Evaluation of Blue and Far-Red Dye Pairs in Single-Molecule Förster Resonance Energy Transfer Experiments

Abstract

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.

The Preprotein Binding Domain of SecA Displays Intrinsic Rotational Dynamics

“…Getting enough bursts from doubly hetero-labeled SecAs, in the shortest possible time yields high quality data due to the collection of several thousand single-molecule samples and minimizes potential loss of SecA functionality. Condition optimization included BSA-coated coverslips and soluble agents such as free BSA or Trolox to prevent nonspecific protein absorption and photo-bleaching) ( Figure S2G) (Aitken et al, 2008;Rasnik et al, 2006;Vandenberk et al, 2018;Vogelsang et al, 2008).…”

“…Single-molecule Fö rster resonance energy transfer (smFRET) monitors intramolecular (conformational rearrangements/domain motions) or intermolecular (association/dissociation) dynamic processes in real time. Thus distinct conformational states, commonly lost in ensemble measurements, can be identified (Ha and Tinnefeld, 2012;Roy et al, 2008) and their physical distances derived from the measured Fö rster resonance energy transfer (FRET) quantum efficiency (E) between the probes (Hellenkamp et al, 2018;Kapanidis et al, 2004;Lee et al, 2005;Vandenberk et al, 2018). In solution smFRET, fluorescent molecules diffuse freely through the confocal volume of a microscope (Figures 2A and S1A) and their fluorescent bursts (i.e., the number of photons they emit during the time they stay in focus) are measured (Eggeling et al, 2001;Kellner et al, 2014;Zander et al, 1996).…”

“…These include dye rotation (nanoseconds); unfolded state dynamics (nanoseconds to microseconds); folding/rearrangements (microseconds to seconds), which depend on ultra-fast motions of structured protein parts (picoseconds to microseconds); fast whole-domain motions (microseconds to milliseconds); and misfolding/oligomerization (seconds to minutes or hours) (Schuler and Hofmann, 2013). smFRET, combined with MFD-PIE and proper time-resolved analysis, discriminates the intrinsic protein dynamics that are of biological interest from the chemical properties of the labels and the artifacts and photophysical dynamics that need to be excluded (Cotlet et al, 2005;Hofkens et al, 2003;Vandenberk et al, 2018;Vosch et al, 2003).…”

Substrate binding modulates the conformational kinetics of the secondary multidrug transporter LmrP

“…Cysteines were placed at the extra-or intracellular extremities of chosen TMs while avoiding mutating conserved residues. The expected FRET efficiencies were estimated by simulating the fluorescent probes' accessible volume [33] so that they are separated by a distance within the Förster radius, 56.8 Å in this case [36]. The accessible volume (AV) clouds were simulated using the parameters given for ATTO488 and Alexa647 in [36].…”

“…MFD-PIE allows for each molecule the determination of the E, the labelling stoichiometry, the fluorescence lifetime, and the anisotropy of the donor and the acceptor that are represented in the multi-parameter graphs in Fig S2. The single-burst analysis was performed as described in [36]. Briefly, the absolute burst-averaged FRET efficiency E was calculated with:…”