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 |
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Authors: | Wienk Hans L J Martínez Mitcheell M Yalloway Gary N Schmidt Jürgen M Pérez Carlos Rüterjans Heinz Löhr Frank |
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Affiliation: | (1) Institut für Biophysikalische Chemie, J.W. Goethe-Universität, Marie-Curie-Str. 9, D-60439 Frankfurt am Main, Germany;(2) Research School of Biosciences, University of Kent at Canterbury, Canterbury, Kent, CT2 7NJ, U.K |
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Abstract: | 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 1JNC, 1JNC, and 2JNC coupling constants are obtained for the two [15N;13C]-labeled, medium-sized proteins flavodoxin and xylanase and for the [2H;15N;13C]-labeled, large protein DFPase. A dependence of one-bond and two-bond JNC values on protein backbone 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 15N-13C residual dipolar couplings. |
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Keywords: | DFPase flavodoxin perdeuteration residual dipolar coupling scalar coupling TROSY xylanase |
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