Determination of multiple ***&phi;***-torsion angles in proteins by selective and extensive (13)C labeling and two-dimensional solid-state NMR.
We describe an approach to efficiently determine the backbone conformation of solid proteins that utilizes selective and extensive (13)C labeling in conjunction with two-dimensional magic-angle-spinning NMR. The selective (13)C labeling approach aims to reduce line broadening and other multispin complications encountered in solid-state NMR of uniformly labeled proteins while still enhancing the sensitivity of NMR spectra. It is achieved by using specifically labeled glucose or glycerol as the sole carbon source in the protein expression medium. For amino acids synthesized in the linear part of the biosynthetic pathways, [1-(13)C]glucose preferentially labels the ends of the side chains, while [2-(13)C]glycerol labels the C(alpha) of these residues. Amino acids produced from the citric-acid cycle are labeled in a more complex manner. Information on the secondary structure of such a labeled protein was obtained by measuring multiple backbone torsion angles phi; simultaneously, using an isotropic-anisotropic 2D correlation technique, the HNCH experiment. Initial experiments for resonance assignment of a selectively (13)C labeled protein were performed using (15)N-(13)C 2D correlation spectroscopy. From the time dependence of the (15)N-(13)C dipolar coherence transfer, both intraresidue and interresidue connectivities can be observed, thus yielding partial sequential assignment. We demonstrate the selective (13)C labeling and these 2D NMR experiments on a 8.5-kDa model protein, ubiquitin. This isotope-edited NMR approach is expected to facilitate the structure determination of proteins in the solid state.
Hong
M
M
Department of Chemistry, University of Massachusetts, Amherst, Massachusetts 01003, USA.
D002247:Carbon Isotopes; D005947:Glucose; D005990:Glycerol; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D011506:Proteins; D014452:Ubiquitins
D002247:Carbon Isotopes; D011506:Proteins; D014452:Ubiquitins; D005947:Glucose; D005990:Glycerol