STABILIZATION OF A PROTEIN BY GUANIDINIUM CHLORIDE

Guanidinium chloride is a commonly used denaturant to unfold native pr oteins and to determine their Gibbs free energy of stabilization, DELT AG(stab). Here we show that this denaturant has a dual role for the st ability and the folding of the model protein ribonuclease T1. When pre sent at low concentration (0-0.3 M), guanidinium chloride stabilizes t he folded protein toward thermal and urea-induced unfolding and decrea ses the rate of unfolding. At high concentration the function of guani dinium chloride as a denaturant dominates and ribonuclease T1 is coope ratively unfolded. Ribonuclease T1 is also strongly stabilized by othe r salts, such as NaCl, at low concentrations, and the dependence of th e thermal stability on salt concentration is not linear. Such a comple x behavior was not found in control experiments with pancreatic ribonu clease A. The stabilization in the presence of low concentrations of g uanidinium chloride originates probably from the binding of guanidiniu m ions to one or a few cation binding sites that exist in native ribon uclease T1. It is not observed when an additional salt, NaCl, is prese nt simultaneously. The favorable interaction of guanidinium chloride w ith the native protein leads to increased values for DELTAG(stab), whe n unfolding transitions induced by guanidinium chloride are analyzed o n the basis of the two-state model by the linear extrapolation procedu re. The noncoincidence of these DELTAG(stab) values with stability dat a derived from urea-induced or thermal unfolding transitions does not imply that the two-state model is not appropriate but that the linear extrapolation to zero molar denaturant is incorrect. Such deviations f rom linearity and a stabilization of folded proteins by ionic denatura nts could be fairly common. They can easily be detected by measuring t hermal or urea-induced unfolding transitions in the presence of small concentrations of the denaturant of interest.