Effect of salt on the urea-unfolded form of barstar probed by m value measurements

Abstract

To probe for residual structure present in the urea-unfolded form of the small protein barstar, to determine how salt might modulate such structure, and to determine how such structure might affect the stability of the protein, mutant variants that display m values different from that of the wild-type protein have been studied. The mutant proteins were obtained by site-directed mutagenesis at residue positions located on the surface of the folded protein. The m value, which represents the preferential free energy of interaction of urea with the unfolded form in comparison to that with the folded state, was determined from equilibrium urea-induced unfolding curves. Mutant proteins for which the m values were significantly greater than (m⁺ mutant forms), significantly smaller than (m⁻ mutant forms), or similar to (m⁰ mutant forms) the m value determined for the wild-type protein were studied. The unfolded forms of the m⁰, m⁺ and m⁻ mutant proteins represent different components within the unfolded form ensemble, which differ from each other in their solvent-exposed surface areas. Hence, the m value has been used as a measure of residual structure in the unfolded form. To further understand the nature of structures present in the unfolded form ensemble, the effects of the salt KCl on the stabilities of the wild-type and the mutant proteins, as well as on the structures present in the unfolded form ensemble, were also studied. It was found that the m values of the m⁰, m⁺ and m⁻ mutant proteins all converge to the wild-type m value in the presence of KCl. This result indicates that the salt modulates residual structure in the unfolded form by screening electrostatic interactions that maintain compact and expanded components in the unfolded protein ensemble. The use of free energy cycles has allowed the effect of salt on the structure and free energy of the unfolded protein to be related to the stability of the protein.