NEUTRON DIFFRACTION STUDIES OF MUSCLE CONTRACTION

肌肉收缩的中子衍射研究

• 批准号: 3159925
• 负责人: ROBERT A MENDELSON
• 金额: $ 28.34万
• 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/3159925
• 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; 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 包括理解ATP水解是如何与大的- 规模大分子的变化，负责力的产生， 这些变化是如何被调节的。 这项工作有一个基本的 科学目标，将产生知识， 对心脏和肌肉相关疾病的影响。 我们提出利用中子溶液散射和中子螺旋 衍射研究特定的结构问题至关重要 对肌肉工作原理的理解。 实验将 利用一个或有时两个的选择性氘化， 研究中的大分子复合物的成员。 通过改变 缓冲液D2 O含量、质子化缓冲液的对比度匹配 (H)或氘代（D）蛋白。 这将允许 原位检测不匹配蛋白（H或D）， 很少或没有来自其它（"不可见"）蛋白质的干扰。 氘代蛋白质将由细胞生物学产生， 重组DNA技术。 我们将研究以下问题：（1）在 肌球蛋白调节轻链（RLC）的原位结构， 肌球蛋白头要么在溶液中游离，要么与肌动蛋白结合， 没有Ca2+？ 什么是RLC的分离时，头是 绑定到actin？ (2)肌钙蛋白C的原位结构是什么 (TNC)有和没有结合的Ca2+和什么是交叉螺旋 径向分离的TNC与和没有绑定的Ca 2+？ 跨国公司是- 肌球蛋白与肌丝结合影响TNC分离？ (3)如果有结构上的变化， 当肌球蛋白头结合肌动蛋白在ATP的情况下丝？ (4)在放松、僵硬、 假定的弱束缚态和类比诱导态 被认为类似于产生肌动球蛋白中间体的力？ (5)肌球蛋白的位置和肌球蛋白内分离的变化是什么？ 当肌肉僵硬时会产生轻链吗 (6)是什么 的轻链承载部分的平均径向位置 在肌肉等长收缩期间肌肉中的肌球蛋白头 纤维 14.3 mn的单色光反射的强度 （仅由轻链产生）在收缩期间增加， 它在X射线衍射中的作用 (7)什么是阶段的 僵直脊椎动物赤道中子衍射图 肌肉？ 僵直肌纤维的低分辨率结构 能用中子衍射确定吗 这些问题的答案将测试假设如何 发生肌肉收缩和调节。