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Micromolecular approaches to protein binding

Abdul Aziz, Azrah (2019) Micromolecular approaches to protein binding. PhD thesis, University of Sheffield.

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AZRAH ABD AZIZ (Ph.D).pdf
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Abstract

This research described in this thesis examined two main macromolecules as ligands for protein binding, namely dendrimers and graphene oxide. The architecture of these materials was exploited and functionalised with amino acid to moderate protein binding and recognition. The first area studied involved dendrimers that had been functionalised using non-covalent methods. Specifically, the amino acid was added to the end of a hydrophobic linear chain that could bind to the interior of the dendrimer using hydrophobic and hydrogen bonding interactions. A number of different linear chains were synthesised and added to various dendrimers to give a surface functionalised system. The dendrimers included an anionic dendrimer that was functionalised with linear chains in order to provide secondary interactions that could bind to the positively charged surface of α-chymotrypsin (Chy). Neutral dendrimers that could not bind to the positive surface were also studied. In this case, binding could only occur if the dendrimers were functionalised with the linear chains. The results showed that the highest affinity occurred for the G3.5COOH-Tyr, with the inhibition constant (Ki) of 0.17 µM, which compares to the Ki for non-functionalised dendrimer of 0.31 µM . When the non-binding dendrimer (G3.5-OH) was functionalised with different amino acids (tyrosine, phenylalanine and valine), the G3.5-OH-Tyr was found to bind the most strongly via polyvalent interactions between the amino acids and the surface of the Chy. All the inhibitors studied were found to be competitive inhibitors, with the exception of the valine functionalised dendrimer, which did not bind. Circular Dichroism (CD) spectroscopy confirmed that denaturation of the protein did not occur during dendrimer binding. The next area of study focused on cytochrome- c. The same non-covalent methodology used for α-chymotrypsin binding was utilised for this area. Binding could be measured directly using encapsulated Tetrahydroxyphenyl Porphyrin (THPP) as an internal quench. To coordinate THPP to the dendrimer, this was strengthened by inserting zinc, which could coordinate the dendrimer’s internal amines. The results showed that the G3.5COOH-Tyr was bound with a dissociation constant (Kd) of 5.55 nm. CD spectroscopy showed that the dendrimer-porphyrin-chains did not cause denaturation of the protein during binding. The final area focused on the use of functionalised graphene oxide (GO). This material was synthesised with amino acids to form a monomeric and an oligomeric functionalised surface of GO. The effects of functionalisation on the binding of GO to Chy was also studied. The binding of the functionalised GO was compared with the binding of the unfunctionalised GO and the results showed an inhibition constant (Ki) of 0.14 µg/mL for the monotyrosine, which was higher than either the oligomeric system or GO alone. In all cases, competitive inhibition was observed. CD spectroscopy confirmed that GO does not induce structural changes within the protein when bound and the spectroscopy also demonstrated that the GO inhibitors had no effect on the thermal stability of the enzyme.

Item Type: Thesis (PhD)
Academic Units: The University of Sheffield > Faculty of Science (Sheffield) > Chemistry (Sheffield)
Depositing User: Mrs Azrah Abdul Aziz
Date Deposited: 17 Jul 2019 08:50
Last Modified: 17 Jul 2019 08:50
URI: http://etheses.whiterose.ac.uk/id/eprint/24349

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