Simultaneous measurement of protein one-bond and two-bond nitrogen-carbon coupling constants using an internally referenced quantitative J-correlated [(15)N,(1)H]-TROSY-HNC experiment.
A quantitative J-correlation pulse sequence is described that allows simultaneous determination of one-bond and two-bond nitrogen-carbon coupling constants for protonated or deuterated proteins. Coupling constants are calculated from volume ratios between cross peaks and reference axial peaks observed in a single 3D spectrum. Accurate backbone (1)J(NC'), (1)J(NCalpha), and (2)J(NCalpha) coupling constants are obtained for the two [(15)N;(13)C]-labeled, medium-sized proteins flavodoxin and xylanase and for the [(2)H;(15)N;(13)C]-labeled, large protein DFPase. A dependence of one-bond and two-bond J(NCalpha) values on protein backbone psi torsion angles is readily apparent, in agreement with previously found correlations. In addition, the experiment is performed on isotropic as well as aligned protein to measure associated (15)N-(13)C residual dipolar couplings.
Wienk
Hans L J
HL
Institut für Biophysikalische Chemie, JW Goethe-Universität, Marie-Curie-Str 9, D-60439 Frankfurt am Main, Germany.
Martínez
Mitcheell M
MM
Yalloway
Gary N
GN
Schmidt
Jürgen M
JM
Pérez
Carlos
C
Rüterjans
Heinz
H
Löhr
Frank
F
D001407:Bacillus / Q000201:enzymology; D002244:Carbon; D002247:Carbon Isotopes; D003001:Cloning, Molecular; D003903:Deuterium; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D007553:Isotope Labeling; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D009584:Nitrogen; D011487:Protein Conformation; D011506:Proteins / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification; D014316:Tritium; D043325:Xylan Endo-1,3-beta-Xylosidase; D014995:Xylosidases / Q000737:chemistry* / Q000302:isolation & purification
D002247:Carbon Isotopes; D011506:Proteins; D011994:Recombinant Proteins; D014316:Tritium; D002244:Carbon; D003903:Deuterium; D014995:Xylosidases; D043325:Xylan Endo-1,3-beta-Xylosidase; D009584:Nitrogen