Measurement of one and two bond N-C couplings in large proteins by TROSY-based J-modulation experiments

Residual dipolar couplings (RDCs) between NC′ and NCα atoms in polypeptide backbones of proteins contain information on the orientation of bond vectors that is complementary to that contained in NH RDCs. The 1JNCα and 2JNCα scalar couplings between these atoms also display a Karplus relation with the backbone torsion angles and report on secondary structure. However, these N-C couplings tend to be small and they are frequently unresolvable in frequency domain spectra having the broad lines characteristic of large proteins. Here a TROSY-based J-modulated approach for the measurement of small 15N-13C couplings in large proteins is described. The cross-correlation interference effects inherent in TROSY methods improve resolution and signal to noise ratios for large proteins, and the use of J-modulation to encode couplings eliminates the need to remove frequency distortions from overlapping peaks during data analysis. The utility of the method is demonstrated by measurement of 1JNC′, 1JNCα, and 2JNCα scalar couplings and 1DNC′ and 1DNCα residual dipolar couplings for the myristoylated yeast ARF1·GTPγs protein bound to small lipid bicelles, a system with an effective molecule weight of ∼70 kDa.