Site‐directed spin label electron paramagnetic resonance spectroscopy as a probe of conformational dynamics in the Fe(III) 'locked‐off' state of the CO‐sensing transcription factor CooA
| Autor: | Judith N. Burstyn, Judy P. Hines, Matthew R. Dent, Daniel J. Stevens |
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| Jazyk: | angličtina |
| Rok vydání: | 2018 |
| Předmět: |
0301 basic medicine
Hemeproteins Models Molecular MTSL Stereochemistry Full‐Length Papers Allosteric regulation Biochemistry Ferric Compounds law.invention 03 medical and health sciences chemistry.chemical_compound Bacterial Proteins law Electron paramagnetic resonance Spin label Molecular Biology Heme Carbon Monoxide 030102 biochemistry & molecular biology biology Protein dynamics Electron Spin Resonance Spectroscopy Site-directed spin labeling DNA 030104 developmental biology chemistry cAMP receptor protein biology.protein Trans-Activators |
| Popis: | The transcriptional activator CooA belongs to the CRP/FNR (cAMP receptor protein/fumarate and nitrate reductase) superfamily of transcriptional regulators and uses heme to sense carbon monoxide (CO). Effector‐driven allosteric activation is well understood in CRP, a CooA homologue. A structural allosteric activation model for CooA exists which parallels that of CRP; however, the role of protein dynamics, which is crucial in CRP, is not well understood in CooA. We employed site‐directed spin labeling electron paramagnetic resonance spectroscopy to probe CooA motions on the μs‐ms timescale. We created a series of Cys substitution variants, each with a cysteine residue introduced into a key functional region of the protein: K26C, E60C, F132C, D134C, and S175C. The heme environment and DNA binding affinity of each variant were comparable to those of wild‐type CooA, with the exception of F132C, which displayed reduced DNA binding affinity. This observation confirms a previously hypothesized role for Phe(132) in transmitting the allosteric CO binding signal. Osmolyte perturbation studies of Fe(III) “locked‐off” CooA variants labeled with either MTSL or MAL‐6 nitroxide spin labels revealed that multicomponent EPR spectra report on conformational flexibility on the μs‐ms timescale. Multiple dynamic populations exist at every site examined in the structurally uncharacterized Fe(III) “locked‐off” CooA. This observation suggests that, in direct contrast to effector‐free CRP, Fe(III) “locked‐off” CooA undergoes conformational exchange on the μs‐ms timescale. Importantly, we establish MAL‐6 as a spin label with a redox‐stable linkage that may be utilized to compare conformational dynamics between functional states of CooA. |
| Databáze: | OpenAIRE |
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