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Free Content Manipulation of thrombin exosite I, by ligand-directed covalent modification

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Background: For many enzymes, substrate specificity is directed by secondary binding sites (exosites) that are remote from the active site. Peptide inhibition studies of protein-protein interactions are useful to identify exosite functions. Objective: To develop an approach to manipulate these exosites using ligand-directed covalent modification of the enzyme. Method: To demonstrate this strategy, we have engineered an exosite-deficient variant of human plasma-derived thrombin (FIIa) . Desulfato-hirugen (Hir55-65) analogs were synthesized with a fluorescent label, photocrosslinker, and an optional cleavable linker conjugated to the N-terminus of the peptide, specifically fluorescein-benzoyl-phenylalanyl-(Fl-bF-)glycyl-Hir55-65, Fl-bF-mercaptopropionyl-Hir55-65 and Fl-bF-lactyl-Hir55-65 were synthesized. Each analog was bound and photocrosslinked to FIIa, and the resulting covalent complex was purified. Results: This modified enzyme, FIIa-Hir55-65, hydrolyzed small substrates as efficiently as native FIIa, but was significantly inhibited in fibrinogen clotting and in thrombomodulin-mediated PC activation, implying that the active site was unaffected by labeling but exosite I was blocked. In addition, this approach was used to transfer a fluorescein label from the exosite I binding peptide Hir55-65 to a site proximal to but not obstructing exosite I. The activity of this fluorescently labeled FIIa (Fl-FIIa) could be inhibited by unlabeled Hir55-65, suggesting that exosite I is unmodified. Importantly, this interaction could be followed spectroscopically by fluorescence, demonstrating that the exosite I proximal probe can be used to monitor specific ligand binding interactions. Conclusion: Our results show that exosites of clotting factors (e.g. thrombin) can be specifically inhibited and labeled with fluorescent reporters. This novel technology may have broad applicability for studies of protein-protein interactions that regulate coagulation.
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Keywords: dye transfer; exosites; fluorescence; peptide inhibition; photocrosslinking; thrombin

Document Type: Research Article

Affiliations: 1: Department of Molecular and Experimental Medicine 2: Departments of Cell Biology and Chemistry, The Scripps Research Institute, La Jolla, CA, USA

Publication date: October 1, 2007

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