The broad, long-term objective of the proposed research is understanding of the sequence determinants of protein structure and function. A genetics-intensive approach is proposed, focused on bacteriophage T4 lysozyme as a model system.
Specific aims i nclude: 1 . Isolating revertants of T4 lysozyme mutants bearing deleterious single amino acid substitutions, screening for secondary site revertants among them, and sequencing verified second-site revertants. 2. Characterizing selected mutant lysozymes with regard to stability in vivo and in vitro, purification, and catalytic activity; in collaboration with others, determining their structures. 3. Studying the involvement of two parts of the lysozyme molecule in catalysis: the previously implicated residue AsP20, the nature of whose mechanistic contribution is now called into question; and a substructure, located in the large domain, identified in previous studies, that appears to provide structural stabilization of the key catalytic residue Glu11. Lysozymes bearing substitutions in these positions will be purified, and their catalytic properties (affinity for substrate, catalytic efficiency) will be determined in kinetic experiments. The functional properties of proteins are determined by their three-dimensional structures, which in turn are determined by the sequences of their polypeptide subunits. Knowledge of the rules by which sequences of amino acids fold into unique structures, would greatly aid in the design of new proteins, as well as modification of existing ones, to serve as exquisitely specific antigens or therapeutic agents.

Agency
National Institute of Health (NIH)
Institute
National Institute of Allergy and Infectious Diseases (NIAID)
Type
Research Project (R01)
Project #
5R01AI024083-07
Application #
2062446
Study Section
Microbial Physiology and Genetics Subcommittee 2 (MBC)
Project Start
1988-02-01
Project End
1998-01-31
Budget Start
1994-02-01
Budget End
1995-01-31
Support Year
7
Fiscal Year
1994
Total Cost
Indirect Cost
Name
University of Massachusetts Medical School Worcester
Department
Genetics
Type
Schools of Medicine
DUNS #
660735098
City
Worcester
State
MA
Country
United States
Zip Code
01655
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Jucovic, M; Poteete, A R (1998) Delineation of an evolutionary salvage pathway by compensatory mutations of a defective lysozyme. Protein Sci 7:2200-9
Poteete, A R; Rennell, D; Bouvier, S E et al. (1997) Alteration of T4 lysozyme structure by second-site reversion of deleterious mutations. Protein Sci 6:2418-25
Bouvier, S E; Poteete, A R (1996) Second-site reversion of a structural defect in bacteriophage T4 lysozyme. FASEB J 10:159-63
Poteete, A R; Hardy, L W (1994) Genetic analysis of bacteriophage T4 lysozyme structure and function. J Bacteriol 176:6783-8
Rennell, D; Poteete, A R; Beaulieu, M et al. (1992) Critical functional role of the COOH-terminal ends of longitudinal hydrophobic strips in alpha-helices of T4 lysozyme. J Biol Chem 267:17748-52
Poteete, A R; Rennell, D; Bouvier, S E (1992) Functional significance of conserved amino acid residues. Proteins 13:38-40
Poteete, A R; Sun, D P; Nicholson, H et al. (1991) Second-site revertants of an inactive T4 lysozyme mutant restore activity by restructuring the active site cleft. Biochemistry 30:1425-32
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