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: 6869632
• 负责人: Thomas P Burghardt
• 金额: $ 32.45万
• 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/6869632
• 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）：与肌动蛋白相关的运动蛋白肌球蛋白将ATP中的化学自由能以肌动蛋白翻译的形式转化为功，以对抗相反的力。从生化动力学数据推断，肌动球蛋白相互作用由一系列状态组成，其中ATP降解中的每种中间体对应于一种独特的肌动球蛋白构象。代表ATP水解中间体的肌球蛋白球形头或亚片段1 （S1）的晶体结构显示一个由几乎完整的n端催化和c端杠杆臂结构域组成的铰接分子，由于中间肽的局部变形而改变了它们的相对位置。该系统的工作原理是放大催化结构域活性位点的小结构变化，其中ATP被水解成大的杠杆臂结构域运动。通过构建显式模型来阐明活性位点结构变化在杠杆臂运动中的传播和放大机制是本研究的主要目的。该模型涉及能量转导的个体残基参与者，从而可以实际地分析特定残基修饰或突变的影响。在Aim 1中，从策略定位的外部探针和内在的atp敏感色氨酸的光谱检测转导过程中的肌球蛋白构象。在Aim 2中，生化和分子生物学方法探讨了结构表面环在肌动球蛋白相互作用中的作用。目标3是利用能量转导模型，从蛋白质数据库中挖掘晶体学结构，结合目标1和目标2数据的结构含义，构建代表收缩周期中骨骼肌动球蛋白构象的原子结构。该模型同时满足晶体学，光谱学和生物化学/分子生物学的多个独立要求，以达到肌球蛋白转导中间结构的最佳解决方案。此外，随着新数据的出现，该模型将不断得到改进。目标4是实现可验证的可靠例程，用于将肽结构转换为光谱信号，这一过程的准确性对所提出的模型构建至关重要。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。