover, T46V and F219L contain an N-like conformer in the unphotolyzed state (20,27). These residues are located near D96, and the OH group of T46 is hydrogen-bonded to the carboxyl group of D96 (28,29).…”

Section: Discussionmentioning

confidence: 99%

Time-resolved x-ray diffraction reveals multiple conformations in the M–N transition of the bacteriorhodopsin photocycle

Oka

Yagi

Fujisawa

et al. 2000

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

We measured the M-N transition of wild-type bacteriorhodopsin (pH 9, 10°C) by time-resolved x-ray diffraction study at SPring8 BL45XU-A. We confirmed the accumulation of M and N intermediates by absorbance measurements, and we found that the time resolution of x-ray diffraction experiments (244 ms) was sufficient to resolve the M-N transition. From the x-ray diffraction data, three components were decomposed by singular value decomposition analysis. The existence of three components in the M3 N3 BR reaction revealed that BR changes its structure during the M-N transition. Moreover, the difference Fourier maps of reconstituted fast and slow decay components clearly showed that the electron density distributions of the F helix changes in the M-N transition. The observed structural change at the F helix will increase access of the Schiff base and D96 to the cytoplasmic surface and facilitate the proton transfer steps that begin with the decay of the M state.A n ion pump protein transports ions across the membrane against the chemical potential. A simple idea of the ion pumping mechanism is that the ion-binding states of the ion pump are linked to protein conformations to switch the ion transport pathway from one membrane surface to another surface (1). Bacteriorhodopsin (BR) is one of the proton transport proteins that make use of absorbed photon energy. Its photocycle is characterized by distinct spectra of the metastable intermediate in the visible range as J, K, L, M, N, and O. The most important steps for the proton pump are deprotonation and reprotonation of the Schiff base. The former is the proton transfer from the Schiff base to D85 and the latter is the proton transfer from D96 to the Schiff base. Proton transfer from the Schiff base to D85 occurs in the L-M transition. The structure of the M intermediate was investigated by diffraction methods under stabilizing conditions (2-4). The obtained data showed that the structure of the M intermediate differed largely from the unphotolyzed state. It was shown that this structural change is closely related to the deprotonation of the Schiff base; in the original structure (conformation E) the proton channel is open to the extracellular side, and when an M-type conformation is assumed (conformation C), it is open to the cytoplasmic side (5-7). This structural transition after the first proton transfer prevents reverse proton transfer from D85 to the Schiff base. Thus, the alternating protein conformation model explains the proton transport mechanism (7). The Schiff base is reprotonated from D96 during the M-N transition. Because D96 is located in a hydrophobic environment, it has unusual high pK and it is protonated in an unphotolyzed state. The pK of D96 should be lowered during the M-N transition to transfer a proton. Therefore, an important event in lowering the D96 pK is expected to occur at the M-N transition.Structural studies on photointermediates have been carried out by accumulating specific intermediates using chemical treatments (3, 4), mutations (8, 9),...

show abstract

“…This BR triple mutant reveals a remarkable conformation of its dark state: It was shown to resemble that of the late M intermediate (preceding N in the photocycle) in wildtype BR, but with a conformation that is retained upon illumination (pseudo M state). 24,49,58,59 In our work, the triple mutant was MTS labeled and studied by W-band high-field EPR without and with light irradiation. 24 The goal was to test the sensitivity of selectively spin-labeled helix segments in singly, doubly and triply mutated BR toward changes of the microenvironment in the cytoplasmic region during the photocycle.…”

Section: Conformational Changes During the Br Photocyclementioning

confidence: 99%

Combining high-field EPR with site-directed spin labeling reveals unique information on proteins in action

et al. 2005

View full text Add to dashboard Cite

In the last decade, joint efforts of biologists, chemists and physicists have helped in understanding the dominant factors determining specificity and directionality of transmembrane transfer processes in proteins. In this endeavor, electron paramagnetic resonance (EPR) spectroscopy has played an important role. Characteristic examples of such determining factors are hydrogen-bonding patterns and polarity effects of the microenvironment of protein sites involved in the transfer process. These factors may undergo characteristic changes during the reaction and, thereby, control the efficiency of biological processes, e.g. light-induced electron and proton transfer across photosynthetic membranes or ion-channel formation of bacterial toxins. In case the transfer process does not involve stable or transient paramagnetic species or states, site-directed spin labeling with suitable nitroxide radicals still allows EPR techniques to be used for studying structure and conformational dynamics of the proteins in action. By combining site-directed spin labeling with high-field/high-frequency EPR, unique information on the proteins is revealed, which is complementary to that of X-ray crystallography, solid-state NMR, FRET, fast infrared and optical spectroscopic techniques. The main object of this publication is twofold: (i) to review our recent spin-label high-field EPR work on the bacteriorhodopsin light-driven proton pump from Halobacterium salinarium and the Colicin A ion-channel forming bacterial toxin produced in Escherichia coli, (ii) to report on novel high-field EPR experiments for probing site-specific pK(a) values in protein systems by means of pH-sensitive nitroxide spin labels. Taking advantage of the improved spectral and temporal resolution of high-field EPR at 95 GHz/3.4 T and 360 GHz/12.9 T, as compared to conventional X-band EPR (9.5 GHz/0.34 T), detailed information on the transient intermediates of the proteins in biological action is obtained. These intermediates can be observed and characterized while staying in their working states on biologically relevant timescales. The paper concludes with an outlook of ongoing high-field EPR experiments on site-specific protein mutants in our laboratories at FU Berlin and Osnabrück.

show abstract

Protein conformational changes in the bacteriorhodopsin photocycle 1 1Edited by B. Honig

Cited by 239 publications

References 61 publications

Mechanism of a proton pump analyzed with computer simulations

Mechanism of a proton pump analyzed with computer simulations

Time-resolved x-ray diffraction reveals multiple conformations in the M–N transition of the bacteriorhodopsin photocycle

Combining high-field EPR with site-directed spin labeling reveals unique information on proteins in action

Contact Info

Product

Resources

About