Pulse dipolar EPR spectroscopy reveals buffer modulated cooperativity of metal templated protein dimerization

Oranges, Maria; Wort, Joshua L.; Fukushima, Miki; Fusco, Edoardo; Ackermann, Katrin; Bode, Bela E.

doi:10.26434/chemrxiv-2022-ht2gb

Cited by 2 publications

(1 citation statement)

References 38 publications

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“…[20,21] Further addition of Fdoes not result in a significant change of the tertiary structure but suppresses dynamics involving a lowly populated excited apo state responsible for transcription termination. [10,22] Pulsed electron-electron double resonance (PELDOR, [23,24] aka DEER for double electron-electron resonance [25] ) spectroscopy permits measuring distances ranging from 20 Å to 100 Å and beyond [26][27][28] between paramagnetic centres such as spin labels, [26,[29][30][31] paramagnetic metal ions, [32][33][34][35][36][37] amino acid radicals and radical cofactors. [38][39][40] This provides a unique opportunity for investigating conformational ensembles through their spin-spin distance distributions.…”

Section: Introductionmentioning

confidence: 99%

Pulsed EPR methods in the angstrom to nanometre scale shed light on the conformational flexibility of a fluoride riboswitch

Remmel,

Meyer,

Ackermann

et al. 2024

Preprint

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Riboswitches control gene regulation upon external stimuli such as environmental factors or ligand binding. The fluoride sensing riboswitch from Thermotoga petrophila is a complex regulatory RNA proposed to be involved in resistance to fluoride cytotoxicity. The details of structure and dynamics underpinning the regulatory mechanism are currently debated. Here we demonstrate that a combination of pulsed electron paramagnetic resonance (ESR/EPR) spectroscopies, detecting distances in the angstrom to nanometre range, can probe distinct regions of conformational flexibility in this riboswitch. PELDOR (pulsed electron-electron double resonance) revealed a similar preorganisation of the sensing domain in three forms, i.e. the free aptamer, the magnesium(II)-bound apo, and the fluoride-bound holo form. 19F ENDOR (electron-nuclear double resonance) was used to investigate the active site structure of the fluoride -bound holo form. Distance distributions without a priori structural information were compared with in silico modelling of spin label conformations based on the crystal structure. While PELDOR, probing longer distances, revealed varying conformational flexibility of the RNA backbone, ENDOR indicated low structural heterogeneity at the ligand binding site. Overall, the combination of PELDOR and ENDOR with sub-angstrom precision gave insight into structural organisation and flexibility of a riboswitch, not easily attainable by other biophysical techniques.

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Section: Introductionmentioning

confidence: 99%

Pulsed EPR methods in the angstrom to nanometre scale shed light on the conformational flexibility of a fluoride riboswitch

Remmel,

Meyer,

Ackermann

et al. 2024

Preprint

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Pulse Dipolar Electron Paramagnetic Resonance Spectroscopy Reveals Buffer-Modulated Cooperativity of Metal-Templated Protein Dimerization

Oranges

Wort

Fukushima

et al. 2022

J. Phys. Chem. Lett.

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Self-assembly of protein monomers directed by metal ion coordination constitutes a promising strategy for designing supramolecular architectures complicated by the noncovalent interaction between monomers. Herein, two pulse dipolar electron paramagnetic resonance spectroscopy (PDS) techniques, pulse electron–electron double resonance and relaxation-induced dipolar modulation enhancement, were simultaneously employed to study the Cu II -templated dimerization behavior of a model protein ( Streptococcus sp. group G, protein G B1 domain) in both phosphate and Tris-HCl buffers. A cooperative binding model could simultaneously fit all data and demonstrate that the cooperativity of protein dimerization across α-helical double-histidine motifs in the presence of Cu II is strongly modulated by the buffer, representing a platform for highly tunable buffer-switchable templated dimerization. Hence, PDS enriches the family of techniques for monitoring binding processes, supporting the development of novel strategies for bioengineering structures and stable architectures assembled by an initial metal-templated dimerization.

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Pulse dipolar EPR spectroscopy reveals buffer modulated cooperativity of metal templated protein dimerization

Cited by 2 publications

References 38 publications

Pulsed EPR methods in the angstrom to nanometre scale shed light on the conformational flexibility of a fluoride riboswitch

Pulsed EPR methods in the angstrom to nanometre scale shed light on the conformational flexibility of a fluoride riboswitch

Pulse Dipolar Electron Paramagnetic Resonance Spectroscopy Reveals Buffer-Modulated Cooperativity of Metal-Templated Protein Dimerization

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