Protein stabilization by specific binding of guanidinium to a functional arginine-binding surface on an SH3 domain.
Guanidinium hydrochloride (GuHCl) at low concentrations significantly stabilizes the Fyn SH3 domain. In this work, we have demonstrated that this stabilizing effect is manifested through a dramatic (five- to sixfold) decrease in the unfolding rate of the domain with the folding rate being affected minimally. This behavior contrasts to the effect of NaCl, which stabilizes this domain by accelerating the folding rate. These data imply that the stabilizing effect of GuHCl is not predominantly ionic in nature. Through NMR studies, we have identified a specific binding site for guanidinium, and we have determined a dissociation constant of 90 mM for this interaction. The guanidinium-binding site overlaps with a functionally important arginine-binding pocket on the domain surface, and we have shown that GuHCl is a specific inhibitor of the peptide-binding activity of the domain. A different SH3 domain possessing a similar arginine-binding pocket is also thermodynamically stabilized by GuHCl. These data suggest that many proteins that normally interact with arginine-containing ligands may also be able to specifically interact with guanidinium. Thus, some caution should be used when using GuHCl as a denaturant in protein folding studies. Since arginine-mediated interactions are often important in the energetics of protein-protein interactions, our observations could be relevant for the design of small molecule inhibitors of protein-protein interactions.
Zarrine-Afsar
Arash
A
Department of Biochemistry, University of Toronto, Toronto, Ontario, Canada.
Mittermaier
Anthony
A
Kay
Lewis E
LE
Davidson
Alan R
AR
D000818:Animals; D001120:Arginine / Q000378:metabolism*; D001665:Binding Sites / Q000502:physiology; D019791:Guanidine / Q000378:metabolism*; D007700:Kinetics; D009682:Magnetic Resonance Spectroscopy; D009154:Mutation; D010455:Peptides / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology; D011489:Protein Denaturation; D051076:Proto-Oncogene Proteins c-fyn / Q000235:genetics / Q000378:metabolism; D012965:Sodium Chloride; D013499:Surface Properties; D013816:Thermodynamics; D014508:Urea; D018909:src Homology Domains / Q000235:genetics / Q000502:physiology*
D010455:Peptides; D012965:Sodium Chloride; D014508:Urea; D001120:Arginine; D051076:Proto-Oncogene Proteins c-fyn; D019791:Guanidine