* Muscle Research Unit, Department of Anatomy and Histology, and + Department of Pathology, The University of Sydney, New South Wales 2006.
Mns-Cl is a fluorescent probe that specifically labels lysine residues. In the presence of divalent metal ions it uniquely labels a single lysine located close to the head-rod junction of the pig cardiac myosin heavy chain (MHC). It is then possible to perform fluorescence resonance energy transfer (FRET) from this donor probe site to other locations on the myosin molecule which have been specifically labelled with an acceptor probe. Using the Förster theory it is then possible to "measure" the distance between the donor and acceptor label sites and so obtain a measure of conformational changes in this region of the myosin molecule.
In a previous study we exchanged fluorescein labelled myosin light chain 2 (LC2) from a chicken gizzard myosin source onto Mns-Cl labelled pig cardiac myosin. It was found that the distance between the donor (Mns) probe and the acceptor (fluorescein) probe was less than 3 mm.
We are now undertaking various other FRET measurements from the Mns site on pig cardiac myosin. This will include exchanging a genetically mutated chicken skeletal muscle LC2 onto the MHC. Measurements will then be performed to isolated cysteine residues (Cys125 and Cys155) labelled with acceptor probe. Additionally, functional studies will be performed to assess the effect that nucleotide and actin binding has on the relationship between the two probe sites.