The use of ¹J_CαHα coupling constants as a probe for protein backbone conformation

Summary

Simple pseudo-3D modifications to the constant-time HSQC and HCACO experiments are described that allow accurate (±0.5 Hz) measurement of one bond J_CαHα coupling constants in proteins that are uniformly enriched with ¹³C. An empirical φ,ψ-surface is calculated which describes the deviation of ¹J_CαHα from its random coil value, using 203 ¹J_CαHα values measured for residues in the proteins calmodulin, staphylococcal nuclease, and basic pancreatic trypsin inhibitor, for which φ and ψ are know with good precision from previous X-ray crystallographic studies. Residues in α-helical conformation exhibit positive deviations of 4–5 Hz, whereas deviations in β-sheet are small and, on average, slightly negative. Data indicate that ¹J_CαHα depends primarily on ψ, and that ¹J_CαHα may be useful as a qualitative probe for secondary structure. Comparison of ¹J_CαHα coupling constants measured in free calmodulin and in its complex with a 26-aminoacid peptide fragment of myosin light-chain kinase confirm that the calmodulin secondary structure is retained upon complexation but that disruption of the middle part of the ‘central helix’ is even more extensive than in free calmodulin.