The representation of a protein's spatial sampling at atomic resolution is fundamental for understanding its function. NMR has been established as the best-suited technique toward this goal for small proteins. However, the accessible information content rapidly deteriorates with increasing protein size. We have recently demonstrated that for small proteins distance restraints with an accuracy smaller than 0.1 Å can be obtained by replacing traditional semi-quantitative Nuclear Overhauser Effects (NOEs) with exact NOEs (eNOE). The high quality of the data allowed us to calculate structural ensembles of the small model protein GB3 consisting of multiple rather than a single state. The analysis has been limited to small proteins because NOEs of spins with unresolved diagonal peaks cannot be used. Here we propose a simple approach to translate such NOEs into correct upper distance restraints, which opens access to larger biomolecules. We demonstrate that for 16 kDa cyclophilin A the collection of such restraints extends the original 1254 eNOEs to 3471.

We have recently developed an NMR protocol to extract exact distances between nuclei in proteins from an exact interpretation of NOESY buildup intensities (eNOEs). This enabled us to calculate multistate structural ensembles that exhibit realistic spatial sampling and long-range correlations. Our initial studies were laborious and required a deep understanding of the underlying spin dynamics. Here, we present a MatLab package that integrates all data processing steps required to convert intensities of assigned peaks in NOESY series into upper and lower distance limits for structure calculation. Those steps include organization of the data in object format, extraction of autorelaxation and cross-relaxation rate constants by fitting of diagonal peak decays and cross peak buildups, validation of the data, correction for spin diffusion, graphical display of the results, and generation of distance limits in CYANA compatible format. The analysis may be carried out using a full relaxation matrix or a simplified "divide and conquer" approach that allows for partial deuteration of protons. As the program does not require expertise beyond that of standard resonance assignment/structure calculation, it is suitable for experts and nonexperts alike.

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For enzyme activity, an exact structural and motional orchestration of the active site and its surroundings is believed to be key. In order to reveal such possible phenomena at atomic resolution on the basis of experimental evidence, an experimental restraint driven two-state ensemble of the prototypical enzyme cyclophilin was determined by using a recently introduced exact NOE approach. The ensemble description reveals the presence of an open and a closed state of cyclophilin, which is indicative of large-scale correlated motion. In the open state, the catalytic site is preorganized for catalysis, thus suggesting the mechanism of action to be conformational sampling, while the ligand-binding loop appears to act through an induced fit mechanism. This finding is supported by affinity measurements of a cyclophilin designed to be more open. Overall, more than 60-70 % of the side-chain conformations of cyclophilin appear to be correlated.