Biophysical Society Conference | Tahoe 2024

Molecular Biophysics of Membranes

Wednesday Speaker Abstracts

AN APPROACH TO STUDY FLAVOPROTEINS BY IN CELL ELECTRON SPIN RESONANCE (ESR) SPECTROSCOPY: THE MEMBRANE PROTEIN AEROTAXIS TRANSDUCER AER. Timothée Chauviré 1 ; Siddarth Chandrasekaran 1 ; Robert Dunleavy 1 ; Jack H Freed 1 ; Brian R Crane 1 ; 1 Cornell University, Chemistry and Chemical Biology, Ithaca, NY, USA Most mobile bacteria move towards favorable environments by exhibiting a behavior known as chemotaxis [1]. In Escherichia coli (E. coli), the transmembrane receptor Aerotaxis (Aer) is a primary energy sensor for mobility. Aer responses are determined by the redox state of the flavin adenine dinucleotide (FAD) cofactor, which sends signals from the electron-transport chain to the chemotaxis histidine kinase CheA [2]. In its oxidized state (quinone), Aer activates CheA autophosphorylation, which causes the cell to tumble. When the cell encounters terminal electron acceptors, the FAD cofactor reduces to the neutral semiquinone state (neutral (NSQ•) or anionic semiquinone state (ASQ•-)), which deactivates the CheA autophosphorylation and enables, via a change in flagellar rotation, the “smooth swimming” of the cell. Until now, Aer has been primarily studied by electron spin resonance (ESR) in reconstituted systems by mimicking the cell structure with membranes or nanodiscs [3]. In this study, we aim to investigate Aer in whole-cell by ESR spectroscopy and obtain insight by pulse dipolar spectroscopy on the organization of Aer in chemosensory arrays.In cell ESR spectroscopy of E. coli identify the type of radical created and characterize distances (via pulse dipolar spectroscopy) between the semiquinone radical located in the FAD-containing Per-Arnt-Sim (PAS) domains of associated Aer subunits. Whole cell isotopic labeling (2H and 15N) allows effective characterization of the spin system of the FAD radical by a combination of continuous-wave ESR and advanced pulsed ESR techniques (3P-ESEEM: Electron Spin Echo Envelope Modulation, ENDOR: Electron Nuclear Double Resonance). These live-cell studies have the potential to reveal the flavin redox couples involved in signaling and structural features of the receptor key to its function. References:[1] Taylor B.L., Zhulin I.G., Johnson M.S.; Annu. Rev. Microbiol., 53, 103–128 (1999)[2] Maschmann Z.A., Chua T.K., Chandrasekaran S., Ibáñez H., Crane B.R.; J. Biol. Chem., 298, 102598-25814 (2022)[3] Samanta D.; Widom J.; Borbat P.P.; Freed J.H.; Crane B.R.; J. Biol. Chem., 291, 25809-25814 (2016)

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