Dynamics of Tropomyosin in Muscle Fibers as Monitored by Saturation Transfer EPR of Bi-Functional Probe

The dynamics of four regions of tropomyosin was assessed using saturation transfer electron paramagnetic resonance in the muscle fiber. In order to fully immobilize the spin probe on the surface of tropomyosin, a bi-functional spin label was attached to i,i+4 positions via cysteine mutagenesis. The dynamics of bi-functionally labeled tropomyosin mutants decreased by three orders of magnitude when reconstituted into “ghost muscle fibers”. The rates of motion varied along the length of tropomyosin with the C-terminus position 268/272 being one order of magnitude slower then N-terminal domain or the center of the molecule. Introduction of troponin decreases the dynamics of all four sites in the muscle fiber, but there was no significant effect upon addition of calcium or myosin subfragment-1

Structure and function of the N-terminal region of cardiac Myosin binding protein - C

Myosin binding protein - C (MyBPC) is a multi-domain protein whose role in the sarcomere is complex and not yet fully understood. The cardiac isoform is linked to familial hypertrophic cardiomyopathy (FHC), which is caused by the expression of abnormal sarcomeric proteins, including numerous mutations in MyBPC. The core structure of cardiac MyBPC consists of eight immunoglobulin (IgI) and three fibronectin (FnIII) domains, numbered 0 - 10 from the N-terminus. The C-terminus of MyBPC binds to titin and the myosin backbone and may bind other MyBPC molecules. At its N-terminus, MyBPC binds myosin S2 when dephosphorylated and may bind the myosin head and actin. MyBPC is phosphorylated up to 3 times in the linker between IgI domains 1 and 2, causing dissociation from S2 and increased contractile force. The role of partial phosphorylation remains unclear. The N-terminal construct, C1C2 (two IgI domains linked by the phosphorylatable linker), has been cloned, expressed, and purified. FHC and permanently phosphorylated mutants have also been produced. The constructs were studied by modelling, circular dichroic (CD) spectroscopy and myosin binding assays. Modelling of the C1C2 construct suggests an alpha-helical content within the phosphorylation linker region. CD spectra confirmed that a fraction of the protein is indeed alpha-helix. CD spectral changes for several FHC mutants indicate secondary structural change. C1C2 binding to myosin was confirmed using a sedimentation assay. Surprisingly, C1C2 also binds to actin, but only in its filamentous form, and this binding appears to be phosphorylation dependent

Myosin Regulatory Domain Orientation in Skeletal Muscle Fibers: Application of Novel Electron Paramagnetic Resonance Spectral Decomposition and Molecular Modeling Methods

Reorientation of the regulatory domain of the myosin head is a feature of all current models of force generation in muscle. We have determined the orientation of the myosin regulatory light chain (RLC) using a spin-label bound rigidly and stereospecifically to the single Cys-154 of a mutant skeletal isoform. Labeled RLC was reconstituted into skeletal muscle fibers using a modified method that results in near-stoichiometric levels of RLC and fully functional muscle. Complex electron paramagnetic resonance spectra obtained in rigor necessitated the development of a novel decomposition technique. The strength of this method is that no specific model for a complex orientational distribution was presumed. The global analysis of a series of spectra, from fibers tilted with respect to the magnetic field, revealed two populations: one well-ordered (±15°) with the spin-label z axis parallel to actin, and a second population with a large distribution (±60°). A lack of order in relaxed or nonoverlap fibers demonstrated that regulatory domain ordering was defined by interaction with actin rather than the thick filament surface. No order was observed in the regulatory domain during isometric contraction, consistent with the substantial reorientation that occurs during force generation. For the first time, spin-label orientation has been interpreted in terms of the orientation of a labeled domain. A Monte Carlo conformational search technique was used to determine the orientation of the spin-label with respect to the protein. This in turn allows determination of the absolute orientation of the regulatory domain with respect to the actin axis. The comparison with the electron microscopy reconstructions verified the accuracy of the method; the electron paramagnetic resonance determined that axial orientation was within 10° of the electron microscopy model