Direct and Indirect Models for Protein Chemical Denaturation are Characteristic of Opposite Dynamic Properties

Two major models, namely direct and indirect models, have been proposed for the protein chemical denaturation but it remains challenging to experimentally demonstrate and distinguish between them. Here, by use of CD and NMR spectroscopy, we succeeded in differentiating the effects on a small but well-folded protein WW4, of GdmCl and NaSCN at diluted concentrations (≤200 mM). Both denaturants up to 200 mM have no alternation of its average structure but do reduce its thermodynamic stability to different degrees. Despite acting as the stronger denaturant, GdmCl only weakly interacts with amide protons, while NaSCN shows extensive interactions with both hydrophobic side chains and amide protons. Although both denaturants show no significant perturbation on overall ps-ns backbone dynamics of WW4, GdmCl suppresses while NaSCN enhances its μ s-ms backbone dynamics in a denaturant concentration dependent manner. Quantitative analysis reveals that although they dramatically raise exchange rates, GdmCl slightly increases while NaSCN reduces the population of the major conformational state. Our study represents the first report deciphering that GdmCl and NaSCN appear to destabilize a protein following two models respectively, which are characteristic of opposite μ s-ms dynamics. NaSCN and GdmCl destabilize a protein with opposite dynamics: Chemical denaturants are commonly utilized to study protein folding and stability but it still remains challenging to experimentally demonstrate and distinguish between direct and indirect models for their actions. Here NaSCN is revealed to destabilize WW4 by direct interactions while GdmCl by indirect effects. Most unexpectedly, two models are characteristic of opposite backbone dynamics. The study also suggests the absence of a simple correlation between protein dynamics and stability (see picture). 4 To function properly in vivo, many proteins fold into a specific three-dimensional structure called the native state which is only marginally stable relative to the denatured state. Thermodynamic stability is not only essential for biological functions of proteins, but also a key factor governing protein aggregation responsible for a variety of neurodegenerative diseases, as well as industrial applications. Consequently, the study of protein folding and stability has attracted the attention of protein chemists for about 100 years [1]. One common method to experimentally investigate protein stability and folding process is involved in using chemical denaturants including guanidinium chloride (GdmCl) and sodium thiocyanate (NaSCN) which can modulate protein stability. However, despite extensive investigations with experimental and computational approaches, it still remains largely controversial for the microscopic mechanisms by which denaturants destabilize the native state …