HDO Semi-heavy water HPLC High-performance liquid chromatography HSA Human serum albumin ITC Isothermal titration calorimetry Kpi Single ion partition coefficient LCST Lower critical solution temperature LMWA Law of matching water affinities MD Molecular dynamics MIRIAM Multimode infrared imaging and microspectroscopy MPD 2-methyl-2,4pentanediol MV Megavolts NES Non-electrostatic force NMR Nuclear magnetic resonance OD Oxygen-deuterium PBS Phosphate buffered saline PDB Protein data bank pI Isoelectric point pm picometers PNIPAM Poly (N-isopropylacrylamide) PPC Pressure perturbation calorimetry Raman-MCR Raman-Multivariate curve resolution SAXS Small angle X-ray scattering SIPs Separated ion-pairs SPC Solute partition coefficient SPM Solute partition model SPT Scaled particle theory STI Surface tension increment T Temperature TBA Tert-butyl alcohol TEC Thermal expansion coefficient THz Terahertz THz-TDS Terahertz – Time domain spectroscopy Tm Melting temperature TMAO Trimethylamine N-oxide v/v Volume/ volume VSFS Vibrational sum-frequency spectroscopy ΔCp Change in heat capacity ΔG Change in Gibbs free energy ΔH Change in enthalpy ΔS Change in entropy ΔΔG Change in the change of Gibbs free energy
molecules (; ). The networking effect is seen to a greater extent at higher protein concentrations, but lower phytic acid concentrations (1 – 10 mM). When some sites on a lysozyme molecule are occupied by phytate there will be free sites on another lysozyme molecule that can interact with one of the free phosphate groups on the phytate molecule, as shown in figure 3.2. In this model phytate is able to act as a bridge between two (or potentially more) protein molecules (; ). At certain concentrations that were dependent on the protein being used, protein isoelectric point (pI) and pH of the solution, long chains and clusters of protein can be formed where phytate is acting as a bridge networking the protein molecules. The chain and complex formation starts to break down as the phytic acid concentration is increased further. This is due to all the available sites that phytic acid can interact with becoming saturated by phytate molecules. Once this happens cross-linking cannot take place as all sites are occupied with phytic acid. It is at this point that when the complexes start to break down and protein solubility starts to increase again. This model has been supported by solubility experiments presented in this work.
In these experiments phytic acids ability to network proteins was investigated. Phytic acid was only able to network the lysozyme and not myoglobin or HSA. These results can be explained by taking into account the pI of lysozyme, myoglobin and HSA which are 11.35, 6.8/ 7.4 and 4.7 respectively. At pH 7.0, lysozyme will have a positive charge, myoglobin will be neutrally charged and HSA will have a negative charge. Due the majority of the accessible basic surface residues of lysozyme being charged it means that phytic acid could interact with these electrostatically, linking protein molecules together. Due to myoglobin and HSA being neutrally and negatively charged respectively, it is most likely that there are
decrease in Tm occurred for each protein when phytic acid was added to that protein. A control experiment was carried out for each protein in which the concentration of NaCl was changed from 1 mM – 1 M. This was done to take into account the effect NaCl had on the stability of the proteins being tested. In the second part of the experiment the set amounts of phytic acid was added to each protein stock solution. NaCl concentrations were increased sample to sample for each protein and the effect on Tm for each NaCl concentration was measured on the DSC, all the thermodynamic parameters were calculated as previously. NaCl did have an effect on the stability of the proteins without the presence of phytic acid as shown in figures 3.15, 3.16 and 3.17. There was always an initial decrease in stability, which was very noticeable for myoglobin and lysozyme, but the destabilisation was only very small for HSA. At the higher NaCl concentrations for lysozyme and myoglobin the Tm plateaued off lower than the starting Tm. Addition of the phytic acid caused a large destabilisation for all proteins as expected. As the NaCl concentration was increased the Tm increased for all proteins. This suggests that the Cl- component of the salt was able to successfully compete with phytic acid for positively charged sites on the proteins.
pressurisation than monovalent anions. The gradient of the monovalent ions were similar to each other, indicating that ΔQ versus temperature is not necessarily dependent on ion surface charge density, size or shape. This suggests that the rate at which ionic solutions absorb heat upon pressurisation is dependent on the charge of the ion. The effect different phosphate concentrations had on ΔQ versus temperature is shown in figures 5.7a and 5.7b. Figure 5.7a focuses lower phosphate concentrations (0 – 50 mM) and figure 5.7b focuses on higher phosphate concentrations (50 – 1000 mM). The gradient of ΔQ versus temperature is dependent on phosphate concentration. The ΔQ versus phosphate concentration at certain temperatures is given in figures 5.8a and 5.8b. It should be noted that the relationship between ΔQ and phosphate concentration is not linear at any of the temperatures studied which suggests that ΔQ cannot be explained in terms of a volume excluded effect caused by increasing phosphate concentration as this would give a linear relationship.
influence water dynamics over an extended range it could compete for water with the protein. As a protein unfolds the newly exposed residues have to be hydrated. If the protein is stable enough to reach a temperature where ions electric field can modulate water dynamics and “compete” for water with the unfolding protein the ion could cause protein stabilisation. This suggests that if the thermal stability of a protein is higher than 80 oC, iodide, which is a powerful protein destabiliser, could cause protein stabilisation. If this was observed it would support the hypothesis put forward in chapter 4 that protein stabilisation by ions occurs due to ions competing for water with the unfolding protein by an electrostatic interaction. proteins with melting temperatures > 90 oC would be suitable for these experiments. CHAPTER 6
80 V vacuum interferometer is shown, the upstream Bruker Vertex 80 V vacuum interferometer is not shown. This diagram was adapted from (). 6.2.3. Data Analysis The data analysis and theory used to analyse the data obtained in these experiments is given in section 2.3.2.