Urea orientation at protein surfaces

We have exploited the unique ability of vibrational sum frequency spectroscopy (VSFS) to investigate interfacial urea molecules at protein surfaces. Experiments were carried out at the bovine serum albumin/water interface. The absolute orientation of interfacial urea could be followed directly by VSFS. It was found that urea orients with its NH groups pointing toward the protein at high pH, where the protein is negatively charged. The orientation flips at low pH, where the protein is positively charged. This behavior resembles that of interfacial water. The direct interactions between urea and proteins should be electrostatic in nature and, therefore, very sensitive to the charge state of the protein. Urea denaturation of proteins, however, is not sensitive to charge, which is inconsistent with a direct interaction mechanism.