BPS2026 - Precise determination of protein side chain rotamer populations by NMR and comparison with MD simulation

Protein side chains are more mobile than indicated by high resolution X-ray or cryo-EM structures. In theory, solution NMR could be used to reveal precise side chain structure and dynamics under physiologic conditions, but robust methodology needs to be developed and widely applied. We used a thermostable mutant of the human Pin1 WW domain. A time interval for measuring initial 1 H transverse magnetization relaxation rates is easily appended to any multidimensional NMR pulse sequence starting on 1 H, providing a robust experimental method for gauging mobility at almost every side chain site in a protein. Guided by the 1 H relaxation data, 3 J coupling data are analyzed to obtain precise side chain chi1 values for rigid residues or rotamer populations for mobile residues. Initially, our NMR-derived side chain chi1 values deviated from the high resolution X-ray crystal structure, using Karplus coefficients previously derived from NMR data. However, David case of Rutgers university generated new ab initio Karplus coefficients from DFT calculations, which brought our NMR-derived results into better agreement with the crystal structure. This suggests that the previous empirically derived Karplus coefficients were skewed by conformational averaging, emphasizing the importance of separating out mobile from rigid residues before embarking on J coupling analysis. In cases where the NMR and X-ray structure differed, molecular dynamics (MD) simulations suggested that the NMR answers were reasonable. There were some residues in which NMR and MD pointedly disagreed about side chain mobility. Using new methodology, solution NMR can determine protein side chain rotamer preferences under physiologic conditions with greater accuracy than X-ray crystallography. The methodology needs to be applied to more protein systems and may be useful for parametrizing MD simulations in the future.