Paracrystals of myosin rod

Abstract

To help understand the packing of myosin tails in the backbone of the vertebrate striated muscle thick filament, paracrystals of myosin rod, a proteolytic fragment corresponding to the whole myosin tail, have been examined by electron microscopy and image analysis. Two types of paracrystal were observed. Type I paracrystals were similar to those seen by Moos et al. (1975; J. molec. Biol. 97, 1-9). These showed a 14-nm axial repeat, but yielded no other structural information. Type II paracrystals were long, flexible ribbons with more regularity. When negatively stained they exhibited a weak 43-nm axial striation and appeared to be composed of a layer of narrow filaments. Optical diffraction showed that the paracrystals had a rectangular unit cell of dimensions 43 nm axially and 12.4 nm laterally. Transverse sections indicated a paracrystal depth similar to the lateral dimension of the unit cell. Each unit cell contained two filaments arranged antiparallel and related by a two-fold screw axis perpendicular to the length, and in the plane of the ribbon. The filaments probably consist of parallel rod molecules related by axial displacements of 43 nm and higher multiples of 43 nm. The nature of these paracrystals indicates that the myosin tail alone can form structures like thick filament subfilaments. Their structure, based on distinguishable parallel and antiparallel rod interactions, was sensitive to pH and divalent cations in a similar way to the ionic effects on the structure of thick filaments. This behaviour suggests that some of the interactions present in the paracrystal are the same as those in the thick filament.

Mutations in the β-myosin rod cause myosin storage myopathy via multiple mechanisms

“…WT LMM formed well-ordered paracrystals with a periodicity of 14.0 nm (Ϯ0.44 nm for n ϭ 20 paracrystals; S2). This value is consistent with reported values for LMM paracrystal periodicity and corresponds well to the 14.3 nm axial spacing of full-length ␤-MyHC found in bipolar thick filaments (1,20,21,24,26). Paracrystals formed from L1793P, R1845W, E1886K, and H1901L LMM all had similar periodicities to WT, indicating that these mutations do not alter the gross morphology of the assemblies (S3).…”

“…Paracrystals are well-ordered protein assemblies that provide an established model for thick filament formation (1,(20)(21)(22)(23)(24)(25)(26). Similarly to light scattering experiments, proteins were dialyzed from a high salt buffer into a buffer with physiological salt concentration allowing them to assemble into well-ordered arrays.…”

Three-dimensional structure of frozen-hydrated paracrystals of myosin rod

“…One form, the type II paracrystal, a thin, ribbon-like species, has recently been studied using conventional electron microscopy and was shown to possess two-dimensional order and, further, is likely to be useful in the investigation of the arrangememt of myosin molecules in the backbone of the vertebrate muscle thick filament (Ward & Bennett, 1989). We have now examined this aggregate in the frozen-hydrated state by cryo-electron microscopy.…”

“…In the electron microscope they appeared to be long (up to 50 #m), single-layered and ribbon-like structures with a longitudinal substructure. They closely mimicked several structural responses to changes in ionic conditions shown by the native thick filament (Ward & Bennett, 1989) and were thus considered to be pertinent to the study of the structure of the thick filament backbone. Analysis of images of negatively stained paracrystals showed the presence of two-dimensional order.…”

“…In an investigation of the aggregation of the purified myosin rod fragment, equivalent to the whole tail, a crystalline species was found to be suitable for structural study (Ward & Bennett, 1989). This structure was highly homogeneous and was called the type II rod paracrystal.…”