Structural and orientational constraints of bacteriorhodopsin in purple membranes determined by oriented-sample solid-state NMR spectroscopy.
We report for the first time, oriented-sample solid-state NMR experiments, specifically polarization inversion spin exchange at the magic angle (PISEMA) and 1H-15N heteronuclear chemical shift correlation (HETCOR), applied to an integral seven-transmembrane protein, bacteriorhodopsin (bR), in natural membranes. The spectra of [15N]Met-bR revealed clearly distinguishable signals from the helical and loop regions. By deconvolution of the helix resonances, it was possible to establish constraints for some helix tilt angles. It was estimated that the extracellular section of helix B has a tilt of less than 5 degrees from the membrane normal, while the tilt of helix A was estimated to be 18-22 degrees , both of which are in agreement with most crystal structures. Comparison of the experimental PISEMA spectrum with simulated spectra based on crystal structures showed that PISEMA and HETCOR experiments are extremely sensitive to the polytopic protein structure, and the solid-state NMR spectra for membrane-embedded bR matched most favorably with the recent 1FBB electron crystallography structure. These results suggest that this approach has the potential to yield structural and orientational constraints for large integral polytopic proteins whilst intercalated and functionally competent in a natural membrane.
Kamihira
Miya
M
Biomembrane Structure Unit, Department of Biochemistry, Oxford University, South Parks Road, Oxford OX1 3QU, United Kingdom.
Vosegaard
Thomas
T
Mason
A James
AJ
Straus
Suzana K
SK
Nielsen
Niels Chr
NC
Watts
Anthony
A
D001436:Bacteriorhodopsins / Q000737:chemistry*; D003460:Crystallization; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D008565:Membrane Proteins / Q000737:chemistry; D017433:Protein Structure, Secondary; D018717:Purple Membrane / Q000737:chemistry*; D012399:Rotation
D008565:Membrane Proteins; D001436:Bacteriorhodopsins