Guanidinium chloride is a commonly used denaturant to unfold native proteins and to determine their Gibbs free energy of stabilization, delta Gstab. 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 delta Gstab, 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 delta Gstab 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.(ABSTRACT TRUNCATED AT 250 WORDS) Study holds ProTherm entries: 4556, 4557 Extra Details: guanidinium chloride; cooperative; cation binding sites;,two-state model
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:26 p.m.
|Number of data points
|Guanyl-specific ribonuclease T1 ; Guanyl-specific ribonuclease T1
|Experimental Assay: Tm ; Experimental Assay: dG_H2O
|Mutations for sequence ACDYTCGSNCYSSSDVSTAQAAGYQLHEDGETVGSNSYPHKYNNYEGFDFSVSSPYYEWPILSSGDVYSGGSPGADRVVFNENNQLAGVITHTGASGNNFVECT
|Guanyl-specific ribonuclease T1