Expression in Escherichia coli of fragments of the coiled-coil rod domain of rabbit myosin: influence of different regions of the molecule on aggregation and paracrystal formation.
We have expressed in Escherichia coli a cDNA clone corresponding broadly to rabbit light meromyosin (LMM) together with a number of modified polypeptides and have used this material to investigate the role of different aspects of molecular structure on the solubility properties of LMM. The expressed material was characterized biochemically and structurally to ensure that it retained the coiled-coil conformation of the native molecule. Full-length recombinant LMM retained the general solubility properties of myosin and, although soluble at high ionic strength, precipitated when the ionic strength was reduced below 0.3 M. Constructs in which the 'skip' residues (that disrupt the coiled-coil heptad repeat) were deleted had solubility properties indistinguishable from the wild type, which indicated that the skip residues did not play a major role in determining the molecular interactions involved in assembly. Deletions from the N terminus of LMM did not alter the solubility properties of the expressed material, but deletion of 92 residues from the C terminus caused a large increase in solubility at low ionic strength, indicating that a determinant important for interaction between LMM molecules was located in this region. The failure of deletions from the molecule's N terminus to alter its solubility radically suggested that the periodic variation of charge along the myosin rod may not be as important as proposed for determining the strength of binding between molecules and thus the solubility of myosin.
Atkinson
S J
SJ
MRC Laboratory of Molecular Biology, Cambridge, UK.
Stewart
M
M
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003460:Crystallization; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008854:Microscopy, Electron; D008969:Molecular Sequence Data; D009154:Mutation; D015879:Myosin Subfragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism / Q000648:ultrastructure; D009994:Osmolar Concentration; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012995:Solubility
D015879:Myosin Subfragments; D011994:Recombinant Proteins