Specific protein-urea interactions

Abstract

The mechanism of urea's action in protein denaturation remains largely unknown. To provide an experimental basis for molecular dynamics (MD) simulations on urea-protein interactions, we investigated the effect of urea on human intestinal fatty acid binding protein (hIFABP) by nuclear magnetic resonance (NMR). Hydrogen-deuterium exchange (HDX) rates at ≤ 2 M urea indicate that urea affects hIFABP in a residue-specific manner via direct urea-protein interactions and preferentially weakens hydrogen bonds between highly protected amides. Residue-specific effects of urea on NMR peak intensities and chemical shifts further support the presence of direct urea-protein interactions. Two-dimensional (2D) water-rotating frame Overhauser enhancement (ROE) data shows one protein-bound water molecule in contact with Val66 and Trp82, one putative bound water molecule in interaction with Thr76 and E-F loop, and that urea at low concentrations cannot displace these protein-bound water molecules. Our urea-nuclear Overhauser effect (NOE) experiments using 15N-urea further show no tightly protein-bound urea molecules. Our results thus suggest specific, but weak or transient, urea-protein interactions, supporting the direct interaction model of urea denaturation.

Keywords

• Protein denaturation
• Protein-folding
• Fatty acid binding protein
• Hydrogen-deuterium exchange