STRUCTURE FUNCTION RELATIONS IN MYOSIN

阐明肌肉收缩的机制需要进一步的 关于肌球蛋白结构-功能关系的信息 头部（S1），包含肌动蛋白和核苷酸相互作用的关键区域 网站. 了解细胞内亚基相互作用的精确模式 肌球蛋白分子以及肌球蛋白分子组装过程 它们的卷绕-卷绕部分相互作用成粗丝， 不完整 因此，该应用程序集中在四个领域：（1） 肌球蛋白S1的拓扑结构将通过以下方式进行探索：（a）建立邻近 肌球蛋白S1与光活化交联剂的关系 在各种条件下（在核苷酸、肌动蛋白等存在下）;（B） 阐明了部分条件下多肽链解折叠的过程， 变性条件，如甲醇或热处理;（c）研究 针对特定片段的抗体的作用和位置 S1的肽。(2)肌动蛋白和肌球蛋白的相互作用将被探讨 通过（a）确定肌动蛋白和S1之间界面内的区域， 在对应于强和弱交联的条件下交联后， 结合状态;（B）使用具有相应序列的合成肽 S1和肌动蛋白的推定结合区域;（c）使用肌动蛋白突变体 在假定的界面处通过位点定向引入硫醇 诱变(3)肌球蛋白杆形成粗肌丝的机制 包括平行和反平行包装协会将被研究 通过用零长度交联剂1-乙基-3-甲基丙烯酸酯交联长丝， [3-（二甲基氨基）丙基]碳二亚胺（EDC）或具有硫醇特异性 试剂 新的硫醇将通过基因工程引入， 将允许荧光探针或交联剂在 理想的位置。(4)轻链/重链相互作用将是 通过识别参与交联的区域， 重链和碱性轻链与EDC和N-羟基琥珀酰亚胺。 我们还将用巯基特异性的轻链与重链交联， 使用具有来自不同来源的轻链的杂合S1的光活化试剂 种或轻链，在定点诱变后表达。 的 正常肌肉的知识将作为参考， 证明在疾病的诊断、治疗和预防中是有用的 包括肌肉组织和心血管系统。

Elucidation of the mechanism of muscle contraction requires further information concerning structure- function relationships in the myosin head (S1), the key regions containing the actin and nucleotide interaction sites. Knowledge of the precise mode of subunit interactions within the myosin molecule as well as the process of assembly of myosin molecules by interaction of their coiled-coil portions into thick filaments is also incomplete. Thus the application is focusing on four areas: (1) The topography of myosin S1 will be explored by (a) establishing proximity relationships in myosin S1 with the use of photoactivatable crosslinkers under various conditions (in the presence of nucleotide, actin, etc.); (b) elucidating the process of polypeptide chain unfolding under partial denaturation conditions such as methanol or heat treatment; (c) studying the effects and locations of antibodies made against specific segments of peptides of S1. (2) The interaction of actin and myosin will be explored by (a) identifying the regions within the interface between actin and S1 after crosslinking under conditions corresponding to strong and weak binding states; (b) using synthetic peptides with sequences corresponding to the putative binding regions of S1 and actin; (c) using actin mutants with thiols introduced at the putative interfaces by site directed mutagenesis. (3) The mechanism of thick filament formation by myosin rod association including parallel and anti-parallel packing will be studied by crosslinking the filaments with the zero length crosslinker 1-ethyl-3- [3- (dimethyl amino) propyl] carbodiimide (EDC) or with thiol specific reagents. New thiols will be introduced by genetic engineering, which would permit the attachment of fluorescent probes or crosslinkers at desired locations. (4) The light chain/ heavy chain interaction will be studied by identifying the regions involved in the crosslinking between heavy chain and alkali light chains with EDC and N-hydroxy succinimide. We will also crosslink light chains to heavy chains with thiol specific photoactivatable reagents using hybrid S1 with light chains from different species or light chains, expressed after site directed mutagenesis. The knowledge of nomal muscle will serve as a reference and may eventually prove useful in the diagnosis, treatment, and prevention of disease involving muscle tissues and the cardiovascular system.