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Experimentally validated computational docking to characterize protein- protein interactions

Livoti, Elsa Livoti (2017) Experimentally validated computational docking to characterize protein- protein interactions. Doctor of Philosophy (PhD) thesis, University of Kent,.

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Abstract

Each biomolecule in a living organism needs to adopt a specific threedimensional conformation to function properly. Function itself is usually achieved by specific interactions between biomolecular units. Structural knowledge at atomic level of biomolecules and their interaction is important to understand the mechanisms leading to biological response and to develop strategies to interfere with them when necessary. Antibodies are molecules of the immune system playing an ever more prominent role in basic research as well as in the biotechnology and pharmaceutical sectors. Characterizing their region of interaction with other proteins (epitopes) is useful for purposes ranging from molecular biology research to vaccine design. During my PhD studies I used a combination of solution NMR mapping, molecular biology and computational docking to provide a structural and biophysical characterization of new neutralizing antibodies from Dengue virus recovered subjects, comparing the binding of the same antibody to the four Dengue serotypes and the binding of different antibodies to the same serotype. We were able to rationally mutate an antibody to first alter its selectivity for different viral strains and then increase its neutralization by ~40 folds. For the first time, this was achieved without the availability of an x-ray structure. In a second sub-project, I investigated the interaction of the chemokine CXCL12 with the chromatin-associated protein HMGB1, confirming their direct interaction (only proposed but never proved before) and providing a structural explanation for the HMGB1 dependent increase of CXCL12 cellular activity. High profile publications resulted from the two above projects. The above mentioned projects relied heavily on solution NMR spectroscopy, which is ideally suited to the atomic level characterization of intermolecular interfaces and, as a consequence, to antibody epitope discovery. Having provided a residue-level description of a protein-protein interface by NMR, we subsequently used this experimental information to guide and validate computational docking experiments aimed at providing a three dimensional structure of the protein-protein (or antibody-protein) complex of interest. In collaboration with other members of my research group I validated the use of NMR and computational simulations to study antibody-antigen interactions, publishing two reviews in collaboration with other members of my research group.

Item Type: Thesis (Doctor of Philosophy (PhD))
Thesis advisor: Varani, Luca
Thesis advisor: Sumbayev, Vadim
Uncontrolled keywords: HSQC: Heteronuclear Single Quantum Coherence HMGB1: High Motility Group Box 1 NMR: Nuclear Magnetic Resonance SPR: Surface Plasmon Resonanc
Divisions: Faculties > Sciences > Medway School of Pharmacy
SWORD Depositor: System Moodle
Depositing User: System Moodle
Date Deposited: 29 Jun 2018 09:10 UTC
Last Modified: 29 May 2019 20:40 UTC
Resource URI: https://kar.kent.ac.uk/id/eprint/67450 (The current URI for this page, for reference purposes)
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