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

Valentina Caorsi*, Dmtry S. Ushakov, Timothy G. West, Niovi Setta-Kaffetzi, Michael A. Ferenczi

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

6 Citations (Scopus)

Abstract

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.

Original languageEnglish
Pages (from-to)13-27
Number of pages15
JournalEuropean Biophysics Journal
Volume40
Issue number1
DOIs
Publication statusPublished - 1 Jan 2011

Keywords

  • Acceptor photobleaching
  • Actomyosin interactions
  • FLIM
  • FRET
  • Rigor muscle fibre
  • Spectral analysis

Fingerprint

Dive into the research topics of 'FRET characterisation for cross-bridge dynamics in single-skinned rigor muscle fibres'. Together they form a unique fingerprint.

Cite this