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Author Notes:

Corresponding author: Kurt Warncke, Department of Physics, N201 MSC, 400 Dowman Drive, Emory University, Atlanta, GA 30322-2430, kwarncke@physics.emory.edu.

The authors declare no competing financial interest.

Subjects:

Research Funding:

This work was supported by the National Insitute of Diabetes and Digestive and Kidney Diseases (NIDDK) of the National Institutes of Health (NIH) (grant R01 DK054514).

The Bruker E500 EPR spectrometer was funded by the National Center for Research Resources of the NIH (grant RR17767) and by Emory University.

Keywords:

  • Science & Technology
  • Life Sciences & Biomedicine
  • Biochemistry & Molecular Biology
  • Electron paramagnetic resonance
  • protein conformation
  • Salmonella
  • signal sequence
  • spin labelling
  • SALMONELLA-TYPHIMURIUM
  • SOLVENT ACCESSIBILITY
  • BACTERIAL MICROCOMPARTMENTS
  • ESCHERICHIA-COLI
  • PROTEIN-SEQUENCE
  • SPECTROSCOPY
  • PURIFICATION
  • MODEL
  • WATER
  • FLUCTUATIONS

Electron spin-labelling of the EutC subunit in B-12-dependent ethanolamine ammonia-lyase reveals dynamics and a two-state conformational equilibrium in the N-terminal, signal-sequence-associated domain

Tools:

Journal Title:

Free Radical Research

Volume:

Volume 52, Number 3

Publisher:

, Pages 307-318

Type of Work:

Article | Post-print: After Peer Review

Abstract:

The B12 (adenosylcobalamin)-dependent ethanolamine ammonia-lyase (EAL) is a product of the ethanolamine utilisation (eut) gene cluster, that is involved in human gut microbiome homeostasis and in disease conditions caused by pathogenic strains of Salmonella and Escherichia coli. Toward elucidation of the molecular basis of EAL catalysis, and its intracellular trafficking and targeting to the Eut biomicrocompartment (BMC), we have applied electron spin-labelling and electron paramagnetic resonance spectroscopy to wild-type (wt) EAL from Salmonella typhimurium, by using the sulphydryl-specific, 4-maleimido-TEMPO (4MT) spin label. One cysteine residue per active site displays exceptional reactivity with 4MT. This site is identified as βC37 on the EutC subunit, by using 4MT-labeling of site-specific cysteine-to-alanine mutants, enzyme kinetics, and accessible surface area calculations. Electron paramagnetic resonance (EPR) spectra of 4MT-labelled wt EAL are collected over 200–265 K in frozen, polycrystalline water-only, and 1% v/v DMSO solvents. EPR simulations reveal two mobility components for each condition. Detectable spin probe reorientational motion of the two components occurs at 215 and 225 K with 1% v/v DMSO, relative to the water-only condition, consistent with formation of an aqueous-DMSO solvent mesodomain around EAL. Parallel trends in fast- and slow-reorientational correlation times and interconversion of the two populations with increasing temperature, indicate 4MT labelling of a single site (βC37). A two-state model is proposed, in which the fast and slow motional populations represent EAL-bound and free conformations of the EutC N-terminal domain. The approximately equal proportion of each state may represent a balance between EutC and EAL protein stability and efficient targeting to the BMC.

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© 2017 Informa UK Limited, trading as Taylor & Francis Group.

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