Analysis of Myosin Motor Domain Interactions with its Chaperone UNC-45 at the Single Molecule Level

Abstract

Myosins are actin-based motor proteins that convert chemical energy from ATP hydrolysis into mechanical work. Movement of myosin heads along actin filaments is a result of structurally complex conformational changes in the myosin motor domain induced by ATP binding and hydrolysis. UNC-45, a member of the UCS family of proteins, acts as a chaperone for myosin and is essential for proper folding and assembly of myosin into muscle thick filaments in vivo. The molecular mechanism of the myosin-UNC-45 interaction in the promoting proper folding of the myosin head domain is not known. We have devised a novel approach to elucidate the interaction of the UNC-45 chaperone with the myosin motor domain utilizing single molecule atomic force microscopy (AFM). By chemically coupling a titin I27 polyprotein to the motor domain of myosin we synthesized a chimera protein that possesses the property of a “molecular reporter”. Our new construct provides a specific attachment point and the well-characterized mechanical fingerprint of the titin octamer in the AFM measurements. Refolding experiments of the chimeric S1-I27 molecules showed that the myosin motor domain interfered with the refolding of otherwise robust I27 modules, presumably by recruiting them into a misfolded state. The presence of UNC-45 restored the folding of the titin I27 domains. We identify the canonical UCS domain of UNC-45 as the essential component of chaperone like activity. This approach enables the study the myosin-UNC-45 interactions at a single molecule level and their consequences for motor domain folding and misfolding in mechanistic detail.