Measurement of dipolar couplings in a transducin peptide fragment weakly bound to oriented photo-activated rhodopsin.
Rhodopsin-containing disks, isolated from rod outer segments of bovine retina, align at high magnetic fields with their membrane normal parallel to the magnetic field. After light-activation of rhodopsin, transient binding of the C-terminal transducin undecapeptide, selectively labeled with 15N at Leu5 and Gly9, results in residual dipolar contributions to the 1J(NH) splittings for these two residues. Both residues show 1J(NH) splittings which are smaller than in the dark-adapted or rhodopsin-free sample, and return to their isotropic values at a rate determined by the decay of the meta II state of rhodopsin. The dipolar couplings indicate that in the bound state, N-H vectors of Leu5 and Gly9 make angles of 48+/-4 degrees and 40+/-8 degrees, respectively, with the disk normal. These 'transferred' dipolar couplings potentially offer a useful method for studying the conformation and orientation of flexible, low affinity ligands when bound to oriented integral membrane receptors.
Koenig
B W
BW
Laboratory of Chemical Physics, NIDDK, National Institutes of Health, Bethesda, MD 20892-0520, USA.
Mitchell
D C
DC
König
S
S
Grzesiek
S
S
Litman
B J
BJ
Bax
A
A
D000595:Amino Acid Sequence; D055598:Chemical Phenomena; D002627:Chemistry, Physical; D004058:Diffusion; D008024:Ligands; D008027:Light; D008051:Lipid Bilayers; D008280:Magnetics; D008565:Membrane Proteins / Q000737:chemistry / Q000378:metabolism / Q000528:radiation effects; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010777:Photochemistry; D011485:Protein Binding; D011827:Radiation; D012243:Rhodopsin / Q000737:chemistry / Q000378:metabolism / Q000528:radiation effects; D055672:Static Electricity; D015293:Transducin / Q000737:chemistry* / Q000378:metabolism
D008024:Ligands; D008051:Lipid Bilayers; D008565:Membrane Proteins; D009587:Nitrogen Isotopes; D010446:Peptide Fragments; D012243:Rhodopsin; D015293:Transducin