STABILIZATION OF A PROTEIN BY GUANIDINIUM CHLORIDE

Guanidinium chloride is a commonly used denaturant to unfold native proteins and to determine their Gibbs free energy of stabilization, DELTAG(stab). Here we show that this denaturant has a dual role for the stability and the folding of the model protein ribonuclease T1. When present at low concentration (0-0.3 M), guanidinium chloride stabilizes the folded protein toward thermal and urea-induced unfolding and decreases the rate of unfolding. At high concentration the function of guanidinium chloride as a denaturant dominates and ribonuclease T1 is cooperatively unfolded. Ribonuclease T1 is also strongly stabilized by other salts, such as NaCl, at low concentrations, and the dependence of the thermal stability on salt concentration is not linear. Such a complex behavior was not found in control experiments with pancreatic ribonuclease A. The stabilization in the presence of low concentrations of guanidinium chloride originates probably from the binding of guanidinium ions to one or a few cation binding sites that exist in native ribonuclease T1. It is not observed when an additional salt, NaCl, is present simultaneously. The favorable interaction of guanidinium chloride with the native protein leads to increased values for DELTAG(stab), when unfolding transitions induced by guanidinium chloride are analyzed on the basis of the two-state model by the linear extrapolation procedure. The noncoincidence of these DELTAG(stab) values with stability data 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 from linearity and a stabilization of folded proteins by ionic denaturants could be fairly common. They can easily be detected by measuring thermal or urea-induced unfolding transitions in the presence of small concentrations of the denaturant of interest.