Accurate Determination of Human CPR Conformational Equilibrium by smFRET Using Dual Orthogonal Noncanonical 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 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.

Across kingdom biased CYP-mediated metabolism via small-molecule ligands docking on P450 oxidoreductase

“…POR is a highly dynamic protein oscillating between compact and extended conformations to execute electron transfer to CYPs. This has been verified by ensemble techniques including electron paramagnetic resonance (EPR) 10 , nuclear magnetic resonance (NMR) 6,9 , small-angle X-ray scattering (SAXS) 6 , small-angle neutron scattering (SANS) 5 , fluorescence 24 , and stopped-flow ultraviolet-visible (UV-VIS) spectroscopy 27 , providing insights into POR conformational sampling recently confirmed by smFRET burst analysis 18,28 . Mutations known to control POR specificity and to cause metabolic disorders are often found in the hinge region of POR that controls conformational dynamics 4,12,13 .…”

“…We used Total Internal Reflection Fluorescence (TIRF) microscopy [29][30][31] to record smFRET traces and directly observe conformational sampling of SbPOR2b and its remodeling by ligands. Data were recorded using the Alternating-Laser Excitation (ALEX) methodology 32 that we and others have been using extensively 28,29,33 . POR was site-specifically labelled with Cy3 and Cy5 fluorophores using a minimal cysteine full-length SbPOR2b variant with two solvent accessible cysteines (N181C/C536S/A552C) that we have recently used for smFRET without impairing activity 18 .…”

Structural insights into the semiquinone form of human Cytochrome P450 reductase by DEER distance measurements between a native flavin and a spin labelled non‐canonical amino acid

“…[3] The conformational equilibrium within Homo sapiens CPR has been studied through both spectroscopic and structural approaches, essentially on mutated or truncated proteins. [3][4][5][6][7] Undoubtedly, these studies highlight the immense significance of the connection between dynamics and function, as domain movement can exert precise control over the efficiency of ET and activities of CYPs, and consequently have a substantial impact on CYPmediated drug metabolism. [7,8] Crystal structures of Rattus norvegicus soluble CPR are available in two distinct states: the "locked/closed" (pdb.…”

“…[3][4][5][6][7] Undoubtedly, these studies highlight the immense significance of the connection between dynamics and function, as domain movement can exert precise control over the efficiency of ET and activities of CYPs, and consequently have a substantial impact on CYPmediated drug metabolism. [7,8] Crystal structures of Rattus norvegicus soluble CPR are available in two distinct states: the "locked/closed" (pdb. 1amo, Δ64) and partially "unlocked/open" (pdb.…”

Dissecting conformational rearrangements and allosteric modulation in metabotropic glutamate receptor activation

“…It relies on the incorporation of a reactive non-canonical amino acid (ncAA) by genetic code expansion ( Figure 1b ). In this way FRET-compatible donor and acceptor fluorophores can be installed at desired positions using bioorthogonal labeling strategies 18, 19 . First, we screened for suitable labeling positions based on ncAA incorporation efficiency and functional integrity of modified receptors, followed by the establishment of live cell compatible click chemistry conditions that allow to monitor ligand-induced conformational rearrangements by lanthanide resonance energy transfer (LRET).…”