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...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 at2 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.Twodimensional(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.展开更多
基金supported by a grant to D.Y.from Singapore Ministry of Education Academic Research Fund Tier 1(R154-000-C03e114 or A-0004464-00-00).
文摘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 at2 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.Twodimensional(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.
