Monitoring the Interaction of a Single G-Protein Key Binding Site with Rhodopsin Disk Membranes upon Light Activation

Kim, Tai-Yang; Uji‐i, Hiroshi; Möller, Martina; Muls, Benoît; Hofkens, Johan; Alexiev, Ulrike

doi:10.1021/bi900308c

Cited by 20 publications

(34 citation statements)

References 20 publications

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“…The existence of such precomplexes has been suggested by others (21,37,(64)(65)(66)(67)(68)(69). Here, we found that the existence of precomplexes is consistent with our kinetic data if they are sufficiently transient.…”

Section: Resultssupporting

confidence: 93%

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Explicit Spatiotemporal Simulation of Receptor-G Protein Coupling in Rod Cell Disk Membranes

Schöneberg

Heck

Hofmann

et al. 2014

Biophysical Journal

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Dim-light vision is mediated by retinal rod cells. Rhodopsin (R), a G-protein-coupled receptor, switches to its active form (R(∗)) in response to absorbing a single photon and activates multiple copies of the G-protein transducin (G) that trigger further downstream reactions of the phototransduction cascade. The classical assumption is that R and G are uniformly distributed and freely diffusing on disk membranes. Recent experimental findings have challenged this view by showing specific R architectures, including RG precomplexes, nonuniform R density, specific R arrangements, and immobile fractions of R. Here, we derive a physical model that describes the first steps of the photoactivation cascade in spatiotemporal detail and single-molecule resolution. The model was implemented in the ReaDDy software for particle-based reaction-diffusion simulations. Detailed kinetic in vitro experiments are used to parametrize the reaction rates and diffusion constants of R and G. Particle diffusion and G activation are then studied under different conditions of R-R interaction. It is found that the classical free-diffusion model is consistent with the available kinetic data. The existence of precomplexes between inactive R and G is only consistent with the data if these precomplexes are weak, with much larger dissociation rates than suggested elsewhere. Microarchitectures of R, such as dimer racks, would effectively immobilize R but have little impact on the diffusivity of G and on the overall amplification of the cascade at the level of the G protein.

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Section: Resultssupporting

confidence: 93%

“…1)). Evidence for the existence of such precomplexes has been reported in several studies (21,37,(64)(65)(66)(67)(68)(69), and it has been hypothesized that they are beneficial for G activation (37). Analogous to the formation of the reactive complex R Ã þ G#R Ã G (Eqs.…”

Section: Existence Of Rg Precomplexesmentioning

confidence: 94%

Explicit Spatiotemporal Simulation of Receptor-G Protein Coupling in Rod Cell Disk Membranes

Schöneberg

Heck

Hofmann

et al. 2014

Biophysical Journal

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“…To investigate the consequences of a track structure on reaction kinetics, the encounter rate of G t with rhodopsin and the kinetics of G t activation were simulated for three different structural scenarios ( Figure 5B): (i) rhodopsin and G t are free to diffuse according to the classic collision-coupling model of signaling by random encounters of molecules (control); (ii) immobile rhodopsin is organized in tracks; light activates rhodopsin (R*) either on the outside or inside of a track; and (iii) rhodopsin and G t form a precomplex RG t , with a kinetic stability that is determined by on and off rate constants. Although preassembly has yet to be demonstrated in vivo, at least simulations and studies using solubilized and reconstituted rhodopsin and G t suggest preassembly of signaling components (Alves et al, 2005;Dell'Orco and Koch, 2011;Fanelli and Dell'orco, 2008;Kim et al, 2009).…”

Section: Simulation Of Transducin Activation Kinetics By Different Rhmentioning

confidence: 99%

“…Preassembly of G t has been studied in vitro using solubilized rhodopsin and G t molecules (Alves et al, 2005;Dell'Orco and Koch, 2011;Fanelli and Dell'orco, 2008;Kim et al, 2009). However, high-affinity sites for preassembly might involve several rhodopsin molecules in a track and might require G t tethered to the disk membrane via its isoprenyl modification.…”

Section: Molecule Preassembly On Tracks Does Not Slow Down Activationmentioning

confidence: 99%

Higher-Order Architecture of Rhodopsin in Intact Photoreceptors and Its Implication for Phototransduction Kinetics

et al. 2015

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The visual pigment rhodopsin belongs to the family of G protein-coupled receptors that can form higher oligomers. It is controversial whether rhodopsin forms oligomers and whether oligomers are functionally relevant. Here, we study rhodopsin organization in cryosections of dark-adapted mouse rod photoreceptors by cryoelectron tomography. We identify four hierarchical levels of organization. Rhodopsin forms dimers; at least ten dimers form a row. Rows form pairs (tracks) that are aligned parallel to the disk incisures. Particle-based simulation shows that the combination of tracks with fast precomplex formation, i.e. rapid association and dissociation between inactive rhodopsin and the G protein transducin, leads to kinetic trapping: rhodopsin first activates transducin from its own track, whereas recruitment of transducin from other tracks proceeds more slowly. The trap mechanism could produce uniform single-photon responses independent of rhodopsin lifetime. In general, tracks might provide a platform that coordinates the spatiotemporal interaction of signaling molecules.

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“…Examples are fluorescent dyes such Atto647N, which can be excited at 640 nm where the amount of rhodopsin absorption is negligible [74]. Another approach is to constantly introduce new sample into the measuring beam.…”

Section: Challenges In Performing Fluorescence Experiments With Rementioning

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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Monitoring the Interaction of a Single G-Protein Key Binding Site with Rhodopsin Disk Membranes upon Light Activation

Cited by 20 publications

References 20 publications

Explicit Spatiotemporal Simulation of Receptor-G Protein Coupling in Rod Cell Disk Membranes

Explicit Spatiotemporal Simulation of Receptor-G Protein Coupling in Rod Cell Disk Membranes

Higher-Order Architecture of Rhodopsin in Intact Photoreceptors and Its Implication for Phototransduction Kinetics

Fluorescence spectroscopy of rhodopsins: Insights and approaches

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