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