Probing and engineering mechanostable protein complexes

Liu, Zhaowei. Probing and engineering mechanostable protein complexes. 2021, Doctoral Thesis, University of Basel, Faculty of Science.


Official URL: https://edoc.unibas.ch/87586/

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The mechanostability of proteins plays an important role in various biological processes, for example cell adhesion and pathogen invasion. Single-molecule force spectroscopy (SMFS) is a powerful tool to understand the molecular mechanisms of mechanostable proteins, gain a mechanistic insight into biological systems and also direct the engineering of biomolecules for desirable mechanical properties, for example enhanced mechanostability.
One family of highly mechanostable cell adhesion proteins is the dockerin (Doc)-cohesin (Coh) family from cellulosomes. Cellulosomes are large protein networks used by certain bacteria to bind and digest cellulose fibers. The interaction between Xmodule-dockerin B (XMod-DocB) and cohesin E (CohE) is responsible for attaching the cellulosome of human gut bacterium R. champanellensis (Rc.) to the cell wall and therefore is crucial for cellulosome function. SMFS is used to demonstrate that the XMod-DocB:CohE complex can be formed in two different conformations, a behavior known as ‘dual-binding mode’, and dissociates through three pathways with distinct mechanical stabilities under force. The complex preferably populates a high force pathway under increased force loading rate, precisely resembling a catch bond.
In addition to naturally occurring adhesion proteins, the mechanostability of antibodies and alternative scaffolds is also important for their functions in the context of antibody-coated therapeutic nanoparticles. Anticalin is a type of alternative scaffold developed to target various human cell surface receptors and small molecules related to diseases. One of its targets is cytotoxic T-lymphocyte antigen 4 (CTLA-4), an important target for tumor immunotherapy. Using SMFS combined with non-canonical amino acid incorporation and click chemistry, external pulling forces are applied to anticalin from eight different directions to dissociate it from CTLA-4 and characterize the geometric dependency of the unbinding energy landscape. The highest rupture force which is ~100% higher than the least mechanostable pulling geometry is found when pulling from residue 60 of anticalin.
The anisotropic response of proteins to mechanical forces can also be used to engineer naturally occurring protein-ligand systems and change their mechanical properties. Another Doc:Coh system from Rc., DocG:CohE complex, dissociates in two pathways under force. The pulling geometry affects the rupture force in both pathways as well as the rate of entering each pathway. When pulling from residue 13 of CohE, the complex exhibits a catch bond behavior, which is distinct from other measured pulling geometries, including the native pulling geometry.
In summary, SMFS is used here both to understand the underlying mechanisms of mechanostable protein-ligand complexes and to engineer them for higher mechanical stabilities as well as unique behaviors such as catch bonding.
Advisors:Nash, Michael and Lim, Roderick Y.H. and Alegre-Cebollada, Jorge
Faculties and Departments:05 Faculty of Science > Departement Chemie > Chemie > Synthetic Systems (Nash)
UniBasel Contributors:Nash, Michael and Lim, Roderick Y.H.
Item Type:Thesis
Thesis Subtype:Doctoral Thesis
Thesis no:1458
Thesis status:Complete
Number of Pages:iv, 174
Identification Number:
  • urn: urn:nbn:ch:bel-bau-diss145983
edoc DOI:
Last Modified:30 Jan 2024 02:30
Deposited On:08 Feb 2022 10:40

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