Structural changes in the myosin cross-bridge
The structure of the myosin cross-bridge has been elucidated by means of x-ray diffraction of myosin crystals. Different types of myosins have been crystallized, and by means of ATP or ADP analogues, different conformations of the myosin structure have been proposed, suggesting an important role for the swing of the 'lever arm' during force generation. (see review by Sweeney and Houdusse, 2010). The interaction of the cross-bridge with the thin filament are usually interpolated by 'docking' the myosin structure onto models of the thin filaments using the latest electron microscope tomography techniques (Lorenz and Holmes, 2010). Crystallographic and electron microscopic approaches offer the highest spatial resolution of a few tenth of nanometre, thus providing an atomic view of the muscle machinery. However this view is static and limited to myosin fragments which are not connected to other structural components in muscle and are not responding to changes in environment, such as force, stretch and changing concentrations of substrates and by-products of hydrolysis. In the lab, we explore the possibility of gaining structural information at the nanometre scale using functioning muscle fibres and optical microscopy. Our first approach is the use of a fluorescently-labelled analogue of ATP, DEAC-pda-ATP: (3′-O-{N-[3-(7-diethylaminocoumarin-3-carboxamido)propyl]carbamoyl}ATP). This molecule is a substrate for contraction and labels the ATP binding site of myosin. When incorporated into muscle fibres, the A-bands appear fluorescent (light bands in the image below). Using fluorescence lifetime imaging microscopy (FLIM), we also show that the fluorescence lifetime of the nucleotide is sensitive to the mode of interaction between actin and myosin. The lifetime of the labelled probe bound to the ATPase binding site is different in the actin-overlap region than in the non-overlap region (Ibanez-Garcia et al., 2007).
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DEAC-pda-ATP with the fluorescent probe attached to the 3' position of the ribose on adenosine. |
Image on the left shows the fluorescence intensity of a segment of a muscle fibre from rabbit psoas muscle incubated in 10 μM DEAC-pda-ATP at a sarcomere length of 2.41 μm (90% overlap of the thick filaments with actin). The A-bands are labelled with the ATP analogue. The right image shows the corresponding FLIM image, with coding of the life-time using an arbitrary colour scale, ranging from 1750 ps (blue) to 2170 ps (red). Scale bar, 2 μm. |
We also investigate changes in the environment of the Essential Light Chain (ELC) during contraction. When the lever arm rotates, the interactions between the lever arm and the ELC, and between the ELC and the motor domain of myosin are expected to change. We developed a methodology to investigate these changes in interaction using FLIM of fluorescent probes attached to ELC. Dr. Dmitry Ushakov developed a procedure for exchanging the ELC in permeabilised muscle fibres. Dr. Ushakov has developed a range of ELC mutants which contain a single cysteine placed at critical positions on the ELC. The cysteines are reactive to the coumarin fluorophore 7-diethylamino-3-((((2-iodoacetamido) ethyl)amino) carbonyl)coumarin. The ELC can be prepared in large quantities by expression into bacteria, and when incubated in muscle fibres, the native ELC is replaced by the fluorescently-labelled mutant form. The image below shows the ribbon structure of the myosin head with strategically placed cysteines (yellow) on the ELC (green). Right-click on the image to see a more detailed version. The blue picture is a fluorescence image of a segment of a psoas muscle fibre labelled with fluorescent ELC. The A-bands appear blue. The M-line in the centre of the A-bands are darker, indicating the absence of myosin heads in the bare zone (the scale bar marks 5 μm). 
Development of the technologies for labelling muscle fibres with more than one fluorophore opens the door to the use of FRET (Förster resonance energy transfer) to measure the distance between fluorophores, and the change in distances during contraction(Caorsi et al., 2010). This pioneering work is being developed in the laboratory by Dr. Valentina Caorsi. The prize is to 'see' changes in the distance between two probes during contraction, for example in response to stretches appled to the muscle. The spatial resolution of the technique is 3-10 nm, ideal for detecting the expected changes in the distance between probes on the ELC and on the myosin heavy chain.
