Single amino acid mutant proteins of the lysozyme of bacteriophage T4 will be examined in order to determine the contribution of individual amino acids toward the structure, stability, and folding of the protein. Specifically, a class of temperature-sensitive (ts) mutants with reduced activity at 33 degrees C and no activity at 43 degrees C has been selected after a random, chemical mutagenesis of bacteriophage T4. By comparing the mutant proteins with the wild-type (WT) protein in terms of the three- dimensional structure and the energetics of folding, molecular explanations for the reduced stability of these particular proteins will be possible. Two different mutant lysozymes have been identified so far from this collection of mutants: leucine 91 replaced with proline (L91P) and leucine 66 replaced with proline (L66P). These two mutant proteins in addition to others that have yet to be sequenced will be studied by three experimental methods. 1) The three-dimensional structures of the proteins will be determined by x-ray crystallography. 2) Oligononucleotide, site-directed mutagenesis will be used to make specific substitutions to test hypotheses for stability and instability suggested by the original mutation. 3) The thermodynamics of the folding/unfolding reaction will be studied using urea denaturation monitored by ultraviolet difference spectroscopy. Thermostable mutants will be isolated by selecting for second-site revertants of the already existing ts mutants and separating the second- site mutation from the ts mutant. By examining a large number of mutant proteins, generalizations can be made that ultimately will permit the determination of the structural role of any amino acid in a folded protein. Such capabilities will ultimately contribute to the ability to determine from first principles the three-dimensional structure of a protein from its amino acid sequence. The solution of the protein folding problem is becoming increasingly relevant because protein sequences are being determined by DNA and protein sequencing techniques at a much faster rate that the three-dimensional structures of these molecules. This problem is especially pronounced for the pharmaceutical industry where successful rational drug design demands a knowledge of the conformation of the enzyme to be inhibited by the designed drug.
| Gray, T M; Arnoys, E J; Blankespoor, S et al. (1996) Destabilizing effect of proline substitutions in two helical regions of T4 lysozyme: leucine 66 to proline and leucine 91 to proline. Protein Sci 5:742-51 |
