The interaction between receptors and guanine nucleotide binding (G) proteins leads to G protein activation subsequent regulation of effector enzymes. molecular basis receptor-G has been examined by using the ability from rods (transducin) cause a conformational change in rhodopsin as an assay. Synthetic peptides corresponding two regions near carboxyl terminus α subunit, Glu 311 -Val 328 Ile 340 -Phe 350 , compete with for rhodopsin. Amino acid substitution studies show that Cys 321 is...

Site-directed spin labeling has qualitatively shown that a key event during activation of rhodopsin is rigid-body movement transmembrane helix 6 (TM6) at the cytoplasmic surface molecule. To place this result on quantitative footing, and to identify movements other helices upon photoactivation, double electron-electron resonance (DEER) spectroscopy was used determine distances distance changes between pairs nitroxide side chains introduced in rhodopsin. Sixteen were selected from set nine...

Rhodopsin is a member of family receptors that contain seven transmembrane helices and are coupled to G proteins. The nature the interactions between rhodopsin mutants protein, transduction (Gt), was investigated by flash photolysis in order monitor directly Gt binding dissociation. Three mutant opsins with alterations their cytoplasmic loops bound 11-cis-retinal yield pigments native absorption spectra, but they failed stimulate guanosine triphosphatase activity Gt. opsin mutations included...

In the G protein-coupled receptor rhodopsin, conserved NPxxY(x) 5,6 F motif connects transmembrane helix VII and cytoplasmic 8. The less geometrically constrained retinal analogue 9-demethyl-retinal prevents efficient transformation of rhodopsin to signaling metarhodopsin (Meta) II after photoisomerization. Here, we demonstrate that Ala replacement mutations within domain, which eliminate an interaction between aromatic residues Y306 F313, allow formation Meta despite presence...

Abstract Using suction electrodes, photocurrent responses to 100-ms saturating flashes were recorded from isolated retinal rods of the larval-stage tiger salamander ( Ambystoma tigrinum ). The delay period T e ) that preceded recovery dark current by a criterion amount (3 pA) was analyzed in relation flash intensity I f ), and corresponding fractional bleach R* 0 /R tot visual pigment; compared with s at which peak level activated transducin approaches saturation. Over an approximately 8 In...

G protein-coupled receptors mediate biological signals by stimulating nucleotide exchange in heterotrimeric proteins (Galphabetagamma). Receptor dimers have been proposed as the functional unit responsible for catalytic interaction with Galphabetagamma. To investigate whether a receptor monomer can activate Galphabetagamma, we used retinal photoreceptor rhodopsin and its cognate protein transducin (G(t)) to determine stoichiometry of rhodopsin/G(t) binding rate catalyzed G(t). Purified was...

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTKinetics, binding constant, and activation energy of the 48-kDa protein-rhodopsin complex by extra-metarhodopsin IIAndreas Schleicher, Hermann Kuehn, Klaus Peter HofmannCite this: Biochemistry 1989, 28, 4, 1770–1775Publication Date (Print):February 21, 1989Publication History Published online1 May 2002Published inissue 21 February 1989https://pubs.acs.org/doi/10.1021/bi00430a052https://doi.org/10.1021/bi00430a052research-articleACS...

G protein coupled receptors (GPCRs) transmit extracellular signals into the cell by binding and activating different intracellular signaling proteins, such as proteins (Gαβγ, families Gi, Gs, Gq, G12/13) or arrestins. To address issue of Gs vs Gi coupling specificity, we carried out molecular dynamics simulations lipid-embedded active β2-adrenoceptor (β2AR*) in complex with C-terminal peptides derived from key interaction site Gα (GαCT) surrogate Gαβγ. We find that GiαCT GsαCT exploit...

Significance The existence of multiple conformational substates G-protein–coupled receptors in equilibrium may provide for the interaction with partners at same interface. Here we evidence that photoactivated rhodopsin exists a manifold lipid environment, but not extensively studied dodecyl maltoside detergent micelles. Moreover, state decays spontaneously to inactive on timescale minutes. Remarkably, binding activated receptor cognate G protein strongly biases an interacting retains more...

Rhodopsin−transducin coupling was used as an assay to investigate a laterally patterned membrane reconstituted with receptor and its G protein. It served model system show the feasibility immobilize protein-coupled receptors on solid supports activation interaction proteins by one-dimensional imaging surface plasmon resonance. Supported membranes were formed self-assembly of lipids rhodopsin from detergent solution onto functionalized gold surfaces. They micrometer-sized alternating regions...

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTRod Outer Segment Retinol Dehydrogenase: Substrate Specificity and Role in PhototransductionKrzysztof Palczewski, Stefan Jager, Janina Buczylko, Rosalie K. Crouch, D. Lucille Bredberg, Klaus Peter Hofmann, Mary Ann Asson-Batres, John C. SaariCite this: Biochemistry 1994, 33, 46, 13741–13750Publication Date (Print):November 1, 1994Publication History Published online1 May 2002Published inissue 1 November...

The role of the putative fourth cytoplasmic loop rhodopsin in binding and catalytic activation heterotrimeric G protein, transducin (G(t)), is not well defined. We developed a novel assay to measure ability G(t), or G(t)-derived peptides, inhibit photoregeneration from its active metarhodopsin II state. show that peptide corresponding residues 340-350 alpha subunit cysteinyl-thioetherfarnesyl 50-71 gamma are able interact with photoconversion rhodopsin. Alteration amino acid sequence either...

In rhodopsin, the 11-cis-retinal chromophore forms a complex with Lys296 of opsin via protonated Schiff base. Absorption light initiates activation rhodopsin by cis/trans photoisomerization retinal. Thermal relaxation through different intermediates leads into metarhodopsin states which bind and activate transducin (Gt) kinase (RK). all-trans-Retinal also recombines independent light, forming activating species receptor. this study, we examined mechanism all-trans-retinal activates opsin. To...

Activation of the G protein-coupled receptor rhodopsin involves both motion transmembrane helix 6 (TM6) and proton exchange events. To study how these activation steps relate to each other, spin-labeled in solutions dodecyl maltoside was used so that time-resolved TM6 could be monitored as a function pH temperature after an activating light flash. The results reveal is not synchronized with deprotonation Schiff base binds chromophore protein but order magnitude slower at 30°C. However,...

The G protein coupled receptor rhodopsin contains a pocket within its seven-transmembrane helix (TM) structure, which bears the inactivating 11-cis-retinal bound by protonated Schiff-base to Lys296 in TM7. Light-induced 11-cis-/all-trans-isomerization leads deprotonated active Meta II intermediate. With decay, bond is hydrolyzed, all-trans-retinal released from pocket, and apoprotein opsin reloaded with new 11-cis-retinal. crystal structure of Ops* conformation provides basis for...