Brian W. Matthews, Ph.D., D.Sc. - US grants

1. Our first aim is to better understand the molecular basis of DNA-protein recognition and the regulation of gene expression. Specifically, we will continue studies of the cro repressor protein from bacteriophage lambda, for which we have recently determined the three-dimensional structure. We will attempt to determine the structure of cro complexed with appropriate DNA fragments. Also, we will attempt to determine the structures of the cII gene activator protein and single-strand DNA-binding proteins, as well as complexes of these proteins with DNA. 2. Our second goal is to better understand the factors that determine the structure, stability, dynamics, folding, activity and evolution of proteins. We believe that our ongoing structural and comparative studies of phage lysozyme, goose lysozyme, thermolysin and other proteins provide a very favorable experimental framework within which to address these problem areas.

The three-dimensional structure of the lysozyme from bacteriophage T4 has been determined by X-ray crystallography from a 2.4 A resolution electron density map. Also an extensive library of mutant enzymes is available, and techniques for obtaining additional mutants have been established. A number of mutant lysozymes have been crystallized isomorphously with the native protein. We propose to compare in detail by X-ray crystallography, nuclear magnetic resonance, and various optical methods the three-dimensional structures, the stability, the folding, and the activity of selected mutant enzymes with that of the wild type enzyme. In so doing we expect to obtain detailed information, not previously accessible, concerning the factors influencing the stability and activity of T4 phage lysozyme, and, by inference, of proteins in general. We also propose to compare, insofar as possible, the structure and activity of T4 phage lysozyme with that of hen egg-white lysozyme. The structure of Embden Goose lysozyme, which is not homologous with phage lysozyme or hen egg-white lysozyme, will be determined and compared with other known lysozyme structures in order to determine the evolutionary relation between these molecules.

Our objective is to determine the three-dimensional structure of bacterial luciferase by the method of X-ray crystallography. Recent success in obtaining large crystals of the protein makes this a feasible undertaking. This will be the first known structure of any bioluminescent system and will be a major step toward understanding the phenomenon of light emission. The study will also contribute to our understanding of the structure and function of biological systems in general and flavin-proteins in particular.

This Biological Facilities Center proposal requests funds for the purchase of ancillary protein sequencing equipment, peptide synthesis and purification equipment, and equipment for x-ray structure determination of native and engineered proteins. The equipment will be shared by the staff of the Institute of Molecular Biology, one of the leading centers for the study of macromolecular structure and function, and its subsidiary program in Cell Biology. The equipment will be used to identify and/or study proteins involved in the mechanism and control of DNA replication and transcription, the effect of changes in primary structure on stability and tertiary protein structure, the interaction of subunits within protein complexes and between membrane proteins and lipids, the export of proteins by eukaryotic cells, and the mechanism of signal transduction in bacterial chemotaxis. This instrumentation will facilitate the collaborative research that has resulted in important scientific contributions in areas such as the stability of proteins, protein-DNA interactions, DNA replication and DNA transcription.

The lysozyme from bacteriophage T4 will be used as a model system to understand the factors that determine the folding, stability, activity and three-dimensional structures of proteins. The specific research to be accomplished is as follows: 1. Mutant lysozymes with altered stability (and/or catalytic activity) will be selected and studied in detail. These mutant lysozymes will include not only temperature-sensitive lysozymes but also lysozymes that are more thermally stable than the wild-type enzyme. The three-dimensional structures of these mutant lysozymes will be determined and compared with the wild-type lysozyme structure. Changes in structure will be correlated with changes in stability and activity. 2. Oligonucleotide-directed mutagenesis will be used to introduce designed alterations in the lysozyme structure. By making a series of selected amino acid replacements at one site we will discriminate between the contributions of different interactions at that site. Site-directed mutagenesis will also be used to test the importance of factors such as hydrogen bonding, secondary structure stabilization, hydrophobic interactions, and steric hindrance in protein stabilization. 3. The evolution of lysozyme structure and function will be investigated by determining and comparing the three-dimensional structures of lysozymes that have dissimilar amino acid sequences. In particular, we will continue structural studies of goose egg-white lysozyme and its relationship to chicken and phage lysozymes.

molecular biology; meeting /conference /symposium; travel; spectrometry; X ray crystallography; optics;

The SIR provided (l)-te-met; 1.8g The principal objective of this proposal is to use T4 lysozyme as a model system to better understand the factors that determine the folding, stability, structure and function of proteins. The specific research to be accomplished includes the following: (a) An attempt will be made to simplify the protein folding problem by identifying which residues, or combinations of residues, in T4 lysozyme are critical for folding and stability. We want to understand not only how given residues contribute to stability, but also the signals, if any, that define the elements of secondary structure. Ultimately we would like to reduce the amino acid sequence of T4 lysozyme to the simplest form that will still give a folded, functional protein. (b) Methionine substitution, together with other nonpolar replacements, will be used to better understand the core-packing interactions that are critical to protein folding. (c) Methods will be developed and tested to improve protein stability. (d) Cavities within T4 lysozyme will be exploited both to understand protein-ligand interaction and to engineer novel active sites. (e) The role of strain within the protein will be systematically analyzed.

Address; Amyloid; Amyloid Substance; CRISP; Computer Retrieval of Information on Scientific Projects Database; Dependence; Elements; Funding; Genetic Alteration; Genetic Change; Genetic Engineering of Proteins; Genetic defect; Grant; Institution; Investigators; Lysozyme; Muramidase; Mutation; N-Acetylmuramide Glycanhydrolase; NIH; National Institutes of Health; National Institutes of Health (U.S.); Peptidoglycan N-acetylmuramoylhydrolase; Protein Engineering; Proteins; Research; Research Personnel; Research Resources; Researchers; Resources; Side; Source; Spinal Column; Spine; United States National Institutes of Health; Vertebral column; backbone; design; designing; gene product; genome mutation; protein folding; response; scaffold; scaffolding