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

2 Citations (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

Keywords

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

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