Refinement of the geometry of the retinal binding pocket in dark-adapted bacteriorhodopsin by heteronuclear solid-state NMR distance measurements.
The bacterial proton pump bacteriorhodopsin (BR) is a 26.5 kDa seven-transmembrane helical protein. Several structural models have been published at > or =1.55 A resolution. The initial cis-trans isomerization of the retinal moiety involves structural changes within <1 A. To understand the chromophore-protein interactions that are important for light-driven proton transport, very accurate measurements of the protein geometry are required. To reveal more structural details at the site of the retinal, we have, therefore, selectively labeled the tryptophan side chains of BR with (15)N and metabolically incorporated retinal, (13)C-labeled at position 14 or 15. Using these samples, heteronuclear distances were measured with high accuracy using SFAM REDOR magic angle spinning solid-state NMR spectroscopy in dark-adapted bacteriorhodopsin. This NMR technique is applied for the first time to a high-molecular mass protein. Two retinal conformers are distinguished by their different isotropic 14-(13)C chemical shifts. Whereas the C14 position of 13-cis-15-syn-retinal is 4.2 A from [indole-(15)N]Trp86, this distance is 3.9 A in the all-trans-15-anti conformer. This latter distance allows us to check on the details of the active center of BR in the various published models derived from X-ray and electron diffraction data. The experimental approach and the results reported in this paper enforce the notion that distances between residues of a membrane protein binding pocket and a bound ligand can be determined at subangstrom resolution.
Helmle
M
M
Max-Planck-Institut für Biochemie, Am Klopferspitz 18A, Martinsried, Germany.
Patzelt
H
H
Ockenfels
A
A
Gärtner
W
W
Oesterhelt
D
D
Bechinger
B
B
D001436:Bacteriorhodopsins / Q000737:chemistry* / Q000378:metabolism; D001665:Binding Sites; D003624:Darkness; D006217:Halobacterium; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D017494:Proton Pumps / Q000737:chemistry* / Q000378:metabolism; D012172:Retinaldehyde / Q000737:chemistry* / Q000378:metabolism; D013347:Subcellular Fractions / Q000737:chemistry / Q000378:metabolism
D017494:Proton Pumps; D001436:Bacteriorhodopsins; D012172:Retinaldehyde