STRUCTURAL AND GENETIC-ANALYSIS OF THE FOLDING AND FUNCTION OF T4 LYSOZYME

The combination of directed mutagenesis with high-resolution structure analysis has made it possible to systematically address fundamental q uestions of protein folding and stability, Here we briefly review some recent results in this area based on studies of the lysozyme of bacte riophage T4. Extended segments of the polypeptide chain can be substit uted with alanine, suggesting that about 50%, or perhaps less, of the overall amino acid sequence protein is necessary to define the 3-dimen sional structure of the protein. It is the internal residues that seem to be most important for folding and stability (although not necessar ily for function). Substitutions within the core of the protein of lar ge nonpolar side chains with smaller ones have been used to better und erstand the nature of hydrophobic stabilization. Mutants that produce the largest cavities within the protein tend to be most destabilizing, allowing the energy cost of cavity formation to be estimated, Small, nonpolar ligands bind within such cavities and restore some stability to the protein. Analogous, nonpolar ligands do not bind, however, prov iding evidence that water molecules do not bind with high occupancy wi thin nonpolar cavities. In a further series of studies it has been pos sible to re-engineer the active site region of TL4 lysozyme to change the catalytic mechanism of the enzyme.