Quantitative and mechanistic assessment of thiol switches by stopped-flow kinetic measurements

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Background:Several thiol switch candidates have been identified and characterized during the 1st funding period of the SPP 1710. Such characterizations are often based on redox mobility shift assays and protein-protein interaction studies. Quantitative kinetic and detailed mechanistic data on potential as well as established switches are usually difficult to obtain using such methods. Hence, the underlying rate constants for toggling thiol switches and the physiological relevance of detected redox species often remain unclear. Objectives:During the 2nd funding period of the SPP 1710, we will focus on the quantitative and mechanistic assessment of (candidate) thiol switches by stopped-flow kinetic measurements. We will therefore complement the portfolio of existing key methods within the priority programme by establishing a stopped-flow platform. This platform will be accessible to all SPP members who are interested in the determination of rate constants for (candidate) thiol switches and the elucidation of mechanisms for thiol switching and roGFP2-based redox sensing. For example, we will assess the reactivity of the cysteine residues of the Keap1-like artemisinin-resistance factor PfK13 from the malaria parasite Plasmodium falciparum and characterize the regulatory role of residues C143 & L109 for the Prx5-type model peroxiredoxin PfAOP. Other peroxiredoxins as well as glutaredoxins, roGFP2 probes and flavoenzymes will be studied in the frame of collaborations with SPP 1710 members. Findings from this project will improve our basic knowledge on thiol switches and redox probes and might also help to better understand redox signaling and artemisinin resistance of malaria parasites.