The shift in urea orientation at protein surfaces at low pH is compatible with a direct mechanism of protein denaturation.

Abstract

Surface-specific spectroscopic data has shown that urea undergoes a shift in orientation at protein surfaces in acidic media. Since urea denatures proteins at a wide range of pHs, the variable chemical nature of protein-urea interactions has been used to support an indirect mechanism of urea-induced denaturation. Here, we use molecular dynamics simulations, minimum-distance distribution functions (MDDFs), and hydrogen-bond analysis, to characterize the interactions of urea with proteins at neutral and low pH, as defined by the protonation state of acidic residues. We obtain the expected preferential solvation by urea and dehydration, consistently with urea-induced denaturation, while the MDDFs allow for a solvent-shell perspective of protein-urea interactions. The distribution functions are decomposed into atomic contributions to show that there is indeed a shift in the orientation of urea molecules in the vicinity of acidic side-chains, as shown by the experimental spectroscopic data. However, this effect is local, and the interactions of urea with the other side chains and with the protein backbone are essentially unaffected at low pH. Therefore, hydrophobic solvation and urea-backbone hydrogen bonds can play a role in a direct mechanism of urea-induced protein denaturation without contradicting the observed variations in the chemical nature of protein-urea interactions as a function of the acidity of the solution.