Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.607405
Title: Infrared and UV-visible time-resolved techniques for the study of tetrapyrrole-based proteins
Author: Russell, Henry
Awarding Body: University of Manchester
Current Institution: University of Manchester
Date of Award: 2013
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Abstract:
Tetrapyrrole-based proteins exhibit arguably the most diverse range of biological functions on the planet, facilitating photosynthesis, respiration and neurotransmission, amongst others. By virtue of their common biosynthetic pathway, all tetrapyrroles consist of a four pyrrole ring structure with a centrally chelated metal ion. This characteristic gives rise to absorbance of ultraviolet, visible and infrared radiation and thus allows their direct probing by ultraviolet-visible (UV-vis) and infrared (IR) spectroscopy. However, the surrounding protein structure can often remain spectroscopically silent in the UV-vis. In this thesis, the potential for combining time-resolved UV-vis with complementary IR spectroscopic techniques are presented, by study of two tetrapyrrole-based proteins; coenzyme B12-dependent ethanolamine ammonia lyase (EAL) and haem-dependent cytochrome c’. These findings highlight the potential for probing chemical changes by UV-vis spectroscopy, whilst also identifying any coupled protein motions using IR spectroscopy. Initially, a stopped-flow Fourier transform infrared (SF/FT-IR) instrument was characterised using test chemistry, which is summarised in an application note for use by the developer, TgK Scientific. Gaining greater insight of the SF/FT-IR instrument was essential for understanding the virtues of the technique prior to protein study. The first protein system analysed was EAL; a 5’-adenosylcobalamin (AdoCbl)-dependent eliminase. EAL mediates catalysis via AdoCbl cobalt-carbon bond cleavage, which yields a radical pair species that facilitates the reaction chemistry. The mechanism by which the protein scaffold guides radical pair formation and reactivity upon substrate binding is currently an area of debate. Using time-resolved IR techniques (including SF/FT-IR), in combination with UV-vis methods, real-time monitoring of protein dynamics during reaction has been achieved. The complementary IR and UV-vis signals suggest that protein dynamics guide cobalt-carbon bond cleavage, stabilisation of the adenosyl radical, and termination of turnover. Based on the EAL crystal structure, the contribution of a number of active site residues is discussed, in particular a mobile Glu287 residue that is thought to assist in the substrate trigger mechanism and radical pair stabilisation. The second protein analysed was cytochrome c’; a NO binding haemoprotein with ligand binding properties analogous to the important eukaryotic signalling molecule soluble guanylate cyclase. Previous crystallographic and spectroscopic studies had implicated the importance of the haem binding pocket residues Leu16 and Arg124 during the NO binding mechanism in regulating ligand discrimination and haem stabilisation, respectively. By study of the cytochrome c’ variants L16A and R124A in comparison with the wild-type using a range of UV-vis and IR photoexcitation techniques, the understanding of these residues’ contribution to haem-NO reactivity has been furthered. In particular, Arg124 demonstrates protection against NO solvent escape, and is implicitly involved in haem-NO reactivity. The heightened importance of this Arg124 residue could have mechanistic implications for soluble guanylate cyclase, for which no crystal structure is available, and the protein motions coupled to catalysis remain under discussion. These studies of EAL and cytochrome c’ have not only extended their respective mechanistic understanding, but also demonstrated the power of coupling UV-vis and IR spectroscopy across wide time courses. By monitoring the electronic state of the chromophore by UV-vis, in concert with protein dynamics by infrared, this offers the opportunity to determine the methods by which proteins achieve their function.
Supervisor: Blanch, Ewan; Scrutton, Nigel Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.607405  DOI: Not available
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