Rapid measurement of scalar three-bond 1HN-1H alpha spin coupling constants in 15N-labelled proteins.
Two new 2D NMR experiments, CT-HMQC-HA and CT-HMQC-HN, are proposed for the rapid measurement of homonuclear 3JHNH alpha coupling constants of uniformly 15N-enriched proteins in solution. The experiments are based on the comparison of the signal intensities in a pair of constant-time [15N,1H]-HMQC spectra recorded with and without decoupling of the amide proton-alpha proton coupling. Experimental data recorded with the 78-residue N-terminal domain of the E. coli arginine repressor (ArgR-N) and with oxidized E. coli flavodoxin (176 residues) showed good agreement with 3JHNH alpha coupling constants obtained by fitting of the multiplet fine structure of the amide proton resonances or from a 3D HNHA-J experiment, respectively. Quantitative estimates for the effects from different relaxation rates of in-phase and antiphase magnetization are given.
Ponstingl
H
H
Department of Medical Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden.
Otting
G
G
D000577:Amides / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins; D005418:Flavodoxin / Q000737:chemistry; D006859:Hydrogen / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy / Q000379:methods*; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D011522:Protons; D012097:Repressor Proteins / Q000737:chemistry
D000577:Amides; C087018:ArgR protein, Bacteria; C490177:ArgR protein, E coli; D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D005418:Flavodoxin; D010446:Peptide Fragments; D011506:Proteins; D011522:Protons; D012097:Repressor Proteins; D006859:Hydrogen