Pressure perturbation calorimetry of protein unfolding

Jakus-Pol, Joanna Ewa (2005) Pressure perturbation calorimetry of protein unfolding. PhD thesis, University of Glasgow.

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Thermodynamic and structural characterisation of various interactions stabilising protein native folds is a key part of multidisciplinary research efforts to solve the problems of protein folding and binding. Amongst such interactions the hydrophobic effect has been considered for several years to be the major driving force behind protein folding and main non-covalent interaction in protein stability. The hydrophobic effect arises from solvation effects around non-polar residues that are buried inside protein interior upon protein folding. The change in heat capacity on protein unfolding (DeltaCp) is believed to be one of the "signatures" of the hydrophobic effect. The thermal expansion coefficient (alpha) and protein volume change upon unfolding (DeltaV) are the volumetric parameters sensitive to solvation changes and hence could be utilised in the studies of hydrophobic interactions. Both these parameters have recently become easily available with the development of the new calorimetric method called Pressure Perturbation Calorimetry (PPC). In this research, the aqueous methanol solutions have been used as a solvent system with a modified hydrogen bonded structure in relation to water. Protein thermal unfolding has been studied in these solutions, where the solvation effects associated with exposing protein buried residues upon unfolding are diminished (the hydrophobic effect is weakened). Three small, one domain model proteins used in this study were: ubiquitin, lysozyme and ribonuclease A. DeltaCp of unfolding of those proteins in aqueous methanol solutions has been obtained by Differential Scanning Calorimetry (DSC) and DeltaV of unfolding was obtained by PPC. DeltaCp is a temperature-related thermodynamic parameter, while DeltaV is a pressure-related parameter, the combined determination of both parameters allows a more comprehensive description of protein unfolding transitions to be built than merely the more routinely studied temperature-related characteristics. DeltaCp and DeltaV, taken together, provide complementary data on the solvation changes during the thermal unfolding process, therefore the effect of increasing methanol concentration on the changes in both DeltaCp and DeltaV of unfolding has been investigated here. Additionally, two other applications of PPC were investigated in this thesis. Firstly, PPC was shown to allow straightforward collection of data used to predict the structure making and structure breaking of solutes in water. Amino acids as small molecular model compound have been investigated here, and the determination of structure making and structure breaking of side-chains in amino acid model has been critically reviewed. Importantly, a serious apparent discrepancy in published data was been identified here. Secondly, the study of another small model protein, cytochrome c conformational transitions (native to unfolded, molten globule to unfolded) has been tested here by the PPC method and the results have been shown to be in a good agreement with ones obtained from high-pressure spectroscopic studies. In summary , PPC has been shown to be a useful technique for studying structure breaking and structure making properties of solutes, both small and macromolecular, and for obtaining volumetric properties of protein transitions that complete the picture of already abundantly available temperature-related calorimetric data such as enthalpy, entropy and heat capacity.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Additional Information: Adviser: Alan Cooper
Keywords: Biophysics
Date of Award: 2005
Depositing User: Enlighten Team
Unique ID: glathesis:2005-74065
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 23 Sep 2019 15:33
Last Modified: 23 Sep 2019 15:33

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