RESUMO
Proton magnetic relaxation dispersion investigations with aqueous solutions of lysozyme and bovine serum albumin (BSA) in the 0-5 M range of guanidine hydrochloride (GdnHCl), pH 4.4, 27 degrees C, were taken up with the objective of examining the hydration dynamics of internal cavity waters as the protein is held under increasingly destabilizing conditions. Field cycling NMR and conventional pulsed NMR techniques were employed to cover a frequency range of 100 kHz to 50 MHz. Analyses of dispersion profiles at different concentrations of GdnHCl were carried out considering the contributions from internal and surface waters. The denaturant-dependent variation of internal water contribution indicates that the reorientational disorder of internal waters decreases with increments of the denaturant up to its subdenaturing limit. For both proteins, the variation of effective correlation time with GdnHCl apparently shows a marginal shrink in hydrodynamic volumes under the subdenaturing condition. These results suggest that subdenaturing amounts of GdnHCl restrict the motional freedom of the internal waters, and can have considerable influence on the surface hydration. On increasing the denaturant concentration further, the dispersion amplitude drops sharply, indicating that the chaotropic action of the denaturant now runs over its own cavity water-ordering effect operative in the subdenaturing limit. The results are fundamentally important for the understanding of the susceptibility of protein structure and hydration to denaturants.
