1H NMR assignments of sidechain conformations in proteins using a high-dimensional potential in the simulated annealing calculations

A high-dimensional potential representing distance constraints for stereospecifically assignable diastereotopic proton or methyl pairs was incorporated into the dynamical simulated annealing protocol to calculate structures with stereospecifically determined sidechain conformations. The protocol is tested on nuclear magnetic resonance cross-relaxation data of a trypsin inhibitor from squash seeds, CMTI-I, and compared with two other methods of stereospecific assignment, the floating chirality and coupling constant methods. There is good agreement between the three methods in predicting the same stereospecific assignments. Because the high-dimensional potential uses more relaxed absolute distance constraints and also takes into account the relative distance constraint patterns, it avoids possible overinterpretation of the NOE data.