Probing protein structure by solvent perturbation of NMR spectra: the surface accessibility of bovine pancreatic trypsin inhibitor.

In the absence of specific interactions, the relative attenuation of protein NMR signals due to added stable free radicals such as TEMPOL should reflect the solvent accessibility of the molecular surface. The quantitative correlation between observed attenuation and surface accessibility was investigated with a model system, i.e., the small protein bovine pancreatic trypsin inhibitor. A detailed discussion is presented on the reliability and limits of the approach, and guidelines are provided for data acquisition, treatment, and interpretation. The NMR-derived accessibilities are compared with those obtained from x-ray diffraction and molecular dynamics data. Although the time-averaged accessibilities from molecular dynamics are ideally suited to fit the NMR data, better agreement was observed between the paramagnetic attenuations of the fingerprint cross-peaks of homonuclear proton spectra and the total NH and H alpha accessibilities calculated from x-ray coordinates, than from time-averaged molecular dynamics simulations. In addition, the solvent perturbation response appears to be a promising approach for detecting the thermal conformational evolution of secondary structure elements in proteins.