Demonstration of two independently folding domains in the alpha subunit of bacterial luciferase by preferential ligand binding-induced stabilization.

The alpha subunit of bacterial luciferase unfolds and refolds reversibly by a three-state mechanism in urea-containing buffer. It has been proposed that the three-state unfolding of the alpha subunit arises from a stepwise unfolding of a C-terminal folding domain at lower concentrations of urea, followed by unfolding of the N-terminal domain at higher concentrations of urea (Noland, B. W., Dangott, L. J., and Baldwin, T. O. (1999) Biochemistry 38, 16136-16145). The location of an anion binding site in the proposed N-terminal folding domain allowed the folding mechanism to be probed in the context of the intact polypeptide. Anions preferentially stabilized the N-terminal domain in a concentration-dependent manner. The polyvalent anions sulfate and phosphate were found to be more stabilizing than monovalent chloride ion. Cations did not show a similar stabilizing effect, demonstrating that the stabilization was due to the anions alone. The purified N-terminal domain prepared by limited proteolysis and anion exchange chromatography was found to refold cooperatively with a midpoint approximately that of the second unfolding transition of the alpha subunit. Phosphate ion stabilized this fragment to roughly the same extent as it did the alpha subunit. The results presented are consistent with the proposed two-domain folding model and demonstrate that anion binding to the N-terminal folding domain stabilizes the alpha subunit of bacterial luciferase.