Four simple and sensitive HNCO-based methods for measurement of 1J(C'Calpha), 1J(NCalpha) and 2J(NCalpha) coupling constants in protein main chains are presented. Three of these experiments enable the simultaneous measurement of 1J(C'Calpha), 1J(NCalpha) and 2J(NCalpha) couplings. Exploitation of the E.COSY principle provides excellent dispersion of cross peaks in the resulting 3D spectra. The couplings can be retrieved with good accuracy from peak-to-peak separations. Karplus parameterizations are provided for 1J(NCalpha) and 2J(NCalpha), obtained from a nearly complete set of couplings of human ubiquitin. In addition, feasibility of the proposed methodology for measuring several residual dipolar couplings (RDCs) simultaneously is assessed.
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