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Dive into the research topics where Reiner Vogel is active.

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Featured researches published by Reiner Vogel.


The EMBO Journal | 2004

Electron crystallography reveals the structure of metarhodopsin I

Jonathan Ruprecht; Thorsten Mielke; Reiner Vogel; Claudio Villa; Gebhard F. X. Schertler

Rhodopsin is the prototypical G protein‐coupled receptor, responsible for detection of dim light in vision. Upon absorption of a photon, rhodopsin undergoes structural changes, characterised by distinct photointermediates. Currently, only the ground‐state structure has been described. We have determined a density map of a photostationary state highly enriched in metarhodopsin I, to a resolution of 5.5 Å in the membrane plane, by electron crystallography. The map shows density for helix 8, the cytoplasmic loops, the extracellular plug, all tryptophan residues, an ordered cholesterol molecule and the β‐ionone ring. Comparison of this map with X‐ray structures of the ground state reveals that metarhodopsin I formation does not involve large rigid‐body movements of helices, but there is a rearrangement close to the bend of helix 6, at the level of the retinal chromophore. There is no gradual build‐up of the large conformational change known to accompany metarhodopsin II formation. The protein remains in a conformation similar to that of the ground state until late in the photobleaching process.


Nature | 2010

Tracking G-protein-coupled receptor activation using genetically encoded infrared probes

Shixin Ye; Ekaterina Zaitseva; Gianluigi Caltabiano; Gebhard F. X. Schertler; Thomas P. Sakmar; Xavier Deupi; Reiner Vogel

Rhodopsin is a prototypical heptahelical family A G-protein-coupled receptor (GPCR) responsible for dim-light vision. Light isomerizes rhodopsins retinal chromophore and triggers concerted movements of transmembrane helices, including an outward tilting of helix 6 (H6) and a smaller movement of H5, to create a site for G-protein binding and activation. However, the precise temporal sequence and mechanism underlying these helix rearrangements is unclear. We used site-directed non-natural amino acid mutagenesis to engineer rhodopsin with p-azido-l-phenylalanine residues incorporated at selected sites, and monitored the azido vibrational signatures using infrared spectroscopy as rhodopsin proceeded along its activation pathway. Here we report significant changes in electrostatic environments of the azido probes even in the inactive photoproduct Meta I, well before the active receptor state was formed. These early changes suggest a significant rotation of H6 and movement of the cytoplasmic part of H5 away from H3. Subsequently, a large outward tilt of H6 leads to opening of the cytoplasmic surface to form the active receptor photoproduct Meta II. Thus, our results reveal early conformational changes that precede larger rigid-body helix movements, and provide a basis to interpret recent GPCR crystal structures and to understand conformational sub-states observed during the activation of other GPCRs.


Proceedings of the National Academy of Sciences of the United States of America | 2008

Two protonation switches control rhodopsin activation in membranes

Mohana Mahalingam; Karina Martínez-Mayorga; Michael F. Brown; Reiner Vogel

Activation of the G protein-coupled receptor (GPCR) rhodopsin is initiated by light-induced isomerization of the retinal ligand, which triggers 2 protonation switches in the conformational transition to the active receptor state Meta II. The first switch involves disruption of an interhelical salt bridge by internal proton transfer from the retinal protonated Schiff base (PSB) to its counterion, Glu-113, in the transmembrane domain. The second switch consists of uptake of a proton from the solvent by Glu-134 of the conserved E(D)RY motif at the cytoplasmic terminus of helix 3, leading to pH-dependent receptor activation. By using a combination of UV–visible and FTIR spectroscopy, we study the activation mechanism of rhodopsin in different membrane environments and show that these 2 protonation switches become partially uncoupled at physiological temperature. This partial uncoupling leads to ≈50% population of an entropy-stabilized Meta II state in which the interhelical PSB salt bridge is broken and activating helix movements have taken place but in which Glu-134 remains unprotonated. This partial activation is converted to full activation only by coupling to the pH-dependent protonation of Glu-134 from the solvent, which stabilizes the active receptor conformation by lowering its enthalpy. In a membrane environment, protonation of Glu-134 is therefore a thermodynamic rather than a structural prerequisite for activating helix movements. In light of the conservation of the E(D)RY motif in rhodopsin-like GPCRs, protonation of this carboxylate also may serve a similar function in signal transduction of other members of this receptor family.


Nature Chemical Biology | 2009

FTIR analysis of GPCR activation using azido probes

Shixin Ye; Thomas Huber; Reiner Vogel; Thomas P. Sakmar

We demonstrate the site-directed incorporation of an IR-active amino acid, p-azido-L-phenylalanine (azidoF, 1), into the G protein-coupled receptor rhodopsin using amber codon suppression technology. The antisymmetric stretch vibration of the azido group absorbs at approximately 2,100 cm(-1) in a clear spectral window and is sensitive to its electrostatic environment. We used FTIR difference spectroscopy to monitor the azido probe and show that the electrostatic environments of specific interhelical networks change during receptor activation.


Journal of Biological Chemistry | 2001

Conformations of the Active and Inactive States of Opsin

Reiner Vogel; Friedrich Siebert

The signaling state metarhodopsin II of the visual pigment rhodopsin decays to the apoprotein opsin and all-trans retinal, which are then regenerated to rhodopsin by the visual cycle. Opsin is known to have at neutral pH only a small residual constitutive activity toward its G protein transducin, which is thought to play a considerable role in light adaptation (bleaching desensitization). In this study we show with Fourier-transform infrared spectroscopy that after metarhodopsin II decay, opsin exists in two conformational states that are in a pH-dependent equilibrium at 30 °C with a pK of 4.1 in the presence of hydroxylamine scavenging the endogenous all-trans retinal. Despite the lack of the native agonist in its binding pocket, the low pH opsin conformation is very similar to that of metarhodopsin II and is likewise stabilized by peptides derived from rhodopsins cognate G protein, transducin. The high pH form, on the other hand, has some conformational similarity to the inactive metarhodopsin I state. We therefore conclude that the opsin apoprotein displays intrinsic conformational states that are merely modulated by bound all-trans retinal.


Current Opinion in Chemical Biology | 2000

Vibrational spectroscopy as a tool for probing protein function.

Reiner Vogel; Friedrich Siebert

Vibrational spectroscopy has become increasingly important as a tool for understanding the mechanisms of photosystem II, phytochrome and terminal oxidases. More general enzymatic or receptor systems have been studied, opening a new field of applications. Femtosecond infrared pump/probe studies of the important amide-I band seem to provide a basis for its molecular and structural interpretation.


Proceedings of the National Academy of Sciences of the United States of America | 2010

Highly conserved tyrosine stabilizes the active state of rhodopsin

Joseph A. Goncalves; Kieron South; Shivani Ahuja; Ekaterina Zaitseva; Chikwado A. Opefi; Markus Eilers; Reiner Vogel; Philip J. Reeves; Steven O. Smith

Light-induced isomerization of the 11-cis-retinal chromophore in the visual pigment rhodopsin triggers displacement of the second extracellular loop (EL2) and motion of transmembrane helices H5, H6, and H7 leading to the active intermediate metarhodopsin II (Meta II). We describe solid-state NMR measurements of rhodopsin and Meta II that target the molecular contacts in the region of the ionic lock involving these three helices. We show that a contact between Arg1353.50 and Met2576.40 forms in Meta II, consistent with the outward rotation of H6 and breaking of the dark-state Glu1343.49-Arg1353.50-Glu2476.30 ionic lock. We also show that Tyr2235.58 and Tyr3067.53 form molecular contacts with Met2576.40. Together these results reveal that the crystal structure of opsin in the region of the ionic lock reflects the active state of the receptor. We further demonstrate that Tyr2235.58 and Ala1323.47 in Meta II stabilize helix H5 in an active orientation. Mutation of Tyr2235.58 to phenylalanine or mutation of Ala1323.47 to leucine decreases the lifetime of the Meta II intermediate. Furthermore, the Y223F mutation is coupled to structural changes in EL2. In contrast, mutation of Tyr3067.53 to phenylalanine shows only a moderate influence on the Meta II lifetime and is not coupled to EL2.


Journal of the American Chemical Society | 2010

Sequential Rearrangement of Interhelical Networks Upon Rhodopsin Activation in Membranes: the Meta IIa Conformational Substate

Ekaterina Zaitseva; Michael F. Brown; Reiner Vogel

Photon absorption by rhodopsin is proposed to lead to an activation pathway that is described by the extended reaction scheme Meta I <==>Meta II(a) <==> Meta II(b) <==> Meta II(b)H(+), where Meta II(b)H(+) is thought to be the conformational substate that activates the G protein transducin. Here we test this extended scheme for rhodopsin in a membrane bilayer environment by investigating lipid perturbation of the activation mechanism. We found that symmetric membrane lipids having two unsaturated acyl chains, such as 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), selectively stabilize the Meta II(a) substate in the above mechanism. By combining FTIR and UV-visible difference spectroscopy, we characterized the structural and functional changes involved in the transition to the Meta II(a) intermediate, which links the inactive Meta I intermediate with the Meta II(b) states formed by helix rearrangement. Besides the opening of the Schiff base ionic lock, the Meta II(a) substate is characterized by an activation switch in a conserved water-mediated hydrogen-bonded network involving transmembrane helices H1/H2/H7, which is sensed by its key residue Asp83. On the other hand, movement of retinal toward H5 and its interaction with another interhelical H3/H5 network mediated by His211 and Glu122 is absent in Meta II(a). The latter rearrangement takes place only in the subsequent transition to Meta II(b), which has been previously associated with movement of H6. Our results imply that activating structural changes in the H1/H2/H7 network are triggered by disruption of the Schiff base salt bridge and occur prior to other chromophore-induced changes in the H3/H5 network and the outward tilt of H6 in the activation process.


Biochemistry | 2003

Deactivation of rhodopsin in the transition from the signaling state Meta II to Meta III involves a thermal isomerization of the retinal chromophore C=N double bond

Reiner Vogel; Friedrich Siebert; Gerald Mathias; Paul Tavan; Gui-Bao Fan; Mordechai Sheves

Light-induced isomerization of rhodopsins retinal chromophore to the activating all-trans geometry initializes the formation of the active receptor state, Meta II. In the absence of peripheral regulatory proteins, the activity of Meta II is switched off spontaneously by two independent pathways: either by hydrolysis of the retinal Schiff base and dissociation of the light receptor into apoprotein opsin plus free retinal or by formation of Meta III, an inactive species with intact retinal protonated Schiff base absorbing at 470 nm. By FTIR spectroscopy on rhodopsin reconstituted with isotopically labeled chromophores in combination with quantum mechanical DFT calculations, we show that the deactivating step during formation of Meta III involves a thermal isomerization of the chromophore C[double bond]N, such that the chromophore in Meta III is all-trans-15-syn. This isomerization step is catalyzed by the protein environment and proceeds via Meta I, as suggested by its dependence on pH and on properties of the lipid/detergent environment of the protein. In the long term, Meta III decays likewise to opsin and free retinal by slow hydrolysis of the Schiff base.


Journal of Physical Chemistry B | 2008

Structure and thermotropic phase behavior of fluorinated phospholipid bilayers: a combined attenuated total reflection FTIR spectroscopy and imaging ellipsometry study.

Steffen Schuy; Simon Faiss; Nicholas C. Yoder; Venkateshwarlu Kalsani; Krishna Kumar; Andreas Janshoff; Reiner Vogel

Lipid bilayers consisting of lipids with terminally perfluoroalkylated chains have remarkable properties. They exhibit increased stability and phase-separated nanoscale patterns in mixtures with nonfluorinated lipids. In order to understand the bilayer properties that are responsible for this behavior, we have analyzed the structure of solid-supported bilayers composed of 1,2-dipalmitoyl- sn-glycero-3-phosphocholine (DPPC) and of a DPPC analogue with 6 terminal perfluorinated methylene units (F6-DPPC). Polarized attenuated total reflection Fourier-transform infrared spectroscopy indicates that for F6-DPPC, the tilt of the lipid acyl chains to the bilayer normal is increased to 39 degrees as compared to 21 degrees for native DPPC, for both lipids in the gel phase. This substantial increase of the tilt angle is responsible for a decrease of the bilayer thickness from 5.4 nm for DPPC to 4.5 nm for F6-DPPC, as revealed by temperature-controlled imaging ellipsometry on microstructured lipid bilayers and solution atomic force microscopy. During the main phase transition from the gel to the fluid phase, both the relative bilayer thickness change and the relative area change are substantially smaller for F6-DPPC than for DPPC. In light of these structural and thermotropic data, we propose a model in which the higher acyl-chain tilt angle in F6-DPPC is the result of a conformational rearrangement to minimize unfavorable fluorocarbon-hydrocarbon interactions in the center of the bilayer due to chain staggering.

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Mordechai Sheves

Weizmann Institute of Science

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Thomas P. Sakmar

Laboratory of Molecular Biology

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Gui-Bao Fan

Weizmann Institute of Science

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Steffen Schuy

University of Göttingen

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