The role of ligand binding in the kinetic folding mechanism of human p21(H-ras) protein.

p21(H-ras) plays a critical role in signal transduction pathways by cycling between an active, GTP/Mg2+ ternary complex and an inactive, GDP/Mg2+ complex. Urea-induced equilibrium unfolding studies [Zhang and Matthews (1998) Biochemistry 37, 14881-14890] have shown that GDP and Mg2+ play essential roles in stabilizing the protein. To probe the mechanism of folding and to examine the effects of these ligands on the kinetic folding reaction, unfolding and refolding experiments were performed at a variety of urea and ligand concentrations. A burst phase intermediate with substantial secondary structure and marginal stability was observed during refolding by stopped-flow circular dichroism spectroscopy. Three subsequent refolding phases were detected using a combination of absorbance, circular dichroism, and fluorescence spectroscopy. The fastest phase involves ligand binding and appears to directly form the fully folded enzyme. The intermediate and slow phases do not depend on either urea or ligand concentration under strongly refolding conditions and appear to reflect isomerization or rearrangement reactions. Double- jump experiments demonstrated that the intermediate and slow refolding phases both lead to the native conformation and correspond to parallel rather than sequential reactions. Unfolding is controlled by two phases that involve the release of the ligands when the ligands are in excess. At stoichiometric ligand concentrations, however, the rate-limiting steps in unfolding change from ligand release to isomerization or rearrangement reactions at high urea concentrations. Only the faster unfolding reaction is observed in the absence of Mg2+, suggesting that this reaction corresponds to the unfolding of the binary complex, p21(H-ras)*GDP. The slower unfolding reaction presumably corresponds to the unfolding of the ternary complex, p21(H-ras)*GDP. Mg2+. The kinetic data show that the refolding/unfolding of p21(H-ras) occurs through parallel channels that are strongly influenced by the binding/release of GDP and Mg2+ to/from a pair of native conformers.