Development of techniques for the analysis of protein: glycosaminoglycan interactions in solution.

Hughes, Ashley ORCID: 0000-0001-5432-5271
Development of techniques for the analysis of protein: glycosaminoglycan interactions in solution. PhD thesis, University of Liverpool.

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Glycosaminoglycans (GAGs), such as Heparan Sulfate (HS), are vital components, of all multicellular organisms, with confirmed roles in development, cell-cell communication and diseases such as dementia and cancer. As such, the interaction of this important group of molecules with proteins is an important process to comprehend and improvements to current techniques are sought. To achieve this HS and its structural analogue heparin, were investigated in various biological systems to simultaneously improve our collective knowledge of the biological systems employed and to improve techniques available to study them. Hen Egg White Lysozyme (HEWL), a model amyloid forming protein, is found to be destabilised with a complex of HS in the zinc (HS(Zn)) from 67.7 °C to 57.6 °C compared to 65.3 °C in the presence of HS in the Na form (as measured by differential scanning fluorimetry). These complexes were studied structurally using synchrotron radiation circular dichroism (SRCD) and found to contain extremely similar secondary structure. To differentiate these similar structures principal component analysis (PCA) of thermal and UV degradations of the complexes using CD were analysed. The HS and HS(Zn) ligands were hypothesised to interact with the two tryptophan residues (Trp) in the active site of HEWL. To confirm this, magnetic circular dichroism (MCD) was also developed as a technique to observe direct Trp:ligand interactions. MCD demonstrated an interaction between HS/HS(Zn) and Trp in HEWL using a signal at the novel position of 286 nm. Antithrombin (AT) was studied in relation to the active pentasaccharide drug and inactive shrimp heparinoids (SH) that contain the important 3-O-sulfate, thought to be key for AT activity against factor Xa. These active and inactive complexes were found to be structurally similar again when studied with SRCD so were subjected to UV degradation. The active and non-active compounds were finally differentiated via their induced stability in AT to thermal degradation. This important correlation challenges the current dogma, which relates activity with a particular pentasaccharide sequence. 2D generalised correlation techniques were applied to the data obtained in this thesis increasing the resolution of the data significantly, allowing for

Item Type: Thesis (PhD)
Additional Information: Date: 2014-09-30 (completed)
Subjects: ?? Q1 ??
Depositing User: Symplectic Admin
Date Deposited: 18 Aug 2015 09:16
Last Modified: 17 Dec 2022 01:18
DOI: 10.17638/02007683