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As protein
engineers, we want to know what happens to functional properties
of proteins when their critical amino acid residues are replaced.
Such knowledge aids the design of new proteins with more desirable
properties, for example, more specific drugs that may decrease
side effects. Such knowledge also helps us to better understand
the protein's mechanism of action and to appreciate the changes
in amino acid sequence that occur during a protein's evolution.
For
our studies, we have selected protein inhibitors of serine
proteinases because among them are some of the smallest known
proteins and it is much easier to understand a small protein
than a large one. In particular, we work with ovomucoid third
domains from birds, which consist of 56 amino acid residues
crosslinked by three disulfide bridges. They inhibit (or can
be made to inhibit) enzymes used in food digestion, in blood
clotting and in clot dissolution, in protein hormone production
and in lung tissue damage. This is one of the best characterized
protein systems. The availability of many 3-dimensional structures
and of a detailed mechanism of action allows us to analyze
our results in great detail.
We produce altered ovomucoid third domains by total chemical
synthesis from amino acids or by semi-synthesis, where a small
synthetic peptide is enzymatically joined with a large natural
one. In Addition, we get many altered ovomucoids obtained
by recombinant DNA technology from Prof. S. Anderson at Rutgers.
For the altered ovomucoid third domains, we measure the equilibrium
and rate constants for binding to many enzymes. In this way
we determine the role of each of the critical amino acid residues.
On this basis we can design new inhibitors for newly discovered
enzymes or inhibitors with very narrow inhibitory specificity.
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Selected
Publications
•
Probing Intermolecular Main Chain Hydrogen. Bonding in Serine Proteinase-Protein
Inhibitor Complexes: Chemical Synthesis of Backbone-Engineered Turkey
Ovomucoid Third Domain, W. Lu, M.A. Qasim, M. Laskowski, Jr. & S.
B. H. Kent (1997) Biochemistry, 36, 673-679.
• Interscaffolding
Additivity. Association of P1 Variants
of Eglin c and of Turkey Ovomucoid Third Domain with Serine Proteinases,
M.A. Qasim, P.J. Ganz, C.W. Saunders, K.S. Bateman, M.N.G. James & M.
Laskowski, Jr. (1997) Biochemistry, 36, 1598-1607.
• Binding
of Amino Acid Side Chains to S1 Cavities of Serine
Proteinases, W. Lu, I. Apostol, M. A. Qasim, N. Warne, R. Wynn,
W.L.Zhang, S. Anderson, Y.W. Chiang, E. Ogin, I. Rothberg, K.
Ryan & M. Laskowski, Jr. (1997) J. Mol. Biol., 266, 441-461.
The
amino acid sequence of turkey ovomucoid third domain written
in one letter amino acid code. The arrow indicates the reactive
site peptide bond involved in combination of the inhibitor
and enzyme. Listed at side are its equilibrium constants for
interaction with (1) chymotrypsin, (2) porcine pancreatic
elastase, (3) human leukocyte elastase and with three bacterial
enzymes: (4) subtilisin and (5) (6) Streptomyces grisens
proteinases A and B.
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