The Reversal by Sulfate of the Denaturant Activity of Guanidinium

摘要

Guanidinium (Gdm~+) chloride is a powerful protein denaturant, whereas the sulfate dianion (SO_4~(2-)) is a strong stabilizer of folded protein states; Gdm_2SO_4 is effectively neutral in its effects on protein stability. While the "neutralizing" effects of protein-stabilizing solutes on the activity of denaturants can be broadly interpreted in terms of additive effects of the solutes, recent experimental and simulation studies support a role for hetero-ion interactions in the effect of sulfate on Gdm~+ denaturation [Mason, P. E.; et al. J. Phys. Chem. 62005, 109, 24185-24196]. Here we describe an experimental strategy for testing this mechanism that involves spectroscopic analysis of the separate effects of alkali metal sulfates (Na_2SO_4, Rb_2SO_4), GdmCI, and Gdm_2SO_4 on the folded populations of several peptides chosen to dissect specific noncovalent contributions to the conformational stability of proteins [alanine-based helical peptides stabilized by hydrogen bonds, tryptophan zipper (trpzip) peptides stabilized largely by cross-strand indole-indole interactions]. While the trpzip peptides are highly sensitive to GdmCI denaturation, they are unaffected by NaCI, Na_2SO_4, or Gdm_2SO_4, indicating that the reversal of the denaturant activity of Gdm~+ by sulfate in this case is not due to competing stabilizing (sulfate) and destabilizing (Gdm~+) interactions. Gdm_2SO_4 was found to retain considerable denaturant activity against alanine-based a-helical peptides. The differences in the effects of Gdm_2SO_4 on the two peptide types can be understood in terms of the different mechanisms of Gdm~+ denaturation of trpzip peptides and helical peptides, respectively, and the specific nature of Gdm~+ and SO_4~(2-) ionic "clustering" that differentially affects the ability of Gdm~+ to make the molecular interactions with the peptides that underlie its denaturant activity.