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Solid-State H NMR spectroscopy of retinal proteins in aligned membranes |
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| Authors: | Michael F Brown Maarten P Heyn Constantin Job Stephan Moltke Alexander A Nevzorov Gilmar FJ Salgado Ingrid Wallat |
| Institution: | a Department of Chemistry, University of Arizona, Tucson, Arizona 85721, USA b Department of Physics, University of Arizona, Tucson, Arizona 85721, USA c Department of Biochemistry and Molecular Biophysics, University of Arizona, Tucson, Arizona 85721, USA d Biophysics Group, Department of Physics, Freie Universität Berlin, D-14195 Berlin, Germany e Department of Chemistry and Chemistry Institute for Fundamental Materials, Pusan National University, Busan 609-735, Korea f Department of Chemistry, Columbia University, New York, New York 10027, USA g Department of Chemistry, North Carolina State University, Raleigh, North Carolina 27695, USA |
| Abstract: | Solid-state 2H NMR spectroscopy gives a powerful avenue to investigating the structures of ligands and cofactors bound to integral membrane proteins. For bacteriorhodopsin (bR) and rhodopsin, retinal was site-specifically labeled by deuteration of the methyl groups followed by regeneration of the apoprotein. 2H NMR studies of aligned membrane samples were conducted under conditions where rotational and translational diffusion of the protein were absent on the NMR time scale. The theoretical lineshape treatment involved a static axial distribution of rotating C-C2H3 groups about the local membrane frame, together with the static axial distribution of the local normal relative to the average normal. Simulation of solid-state 2H NMR lineshapes gave both the methyl group orientations and the alignment disorder (mosaic spread) of the membrane stack. The methyl bond orientations provided the angular restraints for structural analysis. In the case of bR the retinal chromophore is nearly planar in the dark- and all-trans light-adapted states, as well upon isomerization to 13-cis in the M state. The C13-methyl group at the “business end” of the chromophore changes its orientation to the membrane upon photon absorption, moving towards W182 and thus driving the proton pump in energy conservation. Moreover, rhodopsin was studied as a prototype for G protein-coupled receptors (GPCRs) implicated in many biological responses in humans. In contrast to bR, the retinal chromophore of rhodopsin has an 11-cis conformation and is highly twisted in the dark state. Three sites of interaction affect the torsional deformation of retinal, viz. the protonated Schiff base with its carboxylate counterion; the C9-methyl group of the polyene; and the β-ionone ring within its hydrophobic pocket. For rhodopsin, the strain energy and dynamics of retinal as established by 2H NMR are implicated in substituent control of activation. Retinal is locked in a conformation that is twisted in the direction of the photoisomerization, which explains the dark stability of rhodopsin and allows for ultra-fast isomerization upon absorption of a photon. Torsional strain is relaxed in the meta I state that precedes subsequent receptor activation. Comparison of the two retinal proteins using solid-state 2H NMR is thus illuminating in terms of their different biological functions. |
| Keywords: | bR bacteriorhodopsin bRall-t bacteriorhodopsin with all-trans 15-anti retinal chromophore bR13-c bacteriorhodopsin with 13-cis 15-syn retinal chromophore CD circular dichroism EFG electric field gradient GPCR G protein-coupled receptor M bacteriorhodopsin M intermediate with 13-cis 15-anti chromophore MAS magic-angle spinning MD molecular dynamics meta I metarhodopsin I meta II metarhodopsin II NMR nuclear magnetic resonance PAS principal axes system PDB Protein Data Bank POPC 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine PSB protonated Schiff base PYP photoactive yellow protein RMSD root mean square deviation RQC residual quadrupolar coupling |
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