Quantitative J correlation methods for the accurate measurement of 13C'-13Cα dipolar couplings in proteins

Methods are described for the precise and accurate measurement of one-bond dipolar 13C'-13-alpha couplings in weakly aligned proteins. The experiments are based on the principle of quantitative J correlation, where 1JC'C-alpha (or JC'C-alpha+1DC'C-alpha) is measured from the relative intensity of two interleaved 3D TROSY-HN(CO)CA or 3D TROSY-HNCO spectra recorded with dephasing intervals of zero (reference spectrum) and approximate 3/(21JC'C-alpha) (attenuated spectrum). In analogy to other quantitative J correlation techniques, the random error in the measured 1JC'C-alpha value is inversely proportional to the signal-to-noise ratio in the reference spectrum. It is shown that for weakly aligned proteins, with the magnitude of the alignment tensor of D-a-NH less-than-or-equal-to 10-15 Hz, the systematic errors are typically negligible. The methods are demonstrated for the third IgG-binding domain of protein G (GB3) and alpha-synuclein in complex with a detergent micelle, where errors in 1DC'C-alpha of less than 0.1 Hz and ca 0.2 Hz, respectively, are estimated. Remarkably, the dipolar couplings determined for GB3 are in even better agreement with the recently refined 1.1-A X-ray structure than the input 13C'-13C-alpha couplings used for the refinement.