Floating stereospecific assignment revisited: Application to an 18 kDa protein and comparison with J-coupling data

Abstract

We report a floating chirality procedure to treat nonstereospecifically assigned methylene orisopropyl groups in the calculation of protein structures from NMR data using restrainedmolecular dynamics and simulated annealing. The protocol makes use of two strategies toinduce the proper conformation of the prochiral centres: explicit atom ‘swapping’ followingan evaluation of the NOE energy term, and atom ‘floating’ by reducing the angle andimproper force constants that enforce a defined chirality at the prochiral centre. The individualcontributions of both approaches have been investigated. In addition, the effects of accuracyand precision of the interproton distance restraints were studied. The model system employedis the 18 kDa single-stranded DNA binding protein encoded by Pseudomonas bacteriophagePf3. Floating chirality was applied to all methylene and isopropyl groups that give rise to non-degenerate NMR signals, and the results for 34 of these groups were compared to J-couplingdata. We conclude that floating stereospecific assignment is a reliable tool in protein structurecalculation. Its use is beneficial because it allows the distance restraints to be extracteddirectly from the measured peak volumes without the need for averaging or addingpseudoatom corrections. As a result, the calculated structures are of a quality almostcomparable to that obtained with stereospecific assignments. As floating chirality furthermoreis the only approach treating prochiral centres that ensures a consistent assignment of the twoproton frequencies in a single structure, it seems to be preferable over using pseudoatoms or(R-6) averaging.