Hot spot mapping of protein surfaces with TEMPOL: Bovine pancreatic RNase A as a model system

- RNase A free backbone amides establish transient HB interactions with TEMPOL.
- MD simulations offer a suitable basis for predicting RNase A-ligand HB interactions.
- TEMPOL PRE data yield information on hot spot distribution on RNase A surface.
- TEMPOL PRE and MSCS studies give a convergent profile of protein surface druggability.

TEMPOL spin-label has been used to identify surface exposure of protein nuclei from NMR analysis of the induced paramagnetic relaxation enhancements (PRE). The absence of linear dependence between atom depths and observed PRE reveals that specific mechanisms drive the approach of the paramagnet to the protein surface. RNase A represents a unique protein system to explore the fine details of the information offered by TEMPOL induced PRE, due to the abundance of previous results, obtained in solution and in the crystal, dealing with surface dynamics behavior of this protein. MD simulations in explicit solvent have been performed, also in the presence of TEMPOL, in order to delineate the role of intermolecular hydrogen bonds (HB) on PRE extents. Comparison of our results with the ones obtained from multiple solvent crystal structure (MSCS) studies yields information on the specificities that these two techniques have for characterizing protein-ligand interactions, a fundamental step in the development of reliable surface druggability predictors.

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