title: Tracking UNC-45 chaperone-myosin interaction with a titin mechanical reporter.
abstract: 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.
authors: Department of Neuroscience and Cell Biology, University of Texas Medical Branch at Galveston, Galveston, TX, USA.
authors: Christian M
authors: CM
authors: Kaiser
authors: Paul J
authors: PJ
authors: Bujalowski
authors: Liang
authors: L
authors: Ma
authors: John
authors: J
authors: Anderson
authors: Henry F
authors: HF
authors: Epstein
authors: Andres F
authors: AF
authors: Oberhauser
mesh_terms: D029742:Caenorhabditis elegans Proteins / Q000737:chemistry* / Q000235:genetics; D064211:Connectin; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D017930:Genes, Reporter; D040542:Mechanotransduction, Cellular / Q000502:physiology*; D008856:Microscopy, Fluorescence / Q000379:methods; D018832:Molecular Chaperones / Q000737:chemistry* / Q000235:genetics; D057054:Molecular Imaging / Q000379:methods*; D020409:Molecular Motor Proteins; D009124:Muscle Proteins / Q000235:genetics / Q000378:metabolism*; D009218:Myosins / Q000235:genetics / Q000378:metabolism*; D025941:Protein Interaction Mapping / Q000379:methods*; D011494:Protein Kinases / Q000235:genetics / Q000378:metabolism*
chemical_list: D029742:Caenorhabditis elegans Proteins; D064211:Connectin; D018832:Molecular Chaperones; D020409:Molecular Motor Proteins; D009124:Muscle Proteins; C107348:unc-45 protein, C elegans; D011494:Protein Kinases; D009218:Myosins