Single-transporter biochemistry: real-time observation of substrate transport and energy conversion of primary-active membrane transporters

• Applicant: Professor Dr. Thorben Cordes
• Subject Area: Biophysics ; Biochemistry
• Term: since 2022
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 494983708

Membrane transporters are prime targets for current and future pharmaceutical drugs due to their involvement in numerous cellular processes that cause human diseases, bacterial pathogenicity, drug resistance, and other medical phenomena. Whereas the identification and structural analysis of transporters has moved at great pace, there is still an urgent need to better understand transport mechanisms to enable the development of novel therapies, e.g., through the identification of inhibitors. Single-molecule-based approaches could facilitate a next breakthrough in mechanistic understanding of transporters via (simultaneous) real-time observation of transport, energy usage and structural changes. Although there have been recent advances into this direction, mainly in the investigations of ion channels by combining optical and electrophysiological methods, similar progress has not been made for active membrane transporters, which are not amenable to electrophysiology. The objectives of this proposal are to develop assays to monitor transport and energy conversion processes of single ATP-binding cassette transporters (ABC transporters) using fluorescent sensors in combination with protein and liposome markers. These novel experimental tools will provide direct access to initial transport rates, functional dynamics (on/off periods) or static heterogeneity of single transporters. The main goals of this project are:Aim 1) Development and characterization of fluorescent sensors for different ABC transporters to monitor substrate translocation, ATP hydrolysis and energy coupling. Aim 2) Establish single-transporter recordings for ABC transporters to observe functional dynamics over time or static differences between individual transporters. Importantly, the initial rates of transport will be accurately quantified via simultaneous liposome size characterization.Aim 3) Solve mechanistic questions with the developed assays. In detail, we will study the difference between bulk and single transporter experiments and characterize the impact of catalytic NBD mutations and different substrates on energy coupling efficiencies.

• DFG Programme: Research Grants