Application of pressure perturbation calorimetric and high pressure EPR spectroscopic methods for studying membrane proteins and lipid-protein interactions
Thesis supervisor: Tibor Páli
Location of studies (in Hungarian): ELKH BRC Abbreviation of location of studies: SZBK
Description of the research topic:
Pressure perturbation calorimetry (PPC) is a new technique to study the volumetric properties of biomolecules in solution. The coefficient of thermal expansion of the partial volume of a biomolecule is deduced from the heat consumed or produced after small isothermal pressure jumps. This strongly depends on the interaction of the biomolecule with the solvent or co-solvent and on its packing and internal dynamic properties. PPC is sensitive to the state of the lipid bilayer too, changes in lipid-protein interaction or the hydration shell of biomolecules, that is a determinant of structure. On the other hand, high pressure (HP) can reveal regions of elevated compressibility of proteins, and thus flexibility, within individual conformational states, but also shifts conformational equilibria such that "invisible" excited states become accessible for spectroscopic characterization. In combination with site-directed spin-labeling EPR on proteins the method can bring data on: flexible regions in a folded protein, conformational states in equilibrium, excited states undetectable at normal pressure, and structural heterogeneity. These techniques found so far little applications on membranes and membrane proteins. We will implement these techniques on our microcalorimenter and EPR spectrometer and further develop them for studying selected water-soluble and membrane proteins and their interaction with membrane lipids. The outline of the work plan is the following:
• Since PPC is not available commercially for our microcalorimeter instrument, we will build a PPC unit based on literature example [Seeger et al., 2007].
• PPC experiments will be carried out on yeast vacuoles, vacuolar vesicles and on reconstituted membranes in order to test how their thermal expansion coefficient is affected when V-ATPase is in a resting or working state, and when regulatory molecules bind to the enzyme.
• High pressure (HP) sample cells and pressure regulators will be installed on our EPR spectrometer for HP EPR studies. Published examples will serve as reference [e.g., Lerch et al., 2015].
• HP EPR measurements will be carried out on: the above samples, pure spin-labeled membranes in the absence and presence of lysozyme enzyme and on spin-labeled V-ATPase subunits reconstituted into membranes.
Required language skills: English Number of students who can be accepted: 2
Deadline for application: 2023-08-31
2024. IV. 17. ODT ülés Az ODT következő ülésére 2024. június 14-én, pénteken 10.00 órakor kerül sor a Semmelweis Egyetem Szenátusi termében (Bp. Üllői út 26. I. emelet).
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