Engineering of a Stable Myosin Neck Fragment for Understanding the Consumption of Energy in Resting Muscle

Myosin is an important protein component of muscle cells which works in conjunction with actin to allow muscles to relax and contract. Myosin consists of three regions, head, neck and tail. There is a relation between the conformation of the neck and relaxed, active, or super-relaxed states of myosin. Super-relaxed myosin has reduced energy consumption. The neck fragment we plan to study contains two molecules of the regulatory light chain (RLC) and two fragments of the heavy chain (HCF), which forms a coiled coil. The engineered fragment was to maintain the three-dimensional structure observed in the full length protein. In the full length protein one molecule of RLC binds one molecule of HCF. Two of these HCF/RLC complexes dimerize via a coiled coil formed by HCF chains, altogether resulting in a functional tetramer complex. We subcloned RLC and introduced two point mutations into the HCF amino acid sequence that increased its ability to form a coiled coil. Engineered RLC and HCF were co-expressed and co-purified on a Ni-NTA resin using the histidine tag located on HCF. This allowed for a 1:1 stoichiometry between the RLC and HCF in the purified RLC/HCF protein complex. Using mass-spectrometry, we have confirmed the presence of both RLC and HCF in the purified protein complex. Circular dichroism (CD) spectra of the purified protein complex indicated the coiled coil formation. Using the glutaraldehyde cross-linking reaction we have also shown that a tetramer complex was formed. Therefore, by engineering these fragments we were able to reconstruct a stable myosin neck fragment, small in size and amenable for further studies.