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PreprintCited by 4Electron Spin Resonance StudiesBacterial Genetics and BiotechnologyAdvanced NMR Techniques and Applications

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Flavoproteins as native and genetically encoded spin probes for in cell ESR spectroscopy

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Chauviré, Timothée, Chandrasekaran, Siddarth, Dunleavy, Robert, Freed, Jack H., Crane, Brian R.·2024·Nature Communications
DOI 10.1101/2024.12.18.628722PMC PMC12214676

Preprint — published as 10.1038/s41467-025-60623-6

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Abstract

Flavin cofactors are attractive Electron Spin Resonance (ESR) probes for proteins because cellular reductants and light can generate their semiquinone states. We have used ESR spectroscopy to study the bacterial transmembrane aerotaxis receptor (Aer) in its native Escherichia coli membrane environment. Optimization of the spectroscopic (electronic relaxation times) and cell growth (isotopic labeling) conditions allowed for measurements of Aer with its partners - the histidine kinase (CheA) and the coupling protein (CheW) - in native signaling arrays. Continuous-wave ESR measurements at room temperature showed a rigid Aer flavin immobilized in the cofactor pocket and Q-band electron nuclear double resonance (ENDOR) measurements identified a predominant anionic semiquinone radical state in cell . Q-band four-pulse double electron-electron resonance (4P-DEER) measurements indicated a 4.1 nm distance between the two flavins of an Aer homodimer, consistent with previous in vitro measurements, but also revealed additional separations in cell indicative of chemoreceptor arrays, not previously observed for Aer. For general application, we further developed a genetically encoded Light-Oxygen and Voltage (LOV) domain for incorporation into target proteins as an ESR probe of structural properties in cell . This approach provides a framework to elucidate protein oligomeric states and conformations that are difficult to reproduce in vitro .

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Metadata

Authors
Chauviré, T., Chandrasekaran, S., Dunleavy, R., Freed, J. H., & Crane, B. R.
Affiliations
Cornell University (US)
Corresponding author
Crane, Brian R.
ORCID
Venue
Nature Communications
Publisher
openRxiv
Published
2024-12-21
Volume
16
Issue
1
Pages
5406-5406
ISSN
2041-1723 (ISSN-L)
DOI
10.1101/2024.12.18.628722
PMC
PMC12214676
OpenAlex
W4411900525
Cited by
4
References
1
Citations by year
2026: 4
FWCI
1.51
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by-nc-nd)
  • bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion)
  • PubMed Central (publishedVersion, cc-by-nc-nd)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • National Science Foundation (MCB 2129729)
  • U.S. Department of Health and Human Services (R35GM148272, P41GM103521, R35GM122535, R24GM146107)
  • National Institutes of Health
  • National Institute of General Medical Sciences (P41GM103521, R24GM146107, R35GM122535, R35GM148272)
Topics
  • Physical Sciences › Chemistry › Spectroscopy › Electron Spin Resonance Studies
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
  • Physical Sciences › Chemistry › Spectroscopy › Advanced NMR Techniques and Applications
Keywords
flavoprotein, flavin semiquinone, bacterial chemotaxis, CheA histidine kinase, LOV domain, protein oligomerization, Escherichia coli membranes
MeSH terms
Histidine Kinase, Methyl-Accepting Chemotaxis Proteins, Electron Spin Resonance Spectroscopy, Escherichia coli, Flavins, Flavoproteins, Spin Labels, Escherichia coli Proteins

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