Determination of Multiple f-Torsion Angles in Proteins by Selective

a a s b i l p ( b b onformation of solid proteins that utilizes selective and extensive C labeling in conjunction with two-dimensional magic-anglepinning NMR. The selective C labeling approach aims to reduce ine broadening and other multispin complications encountered in olid-state NMR of uniformly labeled proteins while still enhancng the sensitivity of NMR spectra. It is achieved by using specifcally labeled glucose or glycerol as the sole carbon source in the rotein expression medium. For amino acids synthesized in the inear part of the biosynthetic pathways, [1-C]glucose preferenially labels the ends of the side chains, while [2-C]glycerol labels he C of these residues. Amino acids produced from the citriccid cycle are labeled in a more complex manner. Information on he secondary structure of such a labeled protein was obtained by easuring multiple backbone torsion angles f simultaneously, sing an isotropic–anisotropic 2D correlation technique, the NCH experiment. Initial experiments for resonance assignment f a selectively C labeled protein were performed using N–C D correlation spectroscopy. From the time dependence of the N–C dipolar coherence transfer, both intraresidue and interesidue connectivities can be observed, thus yielding partial seuential assignment. We demonstrate the selective C labeling nd these 2D NMR experiments on a 8.5-kDa model protein, biquitin. This isotope-edited NMR approach is expected to failitate the structure determination of proteins in the solid tate. © 1999 Academic Press