Structural Features and Light-Dependent Changes in the Sequence 59−75 Connecting Helices I and II in Rhodopsin:  A Site-Directed Spin-Labeling Study<sup>,</sup>

Altenbach, Christian; Klein‐Seetharaman, Judith; J, Hwa; Hg, Khorana; Wl, Hubbell

doi:10.1021/bi990014l

Cited by 99 publications

(92 citation statements)

References 32 publications

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“…Models of the R1 nitroxide side chain (see Methods) are shown at each of the sites selected on the outer surfaces of the TM helices and site 326 in the C terminus. The EPR spectra for the selected sites show little or no change upon photoactivation, as previously reported (12,(37)(38)(39)(40). Because the spectra are sensitive to changes in backbone dynamics (41,42) and local interactions within the protein (29)(30)(31)43), the lack of spectral change upon photoactivation strongly suggests that the conformation of R1, the immediate local environment, and the secondary structure to which it is attached remain the same under rhodopsin activation.…”

Section: Experimental Design and Resultssupporting

confidence: 71%

“…Moreover, at buried sites, protein conformational changes could lead to rotamer changes in R1 that are not revealed in the strongly immobilized EPR spectra, giving rise to distance changes unrelated to backbone movement. Even at solvent-exposed sites, rotamer changes could contribute to distance changes, but this possibility is minimized by selecting sites where the EPR spectra do not change significantly, as is the case for the sites selected here (12,(37)(38)(39)(40). In this case, distance changes for the nitroxide are expected to reflect positional changes of the backbone.…”

Section: Discussionmentioning

confidence: 99%

“…For the relatively short-range (10-20 Å) CW method used in earlier distance mapping of rhodopsin (13,37,38), it was not possible to avoid the use of buried sites, and the results are subject to the concerns outlined above. Moreover, it was not possible to measure distances across the molecule to determine the global pattern of helix motion.…”

Section: Discussionmentioning

confidence: 99%

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High-resolution distance mapping in rhodopsin reveals the pattern of helix movement due to activation

Altenbach

Kusnetzow

Ernst

et al. 2008

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

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431

454

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Site-directed spin labeling has qualitatively shown that a key event during activation of rhodopsin is a rigid-body movement of transmembrane helix 6 (TM6) at the cytoplasmic surface of the molecule. To place this result on a quantitative footing, and to identify movements of other helices upon photoactivation, double electron-electron resonance (DEER) spectroscopy was used to determine distances and distance changes between pairs of nitroxide side chains introduced in helices at the cytoplasmic surface of rhodopsin. Sixteen pairs were selected from a set of nine individual sites, each located on the solvent exposed surface of the protein where structural perturbation due to the presence of the label is minimized. Importantly, the EPR spectra of the labeled proteins change little or not at all upon photoactivation, suggesting that rigid-body motions of helices rather than rearrangement of the nitroxide side chains are responsible for observed distance changes. For inactive rhodopsin, it was possible to find a globally minimized arrangement of nitroxide locations that simultaneously satisfied the crystal structure of rhodopsin (Protein Data Bank entry 1GZM), the experimentally measured distance data, and the known rotamers of the nitroxide side chain. A similar analysis of the data for activated rhodopsin yielded a new geometry consistent with a 5-Å outward movement of TM6 and smaller movements involving TM1, TM7, and the C-terminal sequence following helix H8. The positions of nitroxides in other helices at the cytoplasmic surface remained largely unchanged.DEER ͉ G protein-coupled receptor ͉ photoreceptor ͉ spin labeling G protein-coupled receptors (GPCRs) constitute a large family of membrane proteins that mediate cellular responses to a variety of both physical and chemical signals.Interaction of a GPCR with a cognate signal produces a conformational change leading to an activated receptor. The activated state subsequently binds to and activates a corresponding heterotrimeric G protein, which in turn triggers events that ultimately modulate an amplified cellular response (for recent reviews, see refs. 1 and 2). The nature of the conformational switch or switches that underlie receptor activation is a subject of much interest (3, 4). Crystal structures of an inactive state of the GPCRs rhodopsin (5-9) and ␤ 2 -adrenergic receptor (10, 11) have been reported, and they provide a crucial foundation for designing and interpreting the results of spectroscopic experiments aimed at revealing conformational changes associated with activation.Site-directed spin-labeling (SDSL) studies of rhodopsin provided the first direct structural evidence for an outward tilt of the cytoplasmic end of transmembrane helix 6 (TM6) as a major event in the activation of rhodopsin, both in solutions of dodecyl maltoside (DM) and in phospholipid bilayers (12-15). Data from site-directed fluorescent labeling and chemical reactivity (16), UV spectroscopy (17), zinc cross-linking of histidines (18), and disulfide cross-linking (13) offered s...

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Section: Experimental Design and Resultssupporting

confidence: 71%

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

High-resolution distance mapping in rhodopsin reveals the pattern of helix movement due to activation

Altenbach

Kusnetzow

Ernst

et al. 2008

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

Self Cite

431

454

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“…Although a number of different factors can influence the reactivity of any one particular residue in STE2, a drop in reactivity over 3-5 residues is consistent with expectations for an α-helical TMD that is crossing the membrane boundary zone (14). Similar results were observed for the solvent accessibility of Cys-substituted residues in rhodopsin (28)(29)(30)(31), and the results were subsequently found to correlate well with the TMD arrangement observed in the rhodopsin crystal structure (27). The TMD boundaries determined by these experimental results for STE2 were compared to the predictions of TMHMM and SOSUI (Fig.…”

Section: Ste2 Topologysupporting

confidence: 83%

Accessibility of Cysteine Residues Substituted into the Cytoplasmic Regions of the α-Factor Receptor Identifies the Intracellular Residues That Are Available for G Protein Interaction

Choi

Konopka

2006

Biochemistry

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The yeast α-factor pheromone receptor (Ste2) belongs to the family of G protein-coupled receptors (GPCRs) that contain seven transmembrane domains. To define the residues that are accessible to the cytoplasmic G protein, Cys scanning mutagenesis was carried out in which each of the residues that span the intracellular loops and the cytoplasmic end of transmembrane domain 7 were substituted with Cys. The 90 different Cys-substituted residues were then assayed for reactivity with MTSEAbiotin (2-([biotinoyl] amino) ethyl methanethiosulfonate), which reacts with solvent accessible sulfhydryl groups. As part of these studies we show that adding free Cys to stop the MTSEA-biotin reactions has potential pitfalls in that Cys can rapidly undergo disulfide exchange with the biotinylated receptor proteins at pH ≥7. The central regions of the intracellular loops of Ste2 were all highly accessible to MTSEA-biotin. Residues near the ends of the loops typically exhibited a drop in the level of reactivity over a consecutive series of residues that was inferred to be the membrane boundary. Interestingly, these boundary residues were enriched in hydrophobic residues, suggesting that they may form a hydrophobic pocket for interaction with the G protein. Comparison with accessibility data from a previous study of the extracellular side of Ste2 indicates that the transmembrane domains vary in length, consistent with some transmembrane domains being tilted relative to the plane of the membrane as they are in rhodopsin. Altogether, these results define the residues that are accessible to the G protein and provide an important structural framework for the interpretation of the role of Ste2 residues that function in G protein activation.The S. cerevisiae α-factor pheromone receptor (Ste2) belongs to the large family of Gproteincoupled receptors (GPCRs) that transduce the signals for light, taste, olfaction, and many biomedically important hormones. Although GPCRs are quite diverse in sequence, they function in a similar manner to activate the α subunit of heterotrimeric G proteins to bind GTP and they also show similar structural architecture in that they are composed of a bundle of seven transmembrane domains (TMDs) connected by extracellular loops and intracellular loops (1,2). Ste2 shows overall similarity to many mammalian GPCRs in that the core region containing the TMDs is involved in signal transduction and the C terminal tail is a target for † This work was supported by National Institutes of Health Grant GM55107 awarded to J.B.K. post-translational modifications that regulate receptor desensitization and endocytosis (3). Ste2 activates a heterotrimeric G protein in which the α subunit shows about 45% identity with mammalian Gα proteins and is most closely related to the Gi subfamily (4). In addition, comparison of Ste2 with rhodopsin, a member of the large class A subfamily of mammalian GPCRs, indicates that there are similar microdomains in these divergent receptors (5). These results suggest that there are underlying ...

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“…A molecular description of this conformational change is a long-range goal of studies on signal transduction. Previous work on rhodopsin has given some insights into the tertiary structure in the cytoplasmic domain and its change upon light activation (1)(2)(3)(4). Cysteine scanning mutagenesis, the determination of proximity relationships by disulfide bond formation between pairs of cysteines, and EPR spin label studies have been instrumental in this work.…”

mentioning

confidence: 99%

Solution ¹⁹ F nuclear Overhauser effects in structural studies of the cytoplasmic domain of mammalian rhodopsin

Loewen

Klein‐Seetharaman

Getmanova

et al. 2001

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

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Structural Features and Light-Dependent Changes in the Sequence 59−75 Connecting Helices I and II in Rhodopsin: A Site-Directed Spin-Labeling Study^,

Cited by 99 publications

References 32 publications

High-resolution distance mapping in rhodopsin reveals the pattern of helix movement due to activation

High-resolution distance mapping in rhodopsin reveals the pattern of helix movement due to activation

Accessibility of Cysteine Residues Substituted into the Cytoplasmic Regions of the α-Factor Receptor Identifies the Intracellular Residues That Are Available for G Protein Interaction

Solution ¹⁹ F nuclear Overhauser effects in structural studies of the cytoplasmic domain of mammalian rhodopsin

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Structural Features and Light-Dependent Changes in the Sequence 59−75 Connecting Helices I and II in Rhodopsin: A Site-Directed Spin-Labeling Study,

Cited by 99 publications

References 32 publications

High-resolution distance mapping in rhodopsin reveals the pattern of helix movement due to activation

High-resolution distance mapping in rhodopsin reveals the pattern of helix movement due to activation

Accessibility of Cysteine Residues Substituted into the Cytoplasmic Regions of the α-Factor Receptor Identifies the Intracellular Residues That Are Available for G Protein Interaction

Solution 19 F nuclear Overhauser effects in structural studies of the cytoplasmic domain of mammalian rhodopsin

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Structural Features and Light-Dependent Changes in the Sequence 59−75 Connecting Helices I and II in Rhodopsin: A Site-Directed Spin-Labeling Study^,

Solution ¹⁹ F nuclear Overhauser effects in structural studies of the cytoplasmic domain of mammalian rhodopsin