Q-Band Pulsed EPR Spectrometer

• Subject Area: Basic Research in Biology and Medicine
• Term: Funded in 2020
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 438280639

Our group investigates the structure, function, and dynamics of a few important membrane protein complexes. Electron spin resonance spectroscopy techniques, especially pulsed electron-electron double resonance is used as the major biophysical tool in our research. Such experiments on membrane proteins are very challenging for several reasons. The low expression levels often lead to small sample quantities. In addition, when attached to membrane proteins, the phase memory time of the spin labels get significantly reduced. These limitations combined with the narrow excitation bandwidth of conventional rectangular microwave pulses lead to poor sensitivity and also make the measurement of longer distances (> 5 nm) extremely difficult. For membrane protein structural biology, there is increasing evidence for the vital role of native lipid environment for protein folding, structure, and function. One of the major activities of our working group is to develop and apply pulsed electron-electron double resonance for studying membrane proteins in situ. For a few additional reasons such as the background labeling, instability of the spin labels, and the low protein expression, etc., this is even more challenging. Over the past few years, the availability of a high-performance pulsed spectrometer has greatly enhanced the sensitivity for such experiments with membrane proteins. This device is equipped with a high-power microwave amplifier and an arbitrary waveform generator, which together offer unprecedented opportunities for novel investigations in membrane proteins. Using such a device, we aim to characterize the conformational heterogeneity and the equilibrium dynamics that form the basis of function in different membrane transport protein complexes. New approaches, including different spin labels, labeling strategies, and sample preparation protocols for in situ electron spin resonance spectroscopy will be tested. Later those approached will be applied to study the protein folding beta-barrel assembly machinery complex and the lipopolysaccharide transport systems of Gram-negative bacteria. Both of these systems are essential and conserved in Gram-negative bacteria and therefore are highly sought-after targets for new drugs. In addition, we have been studying the substrate translocation mechanism for primary and secondary active membrane transporters. With the ATP Binding Cassette exporter TmrAB, we showed the feasibility to independently observe the conformational equilibria at three individual domains. With the proton-coupled fumarate symporter SLC26Dg, we determined the dimer structure in proteoliposomes using pulsed electron-electron double resonance constraints. We aim to further elucidate the details of protein-substrate and protein-lipid interactions as well to explore the changes in the thermodynamic parameters during substrate translocation in these transporters.

• DFG Programme: Major Research Instrumentation
• Major Instrumentation: Q-Band gepulstes EPR Spektrometer (Teilfinanzierung)
• Instrumentation Group: 1770 Elektronenspinresonanz-Spektrometer (EPR, ESR)
• Applicant Institution: Goethe-Universität Frankfurt am Main
• Leader: Benesh Joseph, Ph.D.