Experimental Approach to Understanding the Principle of Protein Folding

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We have studied mutant proteins in order to elucicate the role of amino acid residues in protein folding, protein stability, and enzymatic function. In this report, we describe the outline of two papers.1. In order to elucidate the role of a proline residue in protein folding, the unfolding and refolding kinetics of six proline mutants of the human lysozyme (h-lysozyme) were carried out and compared to that of the wild type protein. Our results show that the slow refolding phase observed in the h-lysozyme refolding kinetics cannot be ascribed to proline isomerization reactions. The h-lysozyme contains two proline residues at positions 71 and 103. The refolding kinetics of the P71G/PI03G mutant were found to be similar to those of the wild type protein. Other mutants such as P103G or P71G, and A47P with its three prolines, gave identical slow refolding phases.2. To understand how the alpha and beta_2 subunits of tryptophan synthase from Escherichia coli interact to form an alpha_2beta_2 complex and undergo mutual activation, we have investigated alpha subunits with single amino acid replacements at conserved proline residues. Although the activities of alpha_2beta_2 complexes that contain wild type alpha subunits or alpha subunits substituted at positions 28, 62, 96, and 207 are similar, the activities of alpha_2beta_2 complexes that contain alpha subunits substituted at positions 57 and 132 are remarkably altered. Isothermal calorimetric titrations of wild type beta_2 subunit with wild type alpha subunit and a mutant alpha subunit containing a substitution of glycine for proline at position 132 show that both the affinity and the exothermic association enthalpy are greatly reduced in the mutant alpha subunit although the stoichiometry of association is unchanged. We conclude that proline 132 plays a critical role in subunit interaction and in mutual subunit activation.