Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.715090
Title: Molecular mechanisms governing the effects of arginine and other positively charged amino acids on protein thermal stability
Author: Platts, Lauren
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2016
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Abstract:
Arginine is an amino acid that is used extensively as an excipient in therapeutic protein formulations due to its unique ability to solubilise proteins and prevent aggregation without negative effects on protein stability. However, the mechanisms by which it exerts these distinctive effects are still open to conjecture. It is also undecided as to whether arginine is capable of destabilising at least some proteins. A major problem with the existing data on arginine effects on protein stability is the lack of low concentration data, and the presence of salt-containing buffers. The major aim of this thesis was to establish whether arginine acts on protein stability as a combination of its two major functional groups: glycine and guanidinium hydrochloride (GdnHCl). These two molecules are well-known stabilisers and destabilisers respectively. A detailed thermal stability study of three globular proteins in the presence of these three cosolutes demonstrated that arginine affects protein stability as an additive combination of its two functional groups: glycine and GdnHCl, with mechanisms originating from both groups. This results in two stage concentration-dependent stability effects, with low cosolute concentrations (100 mM) causing less severe protein-specific effects. Lysine and histidine, the other positively charged amino acids are also shown to affect protein thermal stability via a combination of their respective functional groups. Glycine-GdnHCl mixtures are shown to also act on protein thermal stability similar to arginine, or in a synergistically stabilising manner (depending on the protein), meaning they have the potential to be developed as ‘designer excipients’ whereby bespoke ratios of the functional groups are used in place of arginine to exert the desired stability effects. Experimental evidence of arginine clustering is also presented, with head-to-tail electrostatic interactions and Gdn-Gdn self-association thought to be responsible. Although no link between clustering and protein stability effects are found.
Supervisor: Falconer, Robert J. Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.715090  DOI: Not available
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