Kanji Oshima

Head-Head Interactions of Resting Myosin Crossbridges in Intact Frog Skeletal Muscles, Revealed by Synchrotron X-Ray Fiber Diffraction

The intensities of the myosin-based layer lines in the x-ray diffraction patterns from live resting frog skeletal muscles with full thick-thin filament overlap from which partial lattice sampling effects had been removed were analyzed to elucidate the configurations of myosin crossbridges around the thick filament backbone to nanometer resolution. The repeat of myosin binding protein C (C-protein) molecules on the thick filaments was determined to be 45.33 nm, slightly longer than that of myosin crossbridges. With the inclusion of structural information for C-proteins and a pre-powerstroke head shape, modeling in terms of a mixed population of regular and perturbed regions of myosin crown repeats along the filament revealed that the myosin filament had azimuthal perturbations of crossbridges in addition to axial perturbations in the perturbed region, producing pseudo-six-fold rotational symmetry in the structure projected down the filament axis. Myosin crossbridges had a different organization about the filament axis in each of the regular and perturbed regions. In the regular region that lacks C-proteins, there were inter-molecular interactions between the myosin heads in axially adjacent crown levels. In the perturbed region that contains C-proteins, in addition to inter-molecular interactions between the myosin heads in the closest adjacent crown levels, there were also intra-molecular interactions between the paired heads on the same crown level. Common features of the interactions in both regions were interactions between a portion of the 50-kDa-domain and part of the converter domain of the myosin heads, similar to those found in the phosphorylation-regulated invertebrate myosin. These interactions are primarily electrostatic and the converter domain is responsible for the head-head interactions. Thus multiple head-head interactions of myosin crossbridges also characterize the switched-off state and have an important role in the regulation or other functions of myosin in thin filament-regulated muscles as well as in the thick filament-regulated muscles.

Structural Alterations of Thin Actin Filaments in Muscle Contraction by Synchrotron X-ray Fiber Diffraction

Strong evidence has been accumulated that the conformational changes of the thin actin filaments are occurring and playing an important role in the entire process of muscle contraction. The conformational changes and the mechanical properties of the thin actin filaments we have found by X-ray fiber diffraction on skeletal muscle contraction are explored. Recent studies on the conformational changes of regulatory proteins bound to actin filaments upon activation and in the force generation process are also described. Finally, the roles of structural alterations and dynamics of the actin filaments are discussed in conjunction with the regulation mechanism and the force generation mechanism.

Modeling Analysis of Myosin-Based Meridional X-Ray Reflections from Frog Skeletal Muscles in Relaxed and Contracting States

Analysis of the myosin-based meridional intensity data in the X-ray diffraction patterns of live frog skeletal muscles was performed to propose a more precise model for a myosin crown periodicity and an axial disposition of two-headed crossbridges along the thick filament in a sarcomere. Modeling studies revealed that the thick filament has a mixed structure of two different periodicities of the myosin crossbridge crown arrangement and that the crown periodicity and the axial disposition of crossbridges are altered when muscle goes from the relaxed state to the contracting state. Factors that primarily affect the meridional intensities were examined.

Deduction of the single-myosin-filament transforms from partially sampled layer lines in the X-ray diffraction pattern from vertebrate striated muscle

A novel method to correct a partial sampling effect, due to the hexagonal filament array of a statistical superlattice form, on the thick (myosin)-filament-based layer lines in X-ray diffraction patterns from higher-vertebrate striated muscle has been developed using the cylindrically averaged difference Patterson function [ΔQ(r, z)]. The method involves cutting off the inter-filament vector peaks that appear in the radial region beyond ∼32 nm on the ΔQ(r, z) map calculated from the observed layer-line intensities, and then deducing the single-myosin-filament transforms by inverse Fourier transformation of the truncated ΔQ(r, z). The accuracy of the cut-off method was tested using a single-myosin-filament model and a hexagonal filament-array model with a size of one superlattice unit cell. The layer-line intensities calculated from the truncated ΔQ(r, z) of the hexagonal filament-array model showed few sampling peaks, the layer lines being effectively coincident with those from the single-filament model except for the intensities close to the meridian. Some residual differences were caused by the face-to-face inter-crossbridge vectors between closest neighboring filaments, which correspond to ∼27.5% of the total number of crossbridge vectors in the truncated ΔQ(r, z) map, but the face-to-face inter-crossbridge vectors contributed mainly to the intensities close to the meridian. Their remnant off-meridional layer-line intensity components did not significantly affect a search for the optimum azimuthal orientation of myosin crossbridges in the resting state of muscle.