Structure and dynamics of retinal in rhodopsin elucidated by deuterium solid state NMR

Persistent Link:
http://hdl.handle.net/10150/280741
Title:
Structure and dynamics of retinal in rhodopsin elucidated by deuterium solid state NMR
Author:
Salgado, Gilmar F.
Issue Date:
2004
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
Rhodopsin is a seven transmembrane helix GPCR found which mediates dim light vision, in which the binding pocket is occupied by the ligand 11- cis-retinal. A site-directed ²H-labeling approach utilizing solid-state ²H NMR spectroscopy was used to investigate the structure and dynamics of retinal within its binding pocket in the dark state of rhodopsin, and as well the MetaI and MetaII. 11-cis-[5-C²H₃]-, 11-cis-[9-C²H₃]-, and 11-cis-[13-C²H₃]-retinal were used to regenerate bleached rhodopsin. Recombinant membranes comprising purified rhodopsin and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) were prepared (1:50 molar ratio). Solid-state ²H NMR spectra were obtained for the aligned rhodopsin/POPC recombinant membranes at temperatures below the order-disorder phase transition temperature of POPC. The solid-state NMR studies of aligned samples, give the orientations of the ²H nuclear coupling tensor relative to the membrane frame, which involve both the conformation and orientation of the bound retinal chromophore. Theoretical simulations of the experimental ²H NMR spectra employed a new lineshape treatment for a semi-random distribution due to static uniaxial disorder. The analysis gives the orientation of the ²H-labeled C-C²H₃ methyl bond axes relative to the membrane plane as well as the extent of three-dimensional alignment disorder (mosaic spread). These results clearly demonstrate the applicability of site-directed ²H NMR methods for investigating conformational changes and dynamics of ligands bound to rhodopsin and other GPCRs in relation to their characteristic mechanisms of action.
Type:
text; Dissertation-Reproduction (electronic)
Keywords:
Chemistry, Biochemistry.; Engineering, Biomedical.; Physics, Molecular.
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Graduate College; Biochemistry and Molecular Biophysics
Degree Grantor:
University of Arizona
Advisor:
Brown, Michael F.

Full metadata record

DC FieldValue Language
dc.language.isoen_USen_US
dc.titleStructure and dynamics of retinal in rhodopsin elucidated by deuterium solid state NMRen_US
dc.creatorSalgado, Gilmar F.en_US
dc.contributor.authorSalgado, Gilmar F.en_US
dc.date.issued2004en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.abstractRhodopsin is a seven transmembrane helix GPCR found which mediates dim light vision, in which the binding pocket is occupied by the ligand 11- cis-retinal. A site-directed ²H-labeling approach utilizing solid-state ²H NMR spectroscopy was used to investigate the structure and dynamics of retinal within its binding pocket in the dark state of rhodopsin, and as well the MetaI and MetaII. 11-cis-[5-C²H₃]-, 11-cis-[9-C²H₃]-, and 11-cis-[13-C²H₃]-retinal were used to regenerate bleached rhodopsin. Recombinant membranes comprising purified rhodopsin and 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) were prepared (1:50 molar ratio). Solid-state ²H NMR spectra were obtained for the aligned rhodopsin/POPC recombinant membranes at temperatures below the order-disorder phase transition temperature of POPC. The solid-state NMR studies of aligned samples, give the orientations of the ²H nuclear coupling tensor relative to the membrane frame, which involve both the conformation and orientation of the bound retinal chromophore. Theoretical simulations of the experimental ²H NMR spectra employed a new lineshape treatment for a semi-random distribution due to static uniaxial disorder. The analysis gives the orientation of the ²H-labeled C-C²H₃ methyl bond axes relative to the membrane plane as well as the extent of three-dimensional alignment disorder (mosaic spread). These results clearly demonstrate the applicability of site-directed ²H NMR methods for investigating conformational changes and dynamics of ligands bound to rhodopsin and other GPCRs in relation to their characteristic mechanisms of action.en_US
dc.typetexten_US
dc.typeDissertation-Reproduction (electronic)en_US
dc.subjectChemistry, Biochemistry.en_US
dc.subjectEngineering, Biomedical.en_US
dc.subjectPhysics, Molecular.en_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.disciplineBiochemistry and Molecular Biophysicsen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorBrown, Michael F.en_US
dc.identifier.proquest3158147en_US
dc.identifier.bibrecord.b48137959en_US
All Items in UA Campus Repository are protected by copyright, with all rights reserved, unless otherwise indicated.