Accurate Determination of Human CPR Conformational Equilibrium by smFRET using Dual Orthogonal Non-Canonical Amino Acid Labeling

Robert Benjamin Quast, Fataneh Fatemi, Michel Kranendonk, Emmanuel Margeat, Gilles Truan

Research output: Contribution to journalArticle

1 Citation (Scopus)

Abstract

Conjugation of fluorescent dyes to proteins - a prerequisite for the study of conformational dynamics by single molecule Förster resonance energy transfer (smFRET) - can lead to substantial changes of the dye's photophysical properties, ultimately biasing the determination of inter-dye distances. In particular, cyanine dyes and their derivatives, the most used dyes in smFRET experiments, exhibit such behavior. To overcome this, we developed a general strategy to site-specifically equip proteins with FRET pairs by chemo-selective reactions using two distinct non-canonical amino acids simultaneously incorporated through genetic code expansion in Escherichia coli. Applied to human NADPH-cytochrome P450 reductase (CPR), the importance of homogenously labeled samples for accurate determination of FRET efficiencies was demonstrated and the effect of NADP+ on the ionic strength dependent modulation of the conformational equilibrium of CPR was unveiled. Given its generality and accuracy, the presented methodology establishes a new benchmark to decipher complex molecular dynamics on single molecules.

Original languageEnglish
Pages (from-to)659-666
JournalChembiochem
Volume20
Issue number5
Early online date14 Nov 2018
DOIs
Publication statusPublished - Mar 2019

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NADPH-Ferrihemoprotein Reductase
Energy Transfer
Energy transfer
Labeling
Coloring Agents
Amino Acids
Molecules
Genetic Code
Benchmarking
Molecular Dynamics Simulation
Ionic strength
NADP
Fluorescent Dyes
Osmolar Concentration
Escherichia coli
Molecular dynamics
Proteins
Modulation
Derivatives
Experiments

Keywords

  • bioorthogonal double labeling
  • biophysics
  • conformation analysis
  • noncanonical amino acids
  • protein engineering

Cite this

Quast, Robert Benjamin ; Fatemi, Fataneh ; Kranendonk, Michel ; Margeat, Emmanuel ; Truan, Gilles. / Accurate Determination of Human CPR Conformational Equilibrium by smFRET using Dual Orthogonal Non-Canonical Amino Acid Labeling. In: Chembiochem. 2019 ; Vol. 20, No. 5. pp. 659-666.
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Accurate Determination of Human CPR Conformational Equilibrium by smFRET using Dual Orthogonal Non-Canonical Amino Acid Labeling. / Quast, Robert Benjamin; Fatemi, Fataneh; Kranendonk, Michel; Margeat, Emmanuel; Truan, Gilles.

In: Chembiochem, Vol. 20, No. 5, 03.2019, p. 659-666.

Research output: Contribution to journalArticle

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AU - Quast, Robert Benjamin

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AU - Truan, Gilles

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AB - Conjugation of fluorescent dyes to proteins - a prerequisite for the study of conformational dynamics by single molecule Förster resonance energy transfer (smFRET) - can lead to substantial changes of the dye's photophysical properties, ultimately biasing the determination of inter-dye distances. In particular, cyanine dyes and their derivatives, the most used dyes in smFRET experiments, exhibit such behavior. To overcome this, we developed a general strategy to site-specifically equip proteins with FRET pairs by chemo-selective reactions using two distinct non-canonical amino acids simultaneously incorporated through genetic code expansion in Escherichia coli. Applied to human NADPH-cytochrome P450 reductase (CPR), the importance of homogenously labeled samples for accurate determination of FRET efficiencies was demonstrated and the effect of NADP+ on the ionic strength dependent modulation of the conformational equilibrium of CPR was unveiled. Given its generality and accuracy, the presented methodology establishes a new benchmark to decipher complex molecular dynamics on single molecules.

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