Understanding protein-solute interactions is one of the sizable challenges of protein chemistry; therefore, numerous experimental studies have attempted to explain the mechanism by which proteins unfold in aqueous urea solutions. On the basis of kinetic evidence at low urea concentrations, (1)H NMR spectroscopic analysis, and molecular orbital calculations, we propose a mechanistic model for the denaturation of RNase A in urea. Our results support a direct interaction between urea and protonated histidine as the initial step for protein inactivation followed by hydrogen bond formation with polar residues, and the breaking of hydrophobic collapse as the final steps for protein denaturation. With the proposed model, we can rationalize apparently conflicting results in the literature about the mechanism of protein denaturation with urea.

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Understanding of protein-urea interactions is one of the greatest challenges to modern structural protein chemistry. Based in enzyme kinetics experiments and (1)H NMR spectroscopic analysis we proposed that urea, at low concentrations, directly interacts with the protonated histidines of the active center of RNase A, following a simple model of competitive inhibition. These results were supported by theoretical analysis based on the frontier molecular orbital theory and suggest that urea might establish a favorable interaction with the cationic amino acids. Our experimental evidence and theoretical analysis indicate that the initials steps of the molecular mechanism of Urea-RNase A interaction passes through the establishment of a three center four electron adduct. Also, our results would explain the observed disruption of the (1)H NMR signals corresponding to H12 and H119 (involved in catalysis) of the RNase A studied in the presence of urea. Our interaction model of urea-amino acids (cationic) can be extended to explain the inactivation of other enzymes with cationic amino acids at the active site.