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

ABSTRACT: 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-A 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.

SUBMITTER: Altenbach C

PROVIDER: S-EPMC2396682 | biostudies-literature | 2008 May

REPOSITORIES: biostudies-literature

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

Altenbach Christian C Kusnetzow Ana Karin AK Ernst Oliver P OP Hofmann Klaus Peter KP Hubbell Wayne L WL

Proceedings of the National Academy of Sciences of the United States of America 20080519 21

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 ...[more]

PMID: 18490656

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Project description:BACKGROUND:Spatial resolution in cardiac activation maps based on voltage measurement is limited by far-field interference. Precise characterization of electrical sources would resolve this limitation; however, practical charge-based cardiac mapping has not been achieved. METHODS:A prototype algorithm, developed from first principles of electrostatic field theory, derives charge density (CD) as a spatial representation of the true sources of the cardiac field. The algorithm processes multiple, simultaneous, noncontact voltage measurements within the cardiac chamber to inversely derive the global distribution of CD sources across the endocardial surface. RESULTS:Comparison of CD to an established computer-simulated model of atrial conduction demonstrated feasibility in terms of spatial, temporal, and morphologic metrics. Inverse reconstruction matched simulation with median spatial errors of 1.73 mm and 2.41 mm for CD and voltage, respectively. Median temporal error was less than 0.96 ms and morphologic correlation was greater than 0.90 for both CD and voltage. Activation patterns observed in human atrial flutter reproduced those established through contact maps, with a 4-fold improvement in resolution noted for CD over voltage. Global activation maps (charge density-based) are reported in atrial fibrillation with confirmed reduction of far-field interference. Arrhythmia cycle-length slowing and termination achieved through ablation of critical points demonstrated in the maps indicates both mechanistic and pathophysiological relevance. CONCLUSION:Global maps of cardiac activation based on CD enable classification of conduction patterns and localized nonpulmonary vein therapeutic targets in atrial fibrillation. The measurement capabilities of the approach have roles spanning deep phenotyping to therapeutic application. TRIAL REGISTRATION:ClinicalTrials.gov NCT01875614. FUNDING:The National Institute for Health Research (NIHR) Translational Research Program at Royal Papworth Hospital and Acutus Medical.

Project description:BackgroundRhodopsin, the prototypic member of G protein-coupled receptors (GPCRs), undergoes isomerization of 11-cis-retinal to all-trans-retinal upon photoactivation. Although the basic mechanism by which rhodopsin is activated is well understood, the roles of whole transmembrane (TM) helix-III during rhodopsin photoactivation in detail are not completely clear.Principal findingsWe herein use single-cysteine mutagenesis technique to investigate conformational changes in TM helices of rhodopsin upon photoactivation. Specifically, we study changes in accessibility and reactivity of cysteine residues introduced into the TM helix-III of rhodopsin. Twenty-eight single-cysteine mutants of rhodopsin (P107C-R135C) were prepared after substitution of all natural cysteine residues (C140/C167/C185/C222/C264/C316) by alanine. The cysteine mutants were expressed in COS-1 cells and rhodopsin was purified after regeneration with 11-cis-retinal. Cysteine accessibility in these mutants was monitored by reaction with 4, 4'-dithiodipyridine (4-PDS) in the dark and after illumination. Most of the mutants except for T108C, G109C, E113C, I133C, and R135C showed no reaction in the dark. Wide variation in reactivity was observed among cysteines at different positions in the sequence 108-135 after photoactivation. In particular, cysteines at position 115, 119, 121, 129, 131, 132, and 135, facing 11-cis-retinal, reacted with 4-PDS faster than neighboring amino acids. The different reaction rates of mutants with 4-PDS after photoactivation suggest that the amino acids in different positions in helix-III are exposed to aqueous environment to varying degrees.SignificanceAccessibility data indicate that an aqueous/hydrophobic boundary in helix-III is near G109 and I133. The lack of reactivity in the dark and the accessibility of cysteine after photoactivation indicate an increase of water/4-PDS accessibility for certain cysteine-mutants at Helix-III during formation of Meta II. We conclude that photoactivation resulted in water-accessible at the chromophore-facing residues of Helix-III.

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

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

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