Alpha-helices as alignment reporters in residual dipolar coupling analysis of proteins.

Inclusion of residual dipolar couplings (RDCs) during the early rounds of protein structure determination requires use of a floating alignment tensor or knowledge of the alignment tensor strength and rhombicity. For proteins with interdomain motion, such analysis can falsely hide the presence of domain dynamics. We demonstrate for three proteins, maltotriose-ligated maltose binding protein (MBP), Ca²⁺-ligated calmodulin, and a monomeric N-terminal deletion mutant of the SARS-CoV-2 Main Protease, MPro, that good alignment tensor estimates of their domains can be obtained from RDCs measured for residues that are identified as α-helical based on their chemical shifts. The program, Helix-Fit, fits the RDCs to idealized α-helical coordinates, often yielding a comparable or better alignment tensor estimate than fitting to the actual high-resolution X-ray helix coordinates. The 13 helices of ligated MBP all show very similar alignment tensors, indicative of a high degree of order relative to one another. By contrast, while for monomeric MPro the alignment strengths of the five helices in the C-terminal helical domain (residues 200-306) are very similar, pointing to a well-ordered domain, the single α-helix Y54-I59 in the N-terminal catalytic domain (residues 10-185) aligns considerably weaker. This result indicates the presence of large amplitude motions of either Y54-I59 or of the entire N-terminal domain relative to the C-terminal domain, contrasting with the high degree of order seen in the native homodimeric structure.