INTERACTION OF ATP ANALOGUES WITH MYOSIN

ATP 类似物与肌球蛋白的相互作用

• 批准号: 3482882
• 负责人: RALPH G YOUNT
• 金额: $ 20.58万
• 依托单位: WASHINGTON STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1976
• 资助国家: 美国
• 起止时间: 1976-05-01 至 1991-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3482882
• 关键词: X ray crystallography; adenosine triphosphate; affinity chromatography; aminoacid analyzer; chemical group; chemical synthesis; crosslink; electron microscopy; fluorescent dye /probe; gel electrophoresis; gel filtration chromatography; high performance liquid chromatography; molecular site; muscle contraction; myocardium; nucleotide analog; photolysis; smooth muscle; striated muscles

The goal is to describe in as complete a manner as possible the chemical nature of force-generation in muscle. Specifically new photoaffinity analogues of ATP will be synthesized and used to label covalently active site residues of myosin from both straited (skeletal and cardiac) and non-straited (stomach and arterial) myosins. The subunit location and the amino acid composition of the labeled peptides will be determined by use of high performance liquid chromatography and gas phase sequencing techniques. The ATP analogues used in each case will be trapped at the active site eigter by chemical cross-linking two kinetically reactive thiols, SH1 and SH2, or by forming a stable transition state complex of ADP analogues with vanadate ions. Either of these two approaches allows the photoaffinity analogue myosin complex to be purified free of extraneous photolabels and greatly increases both the specificity and the extent of labeling. Preliminary results indicate that the subunit composition of the active site of smooth muscle myosin (stomach) is formed by elements of both the essential light chains and the heavy chains. Conversely, the active sites of straited muscle myosins appear to be composed only of heavy chains. This dichotomy will be investigated further utilizing new ATP analogues and myosins from other smooth muscles and from non-muscle tissues. A major goal will be to determine if there is a fundamental difference in the composition and nature of the force generating sites of smooth muscle myosins (and by analogy, non-muscle myosins) from those of straited muscles. This difference is not apparent from the gross morphology and subunit composition of the myosins from different tissues but may reflect the known differences in how contraction is regulated in these muscles. Other goals will be to synthesize new ATP analogues to be used (i) in affinity columns to purify myosin, (ii) as fluorescent probes of the heads of myosin and (iii) as electron-rich probes to aid in the solution of the X-ray structure of the recently crystallized head-regions of myosin. The long-term goal of this research is to provide a molecular explanation for how the chemical energy in ATP is converted into the mechanical energy of muscle contraction.

目标是以尽可能完整的方式描述化学物质 在肌肉中产生力量的本质。特别是新的光亲和性 将合成三磷酸腺苷的类似物并用于标记共价活性 骨骼和心脏肌球蛋白的部位残留量 非束缚(胃和动脉)肌球蛋白。子单元位置和 标记多肽的氨基酸组成将通过使用 高效液相色谱-气相测序法 技巧。每种情况下使用的ATP类似物都会被困在 两个动力学反应的化学交联剂的活性中心 硫醇、SH1和SH2，或通过形成稳定的ADP过渡态络合物 钒酸根离子的类似物。这两种方法中的任何一种都允许 光亲和类似物肌球蛋白复合体将被纯化，不含外来物质 光标记并极大地提高了特异性和范围 贴标签。初步结果表明，亚基组成 平滑肌肌球蛋白(胃)的活性部位是由两者的成分形成的 基本的轻链和重链。相反，活跃的 肌肉肌球蛋白紧张的部位似乎只由重的 锁链。将利用新的三磷酸腺苷进一步研究这种二分法 来自其他肌肉和非肌肉的类似物和肌球蛋白 纸巾。一个主要目标将是确定是否存在一个基本的 产生力量的地点的组成和性质的差异 平滑肌肌球蛋白(以此类推，非肌肉肌球蛋白) 肌肉紧张。从总量来看，这一差异并不明显 不同组织肌球蛋白的形态和亚基组成 但这可能反映了在如何调节收缩方面的已知差异 这些肌肉。其他目标将是合成新的ATP类似物 用于亲和柱纯化肌球蛋白，(Ii)作为荧光探针 和(Iii)作为富含电子的探针，以帮助 新近晶化头区的X射线结构解 肌球蛋白。这项研究的长期目标是提供一种分子 解释三磷酸腺苷中的化学能是如何转化为 肌肉收缩的机械能。