The Exact NOE as an Alternative in Ensemble Structure Determination.

The structure-function paradigm is increasingly replaced by the structure-dynamics-function paradigm. All protein activity is steered by the interplay between enthalpy and entropy. Conformational dynamics serves as a proxy of conformational entropy. Therefore, it is essential to study not only the average conformation but also the spatial sampling of a protein on all timescales. To this purpose, we have established a protocol for determining multiple-state ensembles of proteins based on exact nuclear Overhauser effects (eNOEs). We have recently extended our previously reported eNOE data set for the protein GB3 by a very large set of backbone and side-chain residual dipolar couplings and three-bond J couplings. Here, we demonstrate that at least four structural states are required to represent the complete data set by dissecting the contributions to the CYANA target function, which quantifies restraint violations in structure calculation. We present a four-state ensemble of GB3, which largely preserves the characteristics obtained from eNOEs only. Due to the abundance of the input data, the ensemble and χ(1) angles in particular are well suited for cross-validation of the input data and comparison to x-ray structures. Principal component analysis is used to automatically identify and validate relevant states of the ensembles. Overall, our findings suggest that eNOEs are a valuable alternative to traditional NMR probes in spatial elucidation of proteins.