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Regulation of myosin II bipolar thick filament assembly.

机译：调节肌球蛋白II双极粗丝组件。

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摘要

Thick filament formation in Dictyostelium is an attractive candidate for deciphering determinants of self-assembly as all the information required to form a structure of defined size is contained in the myosin molecule itself. In addition, the assembly state of this complex can be dramatically changed by phosphorylation of only three threonine residues. We characterized bacterially expressed fragments of the coiled-coil tail to define what portion of the myosin molecule is required for regulated self-assembly. Tail fragments form paracrystals, structures that are highly organized but contain an undefined number of elements. While tail fragments do not form bipolar thick filaments, they have similar solubility properties to full-length myosin.; Determination of the solubility of a series of tail fragments identified the C-terminal 68 kD of the tail as the minimal domain for regulated assembly (termed A.D. to C-terminus tail fragment). The charged amino acids in the A.D. to C-terminus tail fragment are organized into repeating blocks of 28 and 196 amino acids. Our analysis suggests that these charge repeats must be delicately balanced for efficient self-assembly and that the introduction of negative charge by phosphorylation is sufficient to regulate assembly.; GFP was fused to the N-terminus of the A.D. to C-terminus tail fragment to determine if a globular head is sufficient to limit the size of a thick filament. The GFP A.D. to C-terminus tail fragment undergoes regulated assembly into bipolar thick filaments that are structurally homologous to thick filaments formed from full-length myosin. The symmetrical distribution of both the 28 amino acid and 196 amino acid charge repeats in the A.D. to C-terminus tail fragment enabled us to test whether thick filament assembly requires specific amino acid sequences proximal to the globular head or an overall charge pattern. The A.D. to C-terminus tail fragment with GFP fused to the C-terminus undergoes regulated assembly into bipolar thick filaments, suggesting that assembly is driven by an overall charge pattern. In summary, we have reduced regulated assembly to a smaller and more manageable model system that promises to be a useful reagent in defining the determinants of myosin self-assembly.
机译：在 Dictyostelium 中，粗丝形成是破译自组装决定因素的诱人候选物，因为形成定义大小的结构所需的所有信息都包含在肌球蛋白分子本身中。另外，仅三个苏氨酸残基的磷酸化可大大改变该复合物的组装状态。我们表征了卷曲螺旋尾部的细菌表达片段，以定义肌球蛋白分子的哪些部分需要调控的自组装。尾巴碎片形成副晶体，这些晶体高度组织，但包含数量不确定的元素。尽管尾部片段不形成双极粗丝，但它们具有与全长肌球蛋白相似的溶解性。确定一系列尾巴片段的溶解度后，将尾巴的C端68 kD确定为调节装配的最小区域（称为A到C尾巴片段）。 A.D.到C端尾片段中带电荷的氨基酸被组织成28和196个氨基酸的重复块。我们的分析表明，必须有效地平衡这些电荷重复，以实现有效的自组装，并且通过磷酸化引入负电荷足以调节组装。将GFP融合到A.D.到C端尾片段的N端，以确定球状头是否足以限制粗丝的大小。 GFP A.D.到C末端的尾巴片段经过调节组装成双极粗丝，其结构与全长肌球蛋白形成的粗丝在结构上同源。 28个氨基酸和196个氨基酸电荷的对称分布在A到C端尾片段中重复出现，这使我们能够测试粗丝组装是否需要球状头附近的特定氨基酸序列或总体电荷模式。具有融合到C端的GFP的A.D.到C端尾部片段经过调节组装成双极粗丝，这表明组装是由整体电荷模式驱动的。总而言之，我们已经将调节装配减少到一个更小，更易于管理的模型系统，该系统有望成为定义肌球蛋白自组装决定因素的有用试剂。

著录项

  • 作者

    Hostetter, Daniel.;

  • 作者单位

    Stanford University.;

  • 授予单位 Stanford University.;
  • 学科 Biology Cell.; Biology Molecular.; Biophysics General.
  • 学位 Ph.D.
  • 年度 2004
  • 页码 225 p.
  • 总页数 225
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类 细胞生物学;分子遗传学;生物物理学;
  • 关键词

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