The overall objective of this research is to understand the relationships between the nature and order of the domains of plasminogen (Pg) and plasmin (Pm) to biological foundation. Our general approaches to accomplish these goals are to characterize the properties of: (a) isolated domains of these and other proteins involved in fibrinolysis, as well as mutant forms of these domains obtained through strategic mutagenesis; (b) intact recombinant proteins that contain these altered domains; and (c) recombinant proteins altered by manipulations of domains, e.g., switches, deletions, etc. The resulting protein domains, protein variants, and protein chimeras are analyzed for ligand binding, structural stability, and functional properties in vitro and in vivo.
Five specific aims are proposed to accomplish these goals: (1) to examine the determinants of the tight (T) and relaxed (R) conformational states within the nonprotease (heavy) chain of hPg, as well as its conformational stability and ligand binding properties, by a combination of molecular biological and physicochemical methods; (2) to incorporate into hPg selected mutants of Aim 1 that are found to affect the adoption of the T and R conformations of the protein and to correlate the conformational changes with the activation properties of hPg; (3) to determine by combinatorial peptide chemistry the sequences of hexapeptides maximally cleaved by hPm and by the Pg activator complexes, streptokinase-hPg, streptokinase-hPm, and staphylokinase-hPm; (4) to conduct an investigation of the nature of the binding to hPg, and its isolated K2 domain (K2Pg), of an internal peptide (VEK30) present on the surface of a group A streptococci. To determine the structure of the K2Pg/VEK30 complex, and backbone dynamical alterations in K2Pg and in VEK30 that accompany their interaction; and (5) to determine the in vivo properties of tumor cell lines transfected with mutant Pg molecules or Pg fragments as potential angiostatin derivatives. This combination of approaches will enable us to test the overall hypothesis that by studying regions of proteins that are assembled at the gene level from organized domains, valuable contributions will be made toward a unified understanding of the structural basis for the functional properties of these proteins. This is significant in that it allows rational design of the modular make-up of proteins that could allow specific properties to be added or deleted without effect on other functions of the targeted protein.
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