NEUTRON DIFFRACTION STUDIES OF MUSCLE CONTRACTION

肌肉收缩的中子衍射研究

• 批准号: 3159926
• 负责人: ROBERT A MENDELSON
• 金额: $ 27.3万
• 依托单位: UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-07-01 至 1994-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3159926
• 关键词: X ray crystallography; actins; adenosine triphosphate; calcium; cytoskeletal proteins; deuterium; genetic manipulation; light scattering; muscle contraction; myosins; neutron diffraction; neutron radiation; protein structure; recombinant DNA; troponin

The long-term objective of the proposed work is to understand the molecular basis of the mechanism of muscle contraction. This will involve understanding how ATP hydrolysis is coupled to the large- scale macro-molecular changes responsible for force generation and how the changes are regulated. This work, which has a basic science objective, will generate knowledge which may have implications for cardiac and muscle-related disease. We proposed using neutron solution scattering and neutron helical diffraction to investigate specific structural questions crucial to the understanding of how muscle works. The experiments will utilize the selective deuteration of one, or sometimes two, of the members of the macromolecular complex under study. By varying buffer D2O content, contrast-matching of the buffer with protonated (H) or deuterated (D) protein will be achieved. This will allow examinations of unmatched protein (either H or D) in situ with little or no interference from the other ("invisible") proteins. Deuterated proteins will be generated by cell biology and recombinant DNA techniques. We will investigate the following questions: (1) What is the in situ structure of regulatory light chains (RLC's) of myosin when the myosin heads are either free in solution or bound to actin with and without Ca2+? What is the separation of RLC's when heads ar bound to actin? (2) What is the in situ structure of troponin-C (TNC) with and without bound Ca2+ and what is the cross-helix radial separation of TNC with and without bound Ca2+? Is the TNC- TNC separation affected by myosin binding to the thin filament? (3) What are the structural changes, if any, that occur in the thin filament when myosin heads bind to actin in the absence of ATP? (4) What is the position and separation of RLC in relaxed, rigor, the putative weak-binding state and in an analog-induced state thought to resemble a force generating acto-myosin intermediate? (5) What is the change in position and intra-myosin separation of light chains when muscles are stretched in rigor? (6) What is the average radial position of the light chain-bearing portion of the myosin head in a muscle during isometric contraction of muscle fibers? Does the intensity of the 14.3 mn meridional reflection (arising from light chains alone) increase during contraction as it does in X-ray diffraction? (7) What are the phases of the equatorial neutron diffraction pattern from rigor vertebrate muscle? Can the low-resolution structure of rigor muscle fibers be determined from neutron diffraction? The answers to these questions will test hypotheses about how muscle contraction and regulation occur.

拟议工作的长期目标是了解 肌肉收缩机制的分子基础。这将是 包括了解ATP水解酶是如何与大的- 对力的产生和产生负责的尺度大分子变化 这些变化是如何受到监管的。这部作品，它有一个基本的 科学目标，将产生知识，可能有 对心脏和肌肉相关疾病的影响。 我们提出了用中子溶液散射和中子螺旋线 用衍射法研究特定的结构问题至关重要 了解肌肉是如何工作的。这些实验将会 利用一种，有时是两种有选择地去氢化 正在研究的大分子复合体的成员。通过改变 缓冲液D2O含量，质子化缓冲液的对比度匹配 (H)或氚(D)蛋白将被实现。这将允许 不匹配蛋白质(H或D)的原位检测 很少或根本没有其他(看不见的)蛋白质的干扰。 氚蛋白将由细胞生物学和 重组DNA技术。 我们将调查以下问题：(1)什么是最重要的 肌球蛋白调节轻链(RLC)的原位结构 肌球蛋白头在溶液中是游离的，或者与肌动蛋白结合。 如果没有钙离子呢？当头部出现时，RLC的间隔是什么？ 结合肌动蛋白？(2)肌钙蛋白-C的原位结构是什么 (TNC)结合和不结合钙离子，以及什么是交叉螺旋 径向分离有无结合钙离子的TNC？是全国过渡委员会-- 肌球蛋白与细丝结合对TNC分离的影响？ (3)如果有的话，公司内部会发生哪些结构性变化 肌球蛋白头部在没有三磷酸腺苷的情况下与肌动蛋白结合时的细丝？ (4)RLC在宽松、严谨、 假定的弱结合态和处于模拟诱导态 被认为类似于一种产生肌动蛋白中间体的力量？ (5)肌球蛋白在肌球蛋白内的分离和位置有何变化 肌肉僵硬拉伸时的轻链？(6)什么是 的轻链支承部分的平均径向位置 肌肉等长收缩时肌肉中的肌球蛋白头 纤维？14.3mN经向反射的强度 (仅由轻链引起)在收缩过程中增加为 在X射线衍射法中是这样吗？(7)晶相是什么？ 脊椎动物赤道中子衍射图 肌肉？僵直肌肉纤维的低分辨率结构 是从中子衍射中确定的吗？ 这些问题的答案将检验关于如何 肌肉收缩和调节就会发生。