FRET characterisation for cross-bridge dynamics in single-skinned rigor muscle fibres

In this work we demonstrate for the first time the use of Förster resonance energy transfer (FRET) as an assay to monitor the dynamics of cross-bridge conformational changes directly in single muscle fibres. The advantage of FRET imaging is its ability to measure distances in the nanometre range, relevant for structural changes in actomyosin cross-bridges. To reach this goal we have used several FRET couples to investigate different locations in the actomyosin complex. We exchanged the native essential light chain of myosin with a recombinant essential light chain labelled with various thiol-reactive chromophores. The second fluorophore of the FRET couple was introduced by three approaches: labelling actin, labelling SH1 cysteine and binding an adenosine triphosphate (ATP) analogue. We characterise FRET in rigor cross-bridges: in this condition muscle fibres are well described by a single FRET population model which allows us to evaluate the true FRET efficiency for a single couple and the consequent donor–acceptor distance. The results obtained are in good agreement with the distances expected from crystallographic data. The FRET characterisation presented herein is essential before moving onto dynamic measurements, as the FRET efficiency differences to be detected in an active muscle fibre are on the order of 10–15% of the FRET efficiencies evaluated here. This means that, to obtain reliable results to monitor the dynamics of cross-bridge conformational changes, we had to fully characterise the system in a steady-state condition, demonstrating firstly the possibility to detect FRET and secondly the viability of the present approach to distinguish small FRET variations

Precision and accuracy of single-molecule FRET measurements - a multi-laboratory benchmark study

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