Single-molecule Förster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between ±0.02 and ±0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods.

Bifunctional Rel stringent factors, the most abundant class of RSHs (RelA/SpoT Homologues), are ribosome-associated enzymes that transfer a pyrophosphate from ATP onto the 3 of GTP/GDP to synthesize the bacterial alarmone (p)ppGpp, and also catalyse the 3 pyrophosphate hydrolysis to degrade it. The regulation of the opposing activities of Rel enzymes is a complex allosteric mechanism that remains an active research topic despite decades of research. We show that a guanine-nucleotide switch mechanism controls catalysis by T. thermophilus Rel (Rel Tt ). The binding of GDP/ATP opens the N-terminal catalytic domains (NTD) of Rel Tt (Rel Tt NTD ) by stretching apart the two catalytic domains. This activates the synthetase domain and allosterically blocks hydrolysis. Conversely, binding of ppGpp to the hydrolase domain closes the NTD, burying the synthetase active site and precluding the binding of synthesis precursors. This allosteric mechanism is an activity switch that safeguards against futile cycles of alarmone synthesis and degradation. Fondation Van Buren to A.G.-P.; the Molecular Infection Medicine Sweden (MIMS), Swedish Research council (grant 2017-03783), and Ragnar Söderberg foundation fellowship to V.H.; J. Hendrix and J. Hofkens are grateful to the Research Foundation Flanders (FWO Vlaanderen, G0B4915N) and large infrastructure grant ZW15_09 GOH6316N) and the KU Leuven Research Fund (C14/16/053); J. Hofkens thanks financial support of the Flemish government through long term structural funding Methusalem (CASAS2, Meth/15/04).

Phosphorylation-induced conformational trap is an essential mechanism for phosphoregulation of bacterial metabolism.

Förster resonance energy transfer (FRET) is a powerful tool to probe molecular interactions, activity, analytes, forces, and structure. Single-molecule (sm)FRET additionally allows real-time quantifications of conformation and conformational dynamics. smFRET robustness critically depends on the employed dyes, yet a systematic comparison of different dye pairs is lacking. Here, we evaluated blue (Atto488 and Alexa488) and far-red (Atto647N, Alexa647, StarRed, and Atto655) dyes using confocal smFRET spectroscopy on freely diffusing double-stranded (ds)DNA molecules. Via ensemble analyses (correlation, lifetime, and anisotropy) of single-labeled dsDNA, we find that Alexa488 and Atto647N are overall the better dyes, although the latter interacts with DNA. Via burstwise analyses of double-labeled dsDNA with interdye distances spanning the complete FRET-sensitive range (3.5-9 nm), we show that none of the dye pairs stands out: distance accuracies were generally <1 nm and precision was ∼0.5 nm. Finally, excitation of photoblinking dyes such as Alexa647 influences their fluorescence quantum yield, which has to be taken into account in distance measurements and leads to FRET dynamics. Although dye performance might differ in experiments on immobilized molecules, our combined ensemble and single-molecule approach is a robust characterization tool for all types of smFRET experiments. This is especially important when smFRET is used for atomic-scale distance measurements.

Graphical AbstractHighlights d The preprotein binding domain of SecA is mobile in solution d PBD swivels around its stem to acquire 3-4 distinct states d Monomerization, but not nucleotides, regulate PBD swiveling d smFRET provides unique dynamics analysis of SecA SUMMARY SecA converts ATP energy to protein translocation work. Together with the membrane-embedded SecY channel it forms the bacterial protein translocase. How secretory proteins bind to SecA and drive conformational cascades to promote their secretion remains unknown. To address this, we focus on the preprotein binding domain (PBD) of SecA. PBD crystalizes in three distinct states, swiveling around its narrow stem. Here, we examined whether PBD displays intrinsic dynamics in solution using single-molecule Fö rster resonance energy transfer (smFRET). Unique cysteinyl pairs on PBD and apposed domains were labeled with donor/acceptor dyes. Derivatives were analyzed using pulsed interleaved excitation and multi-parameter fluorescence detection. The PBD undergoes significant rotational motions, occupying at least three distinct states in dimeric and four in monomeric soluble SecA. Nucleotides do not affect smFRET-detectable PBD dynamics. These findings lay the foundations for single-molecule dissection of translocase mechanics and suggest models for possible PBD involvement during catalysis.