INTERACTION OF ATP ANALOGUES WITH MYOSIN

ATP 类似物与肌球蛋白的相互作用

• 批准号: 3482888
• 负责人: RALPH G YOUNT
• 金额: $ 26.78万
• 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 1994-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3482888
• 关键词: X ray crystallography; adenosine triphosphate; affinity chromatography; aminoacid analyzer; chemical group; chemical synthesis; chickens; cow; crosslink; electron microscopy; fluorescent dye /probe; gel electrophoresis; gel filtration chromatography; high performance liquid chromatography; laboratory rabbit; molecular site; muscle contraction; myocardium; myosins; nucleotide analog; photolysis; protein structure; 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.

目标是尽可能完整地描述化学物质 肌肉中力的性质。 特别是新的光性 ATP的类似物将被合成并用于标记共价活动 来自海峡（骨骼和心脏）的肌球蛋白的位点残留 未征收的（胃和动脉）肌球蛋白。 亚基的位置和 标记肽的氨基酸组成将通过使用 高性能液相色谱和气相测序 技术。 每种情况中使用的ATP类似物将被困在 通过化学交联两个动力学反应性的活性位点特征 硫醇，SH1和SH2，或通过形成ADP稳定的过渡状态复合物 与钒酸盐离子的类似物。 这两种方法中的任何一种允许 光亲和力模拟肌球蛋白复合物将不纯化 光标记，并大大提高了特异性和程度 标签。 初步结果表明 平滑肌肌球蛋白（胃）的活性部位都是由两者的元素形成的 基本的轻链和重链。 相反，活动 海峡肌肉肌球蛋白的部位似乎仅由沉重组成 链。 该二分法将通过新的ATP进行进一步研究 来自其他平滑肌肉和非肌肉的类似物和肌球蛋白 组织。 一个主要目标是确定是否有基本 力的组成和性质的差异 平滑肌肌球蛋白（和类比，非肌肉肌球蛋白） 海峡肌肉。 从总体上看不明显这种差异 来自不同组织的肌醇的形态和亚基组成 但是可能反映了在收缩的调节方式中的已知差异 这些肌肉。 其他目标将是综合新的ATP类似物 在亲和力柱中使用（i）净化肌球蛋白，（ii）作为荧光探针 肌球蛋白的头部和（iii）作为富含电子的探针，以帮助 最近结晶的头部区域的X射线结构解决方案 肌球蛋白。 这项研究的长期目标是提供分子 解释ATP中的化学能如何转化为 肌肉收缩的机械能。