Demonstration of Two Independently Folding Domains in the α Subunit of Bacterial Luciferase by Preferential Ligand Binding-Induced Stabilization

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Abstract
The α 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 α 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 α subunit. Phosphate ion stabilized this fragment to roughly the same extent as it did the α 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 α subunit of bacterial luciferase.