ProfessorRonald Clarke

Project Opportunities

Title: Function and regulation of P-type ATPase ion pumps

Summary of opportunity:

The project aims to understand at the atomic level how the Na+,K+-ATPase and related ion pumps function and how they are regulated.

Opportunity synopsis:

Ion-transporting membrane proteins play a decisive role in the mechanism of all cells and in numerous physiological processes, e.g. ATP production, nerve impulse propagation and muscle contraction. A deeper understanding of their mechanisms and regulation can be obtained by the determination of the kinetics of their individual reaction steps. One approach to this research goal is to make use of the electrical current they generate and to apply an electrophysiological method. Another is to convert the electrical voltage that the proteins produce into an optical signal by incorporating a voltage-sensitive dye into the membrane. Voltage-sensitive styrylpyridinium dyes allow a rapid detection (within nanoseconds) of local electric field changes within membranes. Both spectroscopic and electrical detection methods will be applied in this project.A second important aspect of ion pump function is thermodynamics, i.e. the utilisation of energy. To investigate the energy usage of ion pumps the technique of calorimetry will be applied, which allows one to directly measure the release or absorption of heat when an ion pump undergoes a conformational change or substrate binding/release reaction.

The final essential aspect necessary for a complete understanding of ion pump function is structure. Fortunately, over the last 8 years both the Na+,K+-ATPase and its related enzyme, the Ca2+-ATPase, have been crystallized and structures have been determined by x-ray crystallography. In fact, the Ca2+-ATPase has been crystallized in a number of different conformations around its pump cycle. In this project these structures will be used as a template to construct homology models of the Na+,K+-ATPase and then to investigate its substrate binding via theoretical molecular dynamics simulations.    

Techniques to be used in the project:
* Stopped-flow fluorimetry
* Capacitive current biosensor
* Isothermal titration calorimetry
* Protein isolation and purification
* Homology modelling
* Molecular dynamics simulationsInternational collaborators involved in the project:
* Prof. Flemming Cornelius, University of Aarhus, Denmark
* Prof. Hans-Jürgen Apell, University of Constance, Germany
* Dr. Francesco Tadini-Buoninsegni, University of Florence, ItalyScholarships are available to high quality students. Most local students in the laboratory are supported by an Australian or University Postgraduate Award and International students by other scholarships.  Please contact me for further details.

Project Opportunities

Title: Molecular Mechanisms of Ion Pump Regulation via Protein-Lipid Interaction

Summary of opportunity:

Ion pumps determine such fundamental processes as nerve function, muscle contraction and digestion, and constitute a sizeable proportion of all drug targets. While major progress has been made in revealing the structures of specific ion pumps, the puzzle is still incomplete, and we do not know how the pumps actually work in a living organism. This is a key knowledge gap, which if tackled, will feed into structure-activity studies for new drugs. With drug resistance an ever-increasing problem, the identification of new drug targets is of vital importance.

In this project, the focus of attention will be the termini of the catalytic subunit of P-type ATPases, e.g., the Na+,K+,-ATPase and the H+, K+-ATPase. Prior research from our group suggests that the pumps’ N-termini undergo an electrostatic interaction with the surface of the surrounding lipid membrane and that this interaction controls the pumps’ conformational state and hence their activity. The hypothesis to be investigated in this project is that ion pump regulation occurs via an electrostatic-switch mechanism, in which the interaction of the N-terminus with the surrounding membrane is electrostatically controlled by phosphorylation of the N-termini by protein kinases. To investigate this interaction, we will utilise both purified ion pump preparations and synthetic peptides with amino acid sequences corresponding to those of the N-termini. To detect the strength, specificity and site of the interaction we will use a range of different spectroscopic techniques, i.e., fluorescence spectroscopy, circular dichroism, solid-state nuclear magnetic resonance spectroscopy, as well as quartz-crystal microbalance with dissipation monitoring.