The overall objective of our research is to understand the structure- function relationships of proteins that are involved in blood coagulation and anticoagulation. We are focusing herein on the structural biochemistry of human protein C (PC) and activated protein C (APC), an anticoagulant zymogen and enzyme, respectively. Our overall hypothesis is that by understanding the structure-function relationships of individual regions of these proteins we can employ this knowledge to effectively up- regulate or downregulate certain functions, and to incorporate features of unrelated proteins. Proteins of the class of PC and APC contain areas classified as domains, which include the gamma-carboxyglutamic acid (Gla) domain (GD), a helical stretch (HS), two consecutive epidermal growth factor-like (EGF) domains, and a serine protease (PD) homology domain. It is hypothesized that these regions incorporate different functions of these proteins. We propose to rigorously define the roles of these individual domains and to assess whether they can function independently.
Five specific aims are proposed: (l) to synthesize polypeptides containing amino acid sequences present in the [GD]-[HS], [HS]-[EGF1] and [PD] domains of PC and APC and to utilize these polypeptides to define details of their Ca2+-related properties; (2) to express cDNAs containing domain combinations of regions of PC and APC, particularly the [GD]-]HS]-[EGF1], [GD]-[HS]-[EGF1]-[EGF2], [HS]-[EGF1]- [EGF2]-[PD], [EGF2]-[PD] and [PD] domains, and to determine whether these constructs possess or inhibit functional Ca2+-dependent properties of the intact proteins; (3) to evaluate the importance of specific amino acid residues in the Ca2+-dependent structural and functional properties of r- PC and r-APC by site-directed mutagenesis strategies; (4) to construct and express r-chimeric PC, thrombomodulin (TM), fvII, and fIX mutants in which [GD] (where relevant) and/or [EGF]-like domains have been exchanged between these proteins, and to determine whether the structural and functional properties of both the chimeric protein and the newly incorporated domain have been gained or lost; and (5) to perform charge- to-alanine replacements of residues within [EGF1], [EGF2], and [PD] of PC in order to reveal locations within these modules that are involved in activation of PC by thrombin and thrombin/TM, and the loci of APC that are responsible for inactivation of fVa and fVIII. The significance of this work lies in development of a rigorous basic understanding of the functions of domains of proteins and, as a result of this knowledge, to potentially incorporate desired features of one protein into another in a rational and predictable manner. This will allow strategic molecular drug design to occur which could result in therapeutic benefits in patients with coagulation abnormalities.
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