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

Abstract

Conjugation of fluorescent dyes to proteins-a prerequisite for the study of conformational dynamics by single-molecule (sm) FRET-can lead to substantial changes in adye's photophysical properties, ultimately biasingt he determination of inter-dye distances. In particular,c yanine dyes andt heir derivatives, the most commonly used dyes in smFRET experiments,e xhibit such behavior.T oo vercome this, we developed ag eneral strategy to equip proteins site-specifically with FRET pairs through chemoselective reactions with two distinct noncanonical amino acids simultaneouslyi ncorporated throughg enetic code expansion in Escherichia coli. Application of this technique to human NADPH-cytochromeP 450 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 itsg enerality and accuracy, the presented methodology establishes an ew benchmark for deciphering of complex molecular dynamics in single molecules.

Site-Specific Incorporation of Two ncAAs for Two-Color Bioorthogonal Labeling and Crosslinking of Proteins on Live Mammalian Cells

“…This feature has been exploited to vary the PylT anticodon loop, thus allowing it to suppress other stop codons, ochre (TAA) and opal (TGA), as well as sense and quadruplet codons (Niu et al, 2013). Suppression of two codons has been exploited for dual fluorescent labeling of proteins recombinantly expressed in Escherichia coli (Quast et al, 2019;Sachdeva et al, 2014;Wang et al, 2014). In principle, it has been shown that two different stop codons within a single mRNA can be suppressed in mammalian cells, site-specifically introducing two distinct ncAAs in a single protein of interest.…”