Tracking UNC-45 Chaperone-Myosin Interaction with a Titin Mechanical Reporter

• Published in: Biophysical journal Vol. 102; no. 9; pp. 2212 - 2219
• Main Authors: Kaiser, Christian M., Bujalowski, Paul J., Ma, Liang, Anderson, John, Epstein, Henry F., Oberhauser, Andres F.
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 02.05.2012; Biophysical Society; The Biophysical Society
• Subjects: atomic force microscopy; Binding sites; Caenorhabditis elegans Proteins - chemistry; Caenorhabditis elegans Proteins - genetics; Connectin; dissociation; energy; Escherichia coli - genetics; Escherichia coli - metabolism; Genes, Reporter; hydrolysis; kinesin; Mechanotransduction, Cellular - physiology; microfilaments; Microscopy, Fluorescence - methods; Molecular Chaperones - chemistry; Molecular Chaperones - genetics; Molecular Imaging - methods; Molecular Motor Proteins; Molecules; Muscle Proteins - genetics; Muscle Proteins - metabolism; muscles; myosin; Myosins - genetics; Myosins - metabolism; Protein folding; Protein Interaction Mapping - methods; Protein Kinases - genetics; Protein Kinases - metabolism; Spectroscopy, Imaging, and Other Techniques
• ISSN: 0006-3495; 1542-0086
• DOI: 10.1016/j.bpj.2012.03.013

Myosins are molecular motors that convert chemical energy into mechanical work. Allosterically coupling ATP-binding, hydrolysis, and binding/dissociation to actin filaments requires precise and coordinated structural changes that are achieved by the structurally complex myosin motor domain. UNC-45, a member of the UNC-45/Cro1/She4p 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 mechanisms by which UNC-45 interacts with myosin to promote proper folding of the myosin head domain are not known. We have devised a novel approach, to our knowledge, to analyze the interaction of UNC-45 with the myosin motor domain at the single molecule level using atomic force microscopy. By chemically coupling a titin I27 polyprotein to the motor domain of myosin, we introduced a mechanical reporter. In addition, the polyprotein provided a specific attachment point and an unambiguous mechanical fingerprint, facilitating our atomic force microscopy measurements. This approach enabled us to study UNC-45–motor domain interactions. After mechanical unfolding, the motor domain interfered with refolding of the otherwise robust I27 modules, presumably by recruiting them into a misfolded state. In the presence of UNC-45, I27 folding was restored. Our single molecule approach enables the study of UNC-45 chaperone interactions with myosin and their consequences for motor domain folding and misfolding in mechanistic detail.