Visualizing Actomyosin Transients by Data Merging

通过数据合并可视化肌动球蛋白瞬变

• 批准号: 7076931
• 负责人: Thomas P Burghardt
• 金额: $ 31.69万
• 依托单位: MAYO CLINIC ROCHESTER
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2009-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7076931
• 关键词: actins; adenosine triphosphate; biological signal transduction; circular dichroism; conformation; fluorescence polarization; gene mutation; hydrolysis; laboratory rabbit; muscle contraction; myocardium; myosins; physical model; protein protein interaction; protein purification; protein structure function; recombinant virus; site directed mutagenesis; spectrometry; striated muscles; tryptophan

DESCRIPTION (provided by applicant): The motor protein myosin in association with actin transduces chemical free energy in ATP into work in the form of actin translation against an opposing force. Inferences from biochemical kinetic data suggest the actomyosin interaction consists of a series of states for which each intermediate in the degradation of ATP corresponds to a unique actomyosin conformation. Crystal structures of the myosin globular head or subfragment 1 (S1) representing the ATP hydrolysis intermediates show an articulated molecule made from nearly intact N-terminal catalytic and C-terminal lever-arm domains that change their relative position due to localized deformations in the intervening peptide. The system works by amplifying small structure changes in the catalytic domain active site where ATP is hydrolyzed into the large lever-arm domain movement. Elucidating the mechanism for propagation and amplification of the active site structure changes into the lever-arm movement is the principal aim of the proposal to be accomplished by construction of an explicit model. The model involves individual residue participants in energy transduction such that the effect of a specific residue modification or mutation may be realistically analyzed. In Aim 1, spectroscopy from strategically located extrinsic probes and an intrinsic ATP-sensitive tryptophan detect myosin conformation during transduction. In Aim 2, biochemical and molecular biological approaches probe the role of a structured surface loop in the actomyosin interaction. Aim 3 is to build the atomic structures representing skeletal actomyosin conformation during the contraction cycle from crystallographic structures mined from the protein database combined with structural implications from Aims 1 and 2 data using the energy transduction model. The model satisfies simultaneously, multiple independent requirements from crystallography, spectroscopy, and biochemistry/molecular-biology to arrive at a best solution for the myosin transduction intermediate structure. Additionally, the model is subject to cumulative improvement as new data becomes available. Aim 4 is to implement verifiably reliable routines for translating peptide structure to a spectroscopic signal, a process whose accuracy is critical to the proposed model building. The structure to spectrum translation routines have broader implications for structure sensing optical spectroscopy in biophysics.

描述（由申请人提供）：与肌动蛋白结合的马达蛋白肌球蛋白将ATP中的化学自由能转化为肌动蛋白翻译形式的功，以对抗相反的力。从生化动力学数据的推断表明，肌动球蛋白的相互作用由一系列的状态，其中每个中间体在ATP的降解对应于一个独特的肌动球蛋白构象。代表ATP水解中间体的肌球蛋白球状头部或亚片段1（S1）的晶体结构显示出由几乎完整的N-末端催化和C-末端β-臂结构域组成的关节式分子，由于介入肽的局部变形，这些结构域改变了它们的相对位置。该系统通过放大催化结构域活性位点中的小结构变化来工作，在催化结构域活性位点中，ATP被水解成大的臂结构域运动。阐明传播和放大的机制的活动场地结构的变化，以手臂运动的主要目的是要完成的建议，通过构建一个明确的模型。该模型涉及能量转导中的单个残基参与者，使得可以现实地分析特定残基修饰或突变的影响。在目的1中，光谱从战略定位的外源性探针和内在的ATP敏感的色氨酸检测肌球蛋白的构象在转导过程中。在目标2中，生物化学和分子生物学方法探测肌动球蛋白相互作用中结构化表面环的作用。目的3是建立代表骨骼肌球蛋白构象的原子结构，在收缩周期中，从蛋白质数据库中挖掘的晶体结构，结合使用能量转导模型的目的1和2数据的结构含义。该模型同时满足多个独立的要求，从晶体学，光谱学，生物化学/分子生物学，以达到最佳的解决方案的肌球蛋白转导中间结构。此外，随着新数据的出现，该模型会不断得到改进。目的4是实现可验证的可靠的例程翻译肽结构的光谱信号，其准确性是关键的建议模型的建立过程。结构到光谱的转换程序对生物物理学中的结构传感光谱学具有更广泛的意义。