A new method to measure 1J(Ni,Calphai) and 2J(Ni,Calpha(i-1)) coupling constants in proteins based on a J-modulated sensitivity enhanced HSQC was introduced. Coupling constants were measured in the denatured and in the native state of ubiquitin and found to depend on the conformation of the protein backbone. Using a combined data set of experimental coupling constants from ubiquitin and staphylococcal nuclease (Delaglio et al., 1991), the angular dependence of the coupling constants on the backbone angles psi and phi was investigated. It was found that the size of 2J(Ni,Calpha(i-1)) correlates strongly with the backbone conformation, while only a weak conformational dependence on the size of 1J(Ni,Calphai) coupling constants was observed. Coupling constants in the denatured state of ubiquitin were uniform along the sequence of the protein and not dependent on a given residue type. Furthermore it was shown that the observed coupling constants were in good agreement with predicted coupling constants using a simple model for the random coil.