Specificity of Urea Binding to Proteins

Abstract

The binding of urea to the small globular proteins bovine pancreatic trypsin inhibitor (BPTI) and PEC-60 was investigated by nuclear magnetic resonance (NMR) spectroscopy. The conformation of the proteins was unaffected in the temperature range 4-36 °C and urea concentrations up to 8 M. Intermolecular nuclear Overhauser effects (NOE) observed between the proteins and urea at 4 °C showed preferential binding of urea to pockets and grooves on the protein surfaces. These NOEs disappear at higher temperatures indicating kinetic lability of the urea binding on a nanosecond time scale. The binding constants were measured by following the amide proton chemical shifts as a function of urea concentration. The average values of the binding constants in the reaction protein + urea 5=» protein-urea were between about 0.1 and 0.3 with a trend to lower values at higher temperatures. Some of the binding sites identified by intermolecular NOEs with urea showed binding constants above the average but never above 1. The data indicate that aqueous urea solutions present a rather uniform environment to proteins, where preferential binding of urea to defined conformational sites occurs, but the interactions are weak and short lived.