Time-Resolved Detection of Structural Changes During the Photocycle of Spin-Labeled Bacteriorhodopsin

Steinhoff, Heinz‐Jürgen; Mollaaghababa, Ramin; Altenbach, Christian; Hideg, Kálmán; Krebs, Mark P.; Khorana, H. G.; Hubbell, W L

doi:10.1126/science.7939627

Cited by 168 publications

(166 citation statements)

References 21 publications

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“…The structural changes near helix B are in approximately the same location as the peaks seen in previous experiments where a significant amount of the M intermediate was accumulated (11,14). Our observations support the conclusion from spin-labeling experiments (27) that there are likely to be conformational changes at the cytoplasmic end of helix C well after the formation of the M intermediate. However, because changes are observed in helices B, C, F, and G, it is clear that the structural changes on helices B and G are not likely to be limited to only the first phase of the conformational switch (13).…”

Section: Discussionsupporting

confidence: 81%

Electron diffraction studies of light-induced conformational changes in the Leu-93 → Ala bacteriorhodopsin mutant

Subramaniam

Faruqi

Oesterhelt

et al. 1997

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

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We previously have presented evidence for prominent structural changes in helices F and G of bacteriorhodopsin during the photocycle. These changes were determined by carrying out electron diffraction analysis of illuminated two-dimensional crystals of wild-type bacteriorhodopsin or the Asp-96 3 Gly mutant that were trapped at a stage in the photocycle after light-driven proton release, but preceding proton uptake from the aqueous medium. Here, we report structural analysis of the long-lived O intermediate observed in the photocycle of the Leu-93 3 Ala mutant, which accumulates after the release and uptake of protons, but before the reisomerization of retinal to its initial all-trans state. Projection Fourier difference maps show that upon illumination of the Leu-93 3 Ala mutant, significant structural changes occur in the vicinity of helices C, B, and G, and to a lesser extent near helix F. Our results suggest that (i) all four helices that line the proton channel (B, C, F, and G) participate in structural changes during the late stages of the photocycle, and (ii) completion of the photocycle involves significant conformational changes in addition to those that are associated with steps in proton transport.Bacteriorhodopsin is a light-driven proton pump (1). Each cycle of proton transport is initiated by the all-trans to 13-cis photoisomerization of retinal and completed with the thermal re-isomerization of retinal and return of the protein to its initial conformation. A number of distinct intermediates that occur in the course of the photocycle have been identified by spectroscopic methods (2-4). Site-specific mutagenesis studies (5) and selection of transport-negative mutants (6) have identified key residues that play important roles in the formation and decay of these spectroscopic intermediates. Electron cryomicroscopic studies of two-dimensional crystals have resulted in the determination of a model for the structure of bacteriorhodopsin at atomic resolution (7,8), allowing a structural interpretation of the spectroscopic and biochemical experiments.To understand chemical aspects of the molecular mechanism of proton transport, it is also necessary to determine structural changes in the protein at different stages of the photocycle. A combination of neutron (9), x-ray (10-13), and electron diffraction (14, 15) experiments have begun to provide such information. Two general strategies have been used in these experiments. In one, structural changes have been observed by collecting diffraction data from wild-type bacteriorhodopsin where the photocycle has been slowed down by lowering the temperature, changing the pH, adding chaotropic reagents, or combining two or more of these variables. In the other strategy, mutants that have pronounced kinetic defects in specific stages of the photocycle have been used to trap and structurally characterize the corresponding intermediates. To a first approximation, the magnitude and nature of the structural changes determined by the different methods and by the diff...

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Section: Discussionsupporting

confidence: 81%

Electron diffraction studies of light-induced conformational changes in the Leu-93 → Ala bacteriorhodopsin mutant

Subramaniam

Faruqi

Oesterhelt

et al. 1997

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

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“…Furthermore, force-induced reversible conformational changes of single loops have been monitored with the AFM (Mü ller et al, 1995a). These results suggest that this technique may permit to directly assign function related conformational changes of these loops indicated by other methods (Steinhoff et al, 1994(Steinhoff et al, , 1995.…”

Section: Fig 10mentioning

confidence: 90%

Adsorption of Biological Molecules to a Solid Support for Scanning Probe Microscopy

Müller

Amrein

Engel

1997

Journal of Structural Biology

286

204

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“…Electron spin echo envelope modulation (ESEEM) spectroscopy, also a pulse EPR technique, yields additional structural information by determining the water accessibility of singly spin-labeled protein domains (15). Continuous-wave (CW) spectroscopy is a widely used technique to monitor changes in the structure of both membrane proteins and watersoluble proteins (16)(17)(18) and additionally in time-resolved studies to analyze the process of protein folding (19).…”

mentioning

confidence: 99%

Refolding of the integral membrane protein light-harvesting complex II monitored by pulse EPR

Dockter

Volkov

Bauer

et al. 2009

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

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The major light-harvesting chlorophyll a/b complex (LHCII) of the photosynthetic apparatus in plants self-organizes in vitro. The recombinant apoprotein, denatured in dodecyl sulfate, spontaneously folds when it is mixed with its pigments, chlorophylls, and carotenoids in detergent solution, and assembles into structurally authentic LHCII in the course of several minutes. Pulse EPR techniques, specifically double-electron-electron resonance (DEER), have been used to analyze protein folding during this process. Pairs of nitroxide labels were introduced site-specifically into recombinant LHCII and shown not to affect the stability and function of the pigment-protein complex. Interspin distance distributions between two spin pairs were measured at various time points, one pair located on either end of the second transmembrane helix (helix 3), the other one located near the luminal ends of the intertwined transmembrane helices 1 and 4. In the dodecyl sulfatesolubilized apoprotein, both distance distributions were consistent with a random-coil protein structure. A rapid freeze-quench experiment on the latter spin pair indicated that 1 s after initiating reconstitution the protein structure is virtually unchanged. Subsequently, both distance distributions monitored protein folding in the same time range in which the assembly of chlorophylls into the complex had been observed. The positioning of the spin pair spanning the hydrophobic core of LHCII clearly preceded the juxtaposition of the spin pair on the luminal side of the complex. This indicates that superhelix formation of helices 1 and 4 is a late step in LHCII assembly.assembly kinetics ͉ DEER ͉ LHCII T he major light-harvesting complex LHCII largely increases the efficiency of the photosynthesis process by collecting light energy and conducting it to a photosynthetic reaction center where light-driven charge separation takes place. The apoprotein of LHCII is one of the most abundant membrane proteins on Earth, but even so, our knowledge is fragmentary of how the LHCII apoprotein folds and assembles with pigments. For studying these questions, LHCII offers several advantages. Its crystal structure is known (1), its apoprotein can be recombinantly expressed in Escherichia coli (2), and the protein spontaneously folds and assembles with pigments in detergent solution (2, 3). This spontaneous self-organization can be triggered by mixing the apoprotein solubilized in the ionic detergent dodecyl sulfate with a non-ionic detergent solution of the pigments. The assembly process can then easily be monitored by time-resolved f luorescence spectroscopy using the Chls as built-in fluorescence labels. Such experiments showed that protein folding is dependent on the binding of pigments, and that LHCII formation in vitro occurred in at least two apparent phases, a faster one in the range of some tens of seconds and a slower one taking several minutes (4, 5). The faster step could be assigned to the binding of mostly Chl a, whereas the slower one represents Chl b binding exclus...

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Time-Resolved Detection of Structural Changes During the Photocycle of Spin-Labeled Bacteriorhodopsin

Cited by 168 publications

References 21 publications

Electron diffraction studies of light-induced conformational changes in the Leu-93 → Ala bacteriorhodopsin mutant

Electron diffraction studies of light-induced conformational changes in the Leu-93 → Ala bacteriorhodopsin mutant

Adsorption of Biological Molecules to a Solid Support for Scanning Probe Microscopy

Refolding of the integral membrane protein light-harvesting complex II monitored by pulse EPR

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