Dipolar assisted rotational resonance NMR of tryptophan and tyrosine in rhodopsin

Crocker, Evan; Patel, Avnish; Eilers, Markus; Jayaraman, Sundararajan; Getmanova, E. V.; Reeves, Philip J.; Ziliox, Martine; Khorana, H G; Sheves, Mordechai; Smith, Steven O.

doi:10.1023/b:jnmr.0000019521.79321.3c

Cited by 50 publications

(63 citation statements)

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“…2D 13 C DARR NMR measurements (28) were performed with 1024 points in the direct (f2) dimension and 64 points in the indirect (f1) dimension as described previously (26). A DARR mixing time of 600 ms was used to maximize homo-nuclear recoupling (29). The 1 H radiofrequency field strength during the mixing period was matched to the MAS speed for each sample, satisfying the n ϭ 1 matching condition.…”

Section: Methodsmentioning

confidence: 99%

Location of the Retinal Chromophore in the Activated State of Rhodopsin

Ahuja

Crocker

Eilers

et al. 2009

Journal of Biological Chemistry

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138

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Rhodopsin is a highly specialized G protein-coupled receptor (GPCR) that is activated by the rapid photochemical isomerization of its covalently bound 11-cis-retinal chromophore. Using two-dimensional solid-state NMR spectroscopy, we defined the position of the retinal in the active metarhodopsin II intermediate. Distance constraints were obtained between amino acids in the retinal binding site and specific 13 C-labeled sites located on the ␤-ionone ring, polyene chain, and Schiff base end of the retinal. We show that the retinal C20 methyl group rotates toward the second extracellular loop (EL2), which forms a cap on the retinal binding site in the inactive receptor. Despite the trajectory of the methyl group, we observed an increase in the C20-Gly 188 (EL2) distance consistent with an increase in separation between the retinal and EL2 upon activation. NMR distance constraints showed that the ␤-ionone ring moves to a position between Met 207 and Phe 208 on transmembrane helix H5. Movement of the ring toward H5 was also reflected in increased separation between the C⑀ carbons of Lys 296 (H7) and Met 44 (H1) and between Gly 121 (H3) and the retinal C18 methyl group. Helix-helix interactions involving the H3-H5 and H4-H5 interfaces were also found to change in the formation of metarhodopsin II reflecting increased retinal-protein interactions in the region of Glu 122 (H3) and His 211 (H5). We discuss the location of the retinal in metarhodopsin II and its interaction with sequence motifs, which are highly conserved across the pharmaceutically important class A GPCR family, with respect to the mechanism of receptor activation.The first step in the activation mechanism of most G proteincoupled receptors (GPCRs) 3 is the binding of a signaling ligand.Ligand binding to the extracellular loops or within the transmembrane helical bundle of these receptors leads to an allosteric conformational change that causes G protein activation. The detailed location of the activating ligand and the conformational changes triggered by ligand binding are still not well established for any GPCR. The visual photoreceptor rhodopsin is a member of the large family of class A GPCRs (1). These receptors have a common architecture consisting of seventransmembrane ␣-helices. Activation of rhodopsin begins with the absorption of light by the photoreactive 11-cis-retinal chromophore. The retinal is covalently bound as a protonated Schiff base to Lys 296 and tightly packed within the bundle of helices (Fig. 1, B and C). The retinal binding site is restricted along the axis of the retinal polyene chain by transmembrane helices H5 and H7 and is enclosed on the extracellular side of the dark, inactive state of rhodopsin by the second extracellular (or intradiscal) loop (EL2). The retinal binding site in the inactive state of rhodopsin can accommodate the 7-cis, 9-cis, and 11-cis isomers of retinal but not the longer all-trans or 13-cis isomers (2). Within this restricted space, the rapid 11-cis-to-all-trans isomerization of the retinal upon abso...

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Section: Methodsmentioning

confidence: 99%

Location of the Retinal Chromophore in the Activated State of Rhodopsin

Ahuja

Crocker

Eilers

et al. 2009

Journal of Biological Chemistry

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138

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“…For dipolar-assisted rotational resonance (DARR) experiments (15), mixing times of 600 ms to 1 s were used to maximize homonuclear recoupling between 13 C labels (16). The 1 H radiofrequency field strength during mixing was matched to the MAS speed to satisfy the n ϭ 1 condition for each sample.…”

Section: Synthesis Of 13 C-labeled Retinals and Regeneration Into Rhomentioning

confidence: 99%

“…The strategy for establishing retinalprotein contacts in the retinal binding site is to incorporate 13 C labels into both the protein and the retinal and to identify close 13 C contacts by 2D solid-state NMR (16). The 2D NMR spectra are obtained by using MAS and DARR (15).…”

Section: Establishing Retinal-protein Contacts In the Retinal Bindingmentioning

confidence: 99%

Coupling of retinal isomerization to the activation of rhodopsin

Patel

Crocker²,

Eilers³

et al. 2004

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

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Activation of the visual pigment rhodopsin is caused by 11-cis to -trans isomerization of its retinal chromophore. High-resolution solid-state NMR measurements on both rhodopsin and the metarhodopsin II intermediate show how retinal isomerization disrupts helix interactions that lock the receptor off in the dark. We made 2D dipolar-assisted rotational resonance NMR measurements between 13 C-labels on the retinal chromophore and specific 13 C-labels on tyrosine, glycine, serine, and threonine in the retinal binding site of rhodopsin. The essential aspects of the isomerization trajectory are a large rotation of the C20 methyl group toward extracellular loop 2 and a 4-to 5-Å translation of the retinal chromophore toward transmembrane helix 5. The retinal-protein contacts observed in the active metarhodopsin II intermediate suggest a general activation mechanism for class A G proteincoupled receptors involving coupled motion of transmembrane helices 5, 6, and 7.G protein-coupled receptors (GPCRs) are a large superfamily of membrane receptors that have seven transmembrane (TM) helices and respond to a wide array of signaling ligands. Similarities in the sequences of these receptors have led to the idea that they share a common activation mechanism, whereas sequence diversity is correlated with the specificity of different receptors for different ligands and G proteins. With the exception of the visual pigment rhodopsin (1, 2), the structures of GPCRs are unknown. The rhodopsin crystal structure shows that the TM helices are locked in an inactive conformation by interhelical interactions involving conserved amino acids. The general model of GPCR activation that has emerged over the past few years is that ligand-binding, or retinal isomerization in the case of the visual pigments, disrupts these interactions and drives a rigid body movement of one or more of the TM helices (3, 4).Helix-helix interactions in rhodopsin are mediated by two sets of conserved amino acids. The highly conserved signature amino acids have sequence identities of Ͼ80% across the family of class A GPCRs. These amino acids are generally highly polar, aromatics, or proline. The group-conserved amino acids are small and͞or weakly polar (Gly, Ala, Ser, Thr, and Cys) (5). They have low individual sequence identities but are highly conserved (Ͼ80%) when considered as a group. These amino acids are involved in mediating close helix contacts and facilitating interhelical hydrogen bonding (6).The location of the group-conserved amino acids largely in the interfaces of TM helices H1-H4 of rhodopsin has suggested that these helices are locked in a stable structure that does not change significantly upon receptor activation (5). There are fewer group-conserved amino acids in H5-H7. H5 and H7 are unusual in containing conserved prolines that facilitate interactions with the H1-H4 core by exposing the i-4 backbone carbonyl for interhelical hydrogen bonding. H6 is unusual in that the TM region is composed of conserved aromatic and large hydrophobic amino acids ...

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“…In the absence of an unequivocal X-ray definition of the chromophore-binding site, the elucidation of the orientation and conformation of the pSB retinyl chromophore in rhodopsin continues to be the subject of much spectroscopic analysis, including rather heroic efforts in NMR spectroscopy 21,[31][32][33][34][35][36][37][38][39][40][41][42][43][44] as well as FTIR [45][46][47] and resonance Raman spectroscopic studies. [48][49][50][51] While the experimental data reported in these studies have been reproduced and are considered unambiguous, the theoretical interpretation of the data does not yet lead to a fully consistent molecular picture in terms of the spatial, electronic, and vibrational structure of the system.…”

Section: Introductionmentioning

confidence: 99%