The nature of protein-protein interactions was explored through the design of polypeptide ligands targeting specifically human a-thrombin. Design strategies ranged from isolation of binding fragments from natural proteins, conformational stabilization using structural scaffold, to bivalent linkage of library-selected components. / The interaction of thrombin with a 28-residue polypeptide from the sixth-epidermal growth factor-like repeat of human thrombomodulin (hTM-EGF6) was characterized in solution by use of NMR spectroscopy. The thrombin-binding region was identified and the thrombin-bound structure of the binding region was determined. The thrombin-bound structure of this fragment of thrombomodulin was then used as a basis for the design of peptide ligands with potentially enhanced thrombin-binding activities. Attempts for affinity enhancement through conformational stabilization by structure-based methods exemplified the need for alternative approaches that exploit the ubiquitous weak molecular interactions. / Bivalent ligands of thrombin were constructed via linkage of natural protein fragments. It was found that bivalent effects can be afforded by the proper linkage of the individually weak binding moieties. The bivalent designs were further generalized to include linkage of novel polypeptides selected from combinatorial libraries as individual binding components. These novel bivalent ligands targeting both the active site and the exosite I of thrombin exhibited up to 110-fold enhancement of binding activity. / The present study renews the interests in weak protein-protein/polypeptide interactions and their use in protein biochemistry. The design of thrombin-targeting bivalent ligands from weakly-binding moieties highlights the nature of bivalent molecular interactions, emphasizing the synergistic interplay between the two binding sites conferred by linker residues with a proper covalent geometry. It also illustrates that bivalent/multivalent binding can be applied as a general and practical approach for the design of high-affinity inhibitors targeting discrete sites on functionally important proteins, especially those involved in biological and cell signaling processes.
| Identifer | oai:union.ndltd.org:LACETR/oai:collectionscanada.gc.ca:QMM.100664 |
| Date | January 2005 |
| Creators | Ng, Yin Dick Andy, 1974- |
| Publisher | McGill University |
| Source Sets | Library and Archives Canada ETDs Repository / Centre d'archives des thèses électroniques de Bibliothèque et Archives Canada |
| Language | English |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Format | application/pdf |
| Coverage | Doctor of Philosophy (Department of Biochemistry.) |
| Rights | © Yin Dick Andy Ng, 2005 |
| Relation | alephsysno: 002326482, proquestno: AAINR25220, Theses scanned by UMI/ProQuest. |
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