Elucidation of the Nature of the Conformational Changes of the EF-interhelical Loop in Bacteriorhodopsin and of the Helix VIII on the Cytoplasmic Surface of Bovine Rhodopsin: A Time-resolved Fluorescence Depolarization Study

Alexiev, Ulrike; Rimke, Ingo; Pöhlmann, Thomas

doi:10.1016/s0022-2836(03)00326-7

Cited by 80 publications

(166 citation statements)

References 44 publications

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“…This faster fluorescence lifetime of fluorescein when bound to the channel surface can be explained mainly by quenching effects from the protein surface. A similar effect on the fluorescence lifetime was found when fluorescein was covalently bound to the surface of bR (34) and visual rhodopsin (16,35). At pH 7.4 we observed reduced quenching rates for the collisional .…”

Section: Iafsupporting

confidence: 82%

“…Time-resolved Fluorescence Anisotropy for Detection of Dynamic Helix B Conformations-We investigated the dynamics and conformational changes of helix B by time-resolved fluorescence depolarization in the dark state and after illumination with blue light (465 nm) in samples consisting of ChR2 CA/C128T -C79-AF/C208 in DM micelles or reconstituted in nanodiscs. These measurements allow for the analysis of the diffusional dynamics of protein segments directly on the nanosecond time scale (16,19). Position Cys-79 is located at the end of the first cytoplasmic loop and the beginning of helix B facing toward the lipid shell and the putative CrChR2 dimer interface (Fig.…”

Section: Resultsmentioning

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: Iafsupporting

confidence: 82%

Section: Resultsmentioning

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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“…Clearly, the linkage of TM5 and TM6 by the IL-3 loop must play some key role in this. A recent study on bacteriorhodopsin showed that a potential-based change in the conformation of the IL-3 loop may be directly responsible for an outward tilting of TM6 occurring at the end of the M segment of the photocycle (28). Although the function of bacteriorhodopsin is quite different from that of the ␤2AR, this experiment illustrates that manipulating the conformation of IL-3 in a seven-TM protein can cause a meaningful spatial movement of TM6.…”

Section: Discussionmentioning

confidence: 66%

Predicted 3D structure for the human β2 adrenergic receptor and its binding site for agonists and antagonists

Freddolino

Kalani

Vaidehi

et al. 2004

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

121

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We report the 3D structure of human ␤2 adrenergic receptor (AR) predicted by using the MembStruk first principles method. To validate this structure, we use the HierDock first principles method to predict the ligand-binding sites for epinephrine and norepinephrine and for eight other ligands, including agonists and antagonists to ␤2 AR and ligands not observed to bind to ␤2 AR. The binding sites agree well with available mutagenesis data, and the calculated relative binding energies correlate reasonably with measured binding affinities. In addition, we find characteristic differences in the predicted binding sites of known agonists and antagonists that allow us to infer the likely activity of other ligands. The predicted ligand-binding properties validate the methods used to predict the 3D structure and function. This validation is a successful step toward applying these procedures to predict the 3D structures and function of the other eight subtypes of ARs, which should enable the development of subtype-specific antagonists and agonists with reduced side effects.T he adrenergic receptors (ARs) are the class of G proteincoupled receptors (GPCR) responsible for mediating the effects of the catecholamines epinephrine and norepinephrine. There are currently nine known human ARs, partitioned into three subclasses: ␣1 (three subtypes located in vascular smooth muscle, the digestive tract, liver, and postsynaptically in the CNS), ␣2 (three subtypes located pre-and postsynaptically in the CNS, and in a wide variety of peripheral sites), and ␤ (three subtypes located primarily in cardiac, vascular, and adipose tissues, respectively).The members of this receptor class mediate a wide variety of physiological responses, including vasodilation and vasoconstriction, heart rate modulation, regulation of lipolysis, and blood clotting. These diverse and important functions make the adrenergic receptors a tempting pharmaceutical target, but attempts to create effective and specific drugs acting on these receptors have been slowed down by the lack of a 3D structure for any GPCR other than the bovine photoreceptor rhodopsin. The focus of this paper is the ␤2AR, which is targeted by agonist therapy in the treatment of asthma. Unfortunately, ␤2 agonists also exhibit crossreactivity with the other ␤ARs, causing side effects such as increased heart rate and blood pressure (1). Three-dimensional models of the ARs would be extremely useful in the design of subtype-specific pharmaceutical compounds. In addition, the ARs have been thoroughly studied experimentally so that there are ample data for validating the structural predictions, which may in turn provide improved understanding for the superfamily of GPCRs.We report here the predicted 3D structure of ␤2AR, which we use to predict detailed binding sites of agonists and antagonists to ␤2AR. This is an excellent case for validation because there is a wealth of experimental data on ligand-binding sites and mutational analysis with which to compare our results (2, 3).We use the MembStruk ...

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“…2A and SI Materials and Methods). The anisotropy of labeled protein frequently decays with two exponential components that correspond to rotational diffusion of the fluorophore and whole protein (45,46). Such two-exponential decay was observed for both Ras(C181) and the Y64A mutant on membranes ( Fig.…”

Section: H-ras Exhibits Reduced Translational and Rotational Mobilitymentioning

confidence: 85%

H-Ras forms dimers on membrane surfaces via a protein–protein interface

Lin

Iversen

et al. 2014

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

150

165

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The lipid-anchored small GTPase Ras is an important signaling node in mammalian cells. A number of observations suggest that Ras is laterally organized within the cell membrane, and this may play a regulatory role in its activation. Lipid anchors composed of palmitoyl and farnesyl moieties in H-, N-, and K-Ras are widely suspected to be responsible for guiding protein organization in membranes. Here, we report that H-Ras forms a dimer on membrane surfaces through a protein-protein binding interface. A Y64A point mutation in the switch II region, known to prevent Son of sevenless and PI3K effector interactions, abolishes dimer formation. This suggests that the switch II region, near the nucleotide binding cleft, is either part of, or allosterically coupled to, the dimer interface. By tethering H-Ras to bilayers via a membrane-miscible lipid tail, we show that dimer formation is mediated by protein interactions and does not require lipid anchor clustering. We quantitatively characterize H-Ras dimerization in supported membranes using a combination of fluorescence correlation spectroscopy, photon counting histogram analysis, time-resolved fluorescence anisotropy, single-molecule tracking, and step photobleaching analysis. The 2D dimerization K d is measured to be ∼1 × 10 3 molecules/μm 2 , and no higher-order oligomers were observed. Dimerization only occurs on the membrane surface; H-Ras is strictly monomeric at comparable densities in solution. Analysis of a number of H-Ras constructs, including key changes to the lipidation pattern of the hypervariable region, suggest that dimerization is a general property of native H-Ras on membrane surfaces.Ras signaling | Ras assay

show abstract

Elucidation of the Nature of the Conformational Changes of the EF-interhelical Loop in Bacteriorhodopsin and of the Helix VIII on the Cytoplasmic Surface of Bovine Rhodopsin: A Time-resolved Fluorescence Depolarization Study

Cited by 80 publications

References 44 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

Predicted 3D structure for the human β2 adrenergic receptor and its binding site for agonists and antagonists

H-Ras forms dimers on membrane surfaces via a protein–protein interface

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