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Title: From carbon nanotubes to zinc porphyrins : engineering proteins to interface with non-biological molecular systems
Author: Bowen, Benjamin James
ISNI:       0000 0005 0285 5333
Awarding Body: Cardiff University
Current Institution: Cardiff University
Date of Award: 2020
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A protein’s function is dictated by its fold and surface chemistry, which are in turn dictated by its primary amino acid sequence. Protein structure and function can be engineered using protein structural data and in silico design methods to impart new functionality to natural proteins, by mutation of the native primary sequence using genetic manipulation techniques. The natural “toolbox” of 20 standard proteinogenic amino acids provides for enormous functional diversity in the context of complex and tightly regulated naturally-selected systems. However, the limited repertoire of amino acid chemistries can be limiting when engineering comparatively simple artificial protein systems. Genetic incorporation of non-natural amino acid (nnAAs) enables us to add new chemistries to engineered proteins, including fluorophores for spectroscopy, heavy elements to aid crystallography or bioorthogonal chemistries for selective reactions. More than 200 non-natural amino acids have been incorporated in proteins to date. This thesis describes the rational design of engineered proteins, which were made using site-directed mutagenesis with both canonical amino acids and the nnAA p-azido-L-phenylalanine (AzF). In chapters 3 and 4 several variants of the β-lactamase binding protein BLIP-II were created, containing AzF at various defined locations. These were used to functionalise carbon nanotube (CNT) based field-effect transistor (NTFET) devices with populations of BLIP-II proteins all bound to the CNT surface in a defined and consistent orientation. These devices functioned as specific sensors for TEM β-lactamase analyte binding to surface-immobilised BLIP-II proteins. Devices functionalised with BLIP-II in different orientations were found to show distinct signal responses on binding of TEM, demonstrating recognition of analyte orientation. CNT-protein functionalisation using AzF was demonstrated via two chemical routes, using copper-free click chemistry (SPAAC) or UV-activated nitrene cycloaddition, demonstrating the versatility of AzF as a protein engineering tool. Surface functionalisation was analysed using atomic force microscopy (AFM). In chapter 5, a variant of the small electron transport haem-protein cytochrome b562 was designed, produced and characterised, with its cofactor porphyrin binding affinity functionally switched from the native haem to the fluorogenic haem analogue zinc protoporphyrin IX (ZnPP). Cyt b562 haem binding was practically abolished while ZnPP affinity was increased 18-fold. On binding cyt b562ZnPP, the fluorescent emission of ZnPP increased ca. 70-fold, making cyt b562ZnPP potentially useful as a switchable, genetically encoded fluorescent label or as an optoelectronic component.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: Q Science (General)