Time‐resolved surface charge change on the cytoplasmic side of bacteriorhodopsin

Alexiev, Ulrike; Scherrer, P; Marti, Thomas; Khorana, H G; Heyn, Maarten P.

doi:10.1016/0014-5793(95)00985-i

Cited by 24 publications

(24 citation statements)

References 15 publications

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“…We speculate that in this fraction fluorescein bound in position 79 is oriented toward the hydrophilic cluster of the inner gate attracting water molecules to the cytoplasmic surface (6) and that positively charged residues, such as Arg-268, locally increase the iodide concentration, leading to an apparent higher quenching compared with the lower bulk I Ϫ concentrations. This effect may also be transient, because transient surface changes (in addition to the discussed helix tilt) are known to occur at the cytoplasmic surface of both bR (38) and visual rhodopsin (39) upon light activation. For instance, a conformational change of the EF-Loop at the cytoplasmic surface of bR was correlated with surface charge changes (16,38).…”

Section: Iafmentioning

confidence: 99%

“…This effect may also be transient, because transient surface changes (in addition to the discussed helix tilt) are known to occur at the cytoplasmic surface of both bR (38) and visual rhodopsin (39) upon light activation. For instance, a conformational change of the EF-Loop at the cytoplasmic surface of bR was correlated with surface charge changes (16,38). Because conformational heterogeneity was not observed at pH 7.4, a structural pH-dependent rearrangement of helix B below pH 7 must occur that affects the region of the inner gate.…”

Section: Iafmentioning

confidence: 99%

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Light and pH-induced Changes in Structure and Accessibility of Transmembrane Helix B and Its Immediate Environment in Channelrhodopsin-2

Volz

Krause

Balke

et al. 2016

Journal of Biological Chemistry

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A variant of the cation channel channelrhodopsin-2 from Chlamydomonas reinhardtii (CrChR2) was selectively labeled at position Cys-79 at the end of the first cytoplasmic loop and the beginning of transmembrane helix B with the fluorescent dye fluorescein (acetamidofluorescein). We utilized (i) time-resolved fluorescence anisotropy experiments to monitor the structural dynamics at the cytoplasmic surface close to the inner gate in the dark and after illumination in the open channel state and (ii) time-resolved fluorescence quenching experiments to observe the solvent accessibility of helix B at pH 6.0 and 7.4. The light-induced increase in final anisotropy for acetamidofluorescein bound to the channel variant with a prolonged conducting state clearly shows that the formation of the open channel state is associated with a large conformational change at the cytoplasmic surface, consistent with an outward tilt of helix B. Furthermore, results from solute accessibility studies of the cytoplasmic end of helix B suggest a pH-dependent structural heterogeneity that appears below pH 7. At pH 7.4 conformational homogeneity was observed, whereas at pH 6.0 two protein fractions exist, including one in which residue 79 is buried. This inaccessible fraction amounts to 66% in nanodiscs and 82% in micelles. Knowledge about pH-dependent structural heterogeneity may be important for CrChR2 applications in optogenetics.Channelrhodopsins are involved in phototaxis and photophobia of unicellular green algae (1). They constitute a new class of light-gated ion channels containing the seven-transmembrane helix motif and the chromophore retinal as the light-sensitive cofactor, which are both common to the other retinal-containing proteins such as bacteriorhodopsin (bR) 4 or visual rhodopsin (2). In CrChR2 the chromophore retinal is bound to Lys-257 (3). Channelrhodopsin activation via lightinduced isomerization of retinal from all-trans to 13-cis is coupled to a transient cofactor deprotonation and a functional protein structural change. In channelrhodopsin, as well as in other retinal-containing photoreceptors, subtle changes in the chromophore vicinity trigger large scale protein conformational changes remote from the chromophore binding pocket. Rearrangement of helix B is hypothesized to constitute a key element in channel opening by allowing the entry of water molecules (4 -6). A continuous water wire is a prerequisite for the formation of the ion-conducting pathway. A hydrophilic pore between helices A, B, C, and G was suggested to serve as the ion permeation pathway based on the high resolution crystal structure of the C1C2 chimera (3 10).The inner gate was hypothesized to be involved in CrChR2 activation (formation of the open conducting channel state) together with the tilt of helix B (5). Evidence for light-induced movements of helix B comes from structural studies using electron crystallography and EPR spectroscopy (double electronelectron resonance) (11-13). Together these data suggest that in contrast to bR and sensory rhod...

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Section: Iafmentioning

confidence: 99%

Section: Iafmentioning

confidence: 99%

Light and pH-induced Changes in Structure and Accessibility of Transmembrane Helix B and Its Immediate Environment in Channelrhodopsin-2

Volz

Krause

Balke

et al. 2016

Journal of Biological Chemistry

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“…For example, a switch between two different conformations of the third cytoplasmic loop connecting helix E and F in bR was observed that was triggered by a change in surface potential [26]. Since helix F (helix F in bR corresponds to TM6 in visual rhodopsin) is known to move in the second part of the bR photocycle and in the same time range a transient surface potential change was observed [55], it was proposed that the electrostatically triggered loop conformational change facilitates the movement of helix F [26]. In visual rhodopsin, loop dynamics and conformational changes were shown to correlate with the different photo-intermediates states and the interaction of affiliated proteins involved in signal transduction [26, 29, 59, 70, 72, 73, 88, 97].…”

Section: Experimental Approaches To Gain Unique Insight Into Rhodomentioning

confidence: 99%

Fluorescence spectroscopy of rhodopsins: Insights and approaches

Alexiev

Farrens

2014

Biochimica et Biophysica Acta (BBA) - Bioenergetics

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Fluorescence spectroscopy has become an established tool at the interface of biology, chemistry and physics because of its exquisite sensitivity and recent technical advancements. However, rhodopsin proteins present the fluorescence spectroscopist with a unique set of challenges and opportunities due to the presence of the light-sensitive retinal chromophore. This review briefly summarizes some approaches that have successfully met these challenges and the novel insights they have yielded about rhodopsin structure and function. We start with a brief overview of fluorescence fundamentals and experimental methodologies, followed by more specific discussions of technical challenges rhodopsin proteins present to fluorescence studies. Finally, we end by discussing some of the unique insights that have been gained specifically about visual rhodopsin and its interactions with affiliate proteins through the use of fluorescence spectroscopy.

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“…This finding is consistent with the previous observations by Sternberg et al (1992Sternberg et al ( , 1993 showing that 4 M NaCl is essential for the formation of the two-dimensional array of bacteriorhodopsin trimer together with lipids from the purple membrane as studied froin reconstituted bilayers. It is also possible that the lowered surface pH in the absence of cations on the cytoplasmic side (Alexiev et al, 1994) results in electrostatic repulsion between bacteriorhodopsin and lipids. Accordingly, increased mobility at the C-terminal residues and head group of lipids is caused by electrostatic repulsion among charged species without salts.…”

Section: Temperature-dependent Change In Organization Of Membrane Lipmentioning

confidence: 99%

Temperature‐Dependent Conformational Change of Bacteriorhodopsin as Studied by Solid‐State ¹³C NMR

Tuzi¹,

Naito²,

Saitô³

1996

European Journal of Biochemistry

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Cross-polarization and dipolar-decoupled magic-angle spinning "C-NMR spectra of [3-'TC]Ala-labelled bacteriorhodopsin were obtained for hydrated purple membrane in the temperatures range 23 "C to -1 10°C. Well-resolved "C-NMR signals were observed either at ambient temperature or at -20°C but were broadened considerably at lower temperature below -40°C. This situation was interpreted in terms of the presence of exchange processes with a rate constant of 10' s-' at ambient temperature among several conformations slightly different from each other. We found that such an exchange process was strongly influenced by the manner of organization of the lipid bilayers depending upon the presence or absence of cations responsible for electric shielding of negative charge at the polar head groups. The manner of organization of the lipid bilayers was conveniently characterized by a characteristic temperature at which the methyl peaks of fatty acyl groups of lipids in the purple membrane were suppressed due to interference of motional frequency with the decoupling frequency (1 0-100 kHz) for preparations containing 10 mM NaCl or CaCl,. No such spectral change in the absence of these cations was noted even if a preparation was cooled to -1 10 "C. The secondary structures of [3-'3C]Ala-labelled bacteriorhodopsin was not always identical at temperatures between ambient and low temperatures, since the "C chemical shifts and relative peak intensities for purple membrane preparations containing these salts changed with temperature in the range -110°C to 23°C. In particular, we found that some residues involving Ala residues at the a,,-helix and loop region were converted at temperatures below -60°C to a conformation involving a,-helix. In other words, some portion of the a-helical conformation of bacteriorhodopsin proposed from results obtained by cryo-electron microscopy, at very low temperatures, is not always retained at ambient temperature.

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Time‐resolved surface charge change on the cytoplasmic side of bacteriorhodopsin

Cited by 24 publications

References 15 publications

Light and pH-induced Changes in Structure and Accessibility of Transmembrane Helix B and Its Immediate Environment in Channelrhodopsin-2

Light and pH-induced Changes in Structure and Accessibility of Transmembrane Helix B and Its Immediate Environment in Channelrhodopsin-2

Fluorescence spectroscopy of rhodopsins: Insights and approaches

Temperature‐Dependent Conformational Change of Bacteriorhodopsin as Studied by Solid‐State ¹³C NMR

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Time‐resolved surface charge change on the cytoplasmic side of bacteriorhodopsin

Cited by 24 publications

References 15 publications

Light and pH-induced Changes in Structure and Accessibility of Transmembrane Helix B and Its Immediate Environment in Channelrhodopsin-2

Light and pH-induced Changes in Structure and Accessibility of Transmembrane Helix B and Its Immediate Environment in Channelrhodopsin-2

Fluorescence spectroscopy of rhodopsins: Insights and approaches

Temperature‐Dependent Conformational Change of Bacteriorhodopsin as Studied by Solid‐State 13C NMR

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Temperature‐Dependent Conformational Change of Bacteriorhodopsin as Studied by Solid‐State ¹³C NMR