Structural studies on folding intermediates of serine hydroxymethyltransferase using single tryptophan mutants

Abstract

Previous studies showed that during the in vitro folding of Escherichia coli serine hydroxymethyltransferase at 4 °C, both monomer and dimer intermediates accumulated and were stable for periods of minutes to hours (Cai, K., Schirch, D., and Schirch, V. (1995) J. Biol. Chem. 270, 19294- 19299). To obtain structural information on these intermediates, two of the three Trp residues in the protein were changed to Phe to generate a set of three single Trp mutant enzymes. These mutant enzymes were purified and characterized and shown to retain essentially all of the properties of the wild-type enzyme. The fluorescence and circular dichroism measurements of each mutant enzyme were studied under unfolding-refolding equilibrium conditions and during refolding. In addition, the sensitivity of the protein to digestion by subtilisin during refolding was investigated. The results of these studies show that the unfolded enzyme has two domains that rapidly fold to form a monomer in which the first 55 amino acids and a segment between residues 225 and 276 remain in a largely disordered form. This partially folded enzyme can form dimers and slowly undergoes a rate determining conformational change in which the unstructured segments assume their native state.