STRUCTURE FUNCTION RELATIONS IN MYOSIN

肌肉收缩的机制还需要进一步的阐明。 肌球蛋白结构-功能关系的信息 Head(S1)，包含肌动蛋白和核苷酸相互作用的关键区域 网站。中亚基相互作用的精确模式的知识 肌球蛋白分子以及肌球蛋白分子的组装过程 它们盘绕的线圈部分与粗丝的相互作用也是 不完整。因此，该应用程序集中在四个方面：(1) 肌球蛋白S1的拓扑学将通过(A)建立近似性 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、肌动蛋白等存在的情况下)； 部分解离条件下多肽链的解离过程 变性条件，如甲醇或热处理；(C)研究 针对特定节段的抗体的作用和定位 S1的多肽。(2)肌动蛋白和肌球蛋白的相互作用 通过(A)确定肌动蛋白和S1之间界面内的区域 在强弱相对应的条件下进行交联后 结合状态；(B)使用具有相应序列的合成肽 到假定的S1和肌动蛋白结合区；(C)使用肌动蛋白突变体 硫醇通过位置定向引入假定的界面 诱变。(3)肌球蛋白棒形成粗丝的机理 将研究包括平行和反平行填料在内的关联性 通过用零长度交联剂1-乙基-3-丙交联剂使长丝交联。 [3-(二甲氨基)丙基]碳二亚胺(EDC)或与硫醇结合 试剂。基因工程将引入新的硫醇，这将是 将允许将荧光探针或交联剂附着在 所需位置。(4)轻链/重链相互作用 通过确定参与相互作用的区域进行研究 含有EDC和N-羟基丁二酰亚胺的重链和碱性轻链。 我们还将用特定于硫醇的方法将轻链与重链交联 含不同来源轻链杂化S1的光活化试剂 物种或轻链，在定点突变后表达。这个 正常肌肉的知识将作为参考，并最终可能 在疾病的诊断、治疗和预防方面证明是有用的 涉及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.