SPECIFICITY OF UREA BINDING TO PROTEINS

The binding of urea to the small globular proteins bovine pancreatic t rypsin inhibitor (BPTI) and PEC-60 was investigated by nuclear magneti c resonance (NMR) spectroscopy. The conformation of the proteins was u naffected in the temperature range 4-36 degrees C and urea concentrati ons up to 8 M. Intermolecular nuclear Overhauser effects (NOE) observe d between the proteins and urea at 4 degrees C showed preferential bin ding of urea to pockets and grooves on the protein surfaces. These NOE s disappear at higher temperatures indicating kinetic lability of the urea binding on a nanosecond time scale. The binding constants were me asured 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 reversible arrow protein urea were between ab out 0.1 and 0.3 with a trend to lower values at higher temperatures. S ome of the binding sites identified by intermolecular NOEs with urea s howed binding constants above the average but never above 1. The data indicate that aqueous urea solutions present a rather uniform environm ent to proteins, where preferential binding of urea to defined conform ational sites occurs, but the interactions are weak and short lived.