Solid-state 2H NMR spectroscopy of retinal proteins in aligned membranes.
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 beta-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.
Brown
Michael F
MF
Department of Chemistry, University of Arizona, Tucson, Arizona 85721, USA. mfbrown@u.arizona.edu
Heyn
Maarten P
MP
Job
Constantin
C
Kim
Suhkmann
S
Moltke
Stephan
S
Nakanishi
Koji
K
Nevzorov
Alexander A
AA
Struts
Andrey V
AV
Salgado
Gilmar F J
GF
Wallat
Ingrid
I
D000818:Animals; D001436:Bacteriorhodopsins / Q000737:chemistry; D006801:Humans; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008565:Membrane Proteins / Q000737:chemistry*; D008958:Models, Molecular; D015394:Molecular Structure; D012172:Retinaldehyde / Q000737:chemistry*; D012243:Rhodopsin / Q000737:chemistry
D008565:Membrane Proteins; D001436:Bacteriorhodopsins; D012243:Rhodopsin; D012172:Retinaldehyde