Research Projects

The sarcomere is the smallest unit of mammalian muscles where myosin heads interact with actin filaments to generate force at the expense of ATP. Due to genetic mutations in myosin, the myosin heads develop a stronger affinity towards actin filaments, resulting in more contraction and a disease called Hypertrophic cardiomyopathy (HCM). These mutations are populated at the myosin heads, the hotspots called myosin mesa. In myosin filaments, myosin is present in a conformation called the "interacting head motif" (IHM). This allows only a small number of myosin molecules to interact with actin simultaneously. The number of accessible heads could be uncontrolled, which could lead to hypercontractility, a condition in which the rate at which muscles contract is faster than the rate at which they relax. The atomic structure of myosin II was solved during my post-doctoral training (Yang S. and Tiwari P., Nature, 2020), which was a breakthrough in the research field in which I am a co-first author and played a key role in achieving this breakthrough the state-of-art cryo-electron microscopy (Cryo-EM). Several key insights about the head and tail interaction and the mutation hotspots were revealed. However, it did not show the mechanism of how the myosin gets activated from its shut-down state. Myosin light chain kinase (MLCK) is believed to phosphorylate myosin's regulatory light chains (RLC), which results in myosin activation. It has been hypothesized, but no structure is available, probably due to the RLC’s disordered N-terminus. This creates a big gap in this field; therefore, no clear experimental details explain the overall mechanism of myosin activation. The N-terminal extension (NTE) of RLC is highly disordered, so it has not been seen earlier. Still, it was stabilized in the myosin structure (Nature, 2020) because all the domains, including the coiled-coil myosin tail are in the complex. While working on my Nature paper, I learned it can be stabilized because I observed the N-terminal extension of the Regulatory Light Chain (RLC) electron density for the first time as it interacted with the myosin tail. In this proposal, I aim to characterize and solve the structure of the MLCK protein and its complex MLCK using Cryo-EM. We will also characterize its biophysical properties using CD, fluorescence spectroscopy, DSC, ITC, and other techniques already in our department at IIT Roorkee. Cryo-EM suits this big protein complex, and I have the right skills and knowledge. The atomic details will reveal the mechanism of myosin activation and the sequence of events. It will help understand how HCM mutation affects contraction, which could be targeted to develop small molecules to restore normal cardiac function.