Microcalorimetry of protein stability and interactions

Robertson, Stephen R. (1995) Microcalorimetry of protein stability and interactions. PhD thesis, University of Glasgow.

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Printed Thesis Information: https://eleanor.lib.gla.ac.uk/record=b1577924


Protein stability and interactions in a number of systems have been examined using a range of calorimetric and spectroscopic methods. The thermodynamics of unfolding of bovine alpha-lactalbumin (BLA) was studied using differential scanning calorimetry (DSC) under a variety of pH and calcium ion concentration conditions. Values for AG2, AH2 and AS2 under each of these conditions were obtained, and these data were used to calculate a value for ACp. The information thus obtained for the wild type (WT) protein was then compared to data obtained under similar conditions for a modified form of the protein (3SS-BLA) which contains only three of the usual four disulphide bonds, the other (C6-C120) having been specifically reduced and blocked. In this way it was possible to gain information on the role of this disulphide bond for the stability of the protein. In line with current theory it was found that the modified form of the protein was less stable and this was accountable for in terms of entropy. However, the difference in unfolding entropy between the two forms (AAS) was less than may be anticipated under the current theory. Fluorescence spectroscopy was employed to show whether or not removal of the disulphide bond caused any significant alteration in the dynamics of the native state. It was found that 3SS-BLA was significantly more flexible than its wild type counterpart, and this may account for the unexpectedly low AAS. DSC was also used to study the effect of heavy and light water on the stability of hen egg white lysozyme (HEWL). It was found in heavy water the protein was enthalpically destabilised, but this was more than offset by an entropic stabilisation, a finding which is in line with model compound studies. Through this work it was also noticed that thermal aggregation of proteins increases with concentration. DSC was further employed to study partially proteolyzed Pf1 gene five protein (Pf1- gVp) and complex (Pf1-gVc). It was found that removal of the flexible N-terminal domain had no apparent effect on thermal stability of the complex and therefore is unlikely to be involved in protein-protein interactions in the complex. The Tm of this complex was found to be concentration dependent which shows that the system undergoes cooperative dissociation and unfolding. Isothermal titration calorimetry (ITC) was used alongside DSC to study the coat protein of the bacteriophage MS2. ITC measurements on the binding of a specific RNA 19-mer to the coat protein were made, forming a basis for future work. The Tm measured in DSC was found to be comparable to literature estimates. ITC was also employed to study the binding of arginine (Arg) to the protein AhrC. It was found that three molecules of Arg bind per protein hexamer to a strong site and that there is a second, much weaker site or sites which bind a large number of molecules.

Item Type: Thesis (PhD)
Qualification Level: Doctoral
Keywords: Biophysics.
Colleges/Schools: College of Medical Veterinary and Life Sciences
Supervisor's Name: Cooper, Dr. Alan
Date of Award: 1995
Depositing User: Enlighten Team
Unique ID: glathesis:1995-71680
Copyright: Copyright of this thesis is held by the author.
Date Deposited: 17 May 2019 09:31
Last Modified: 30 Jun 2022 15:30
Thesis DOI: 10.5525/gla.thesis.71680
URI: https://theses.gla.ac.uk/id/eprint/71680

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