Skip to main content

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

Download Article:

You have access to the full text article on a website external to Ingenta Connect.

Please click here to view this article on Wiley Online Library.

You may be required to register and activate access on Wiley Online Library before you can obtain the full text. If you have any queries please visit Wiley Online Library

Summary. 

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.
No References
No Citations
No Supplementary Data
No Article Media
No Metrics

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: 01 October 2007

  • Access Key
  • Free content
  • Partial Free content
  • New content
  • Open access content
  • Partial Open access content
  • Subscribed content
  • Partial Subscribed content
  • Free trial content
Cookie Policy
X
Cookie Policy
Ingenta Connect website makes use of cookies so as to keep track of data that you have filled in. I am Happy with this Find out more