STRUCTURAL STUDIES ON FOLDING INTERMEDIATES OF SERINE HYDROXYMETHYLTRANSFERASE USING SINGLE TRYPTOPHAN MUTANTS

Previous studies showed that during the in vitro folding of Escherichi a coli serine hydroxymethyltransferase at 4 degrees C, both monomer an d dimer intermediates accumulated and were stable for periods of minut es to hours (Cai, K., Schirch, D., and Schirch, V., (1995) J. Biol. Ch em. 270, 19294-19299). To obtain structural information on these inter mediates, two of the three Trp residues in the protein were changed to Phe to generate a set of three single Trp mutant enzymes. These mutan t enzymes were purified and characterized and shown to retain essentia lly all of the properties of the wild-type enzyme. The fluorescence an d circular dichroism measurements of each mutant enzyme were studied u nder 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 m onomer in which the first 55 amino acids and a segment between residue s 225 and 276 remain in a largely disordered form. This partially fold ed enzyme can form dimers and slowly undergoes a rate determining conf ormational change in which the unstructured segments assume their nati ve state.