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中的化学自由能转化为肌动蛋白翻译的形式，以反对性力。来自生化动力学数据的推论表明，肌动球蛋白相互作用由一系列状态组成，在ATP的降解中，每个状态中的每个状态都对应于独特的Actomyosin构象。代表ATP水解中间体的肌球蛋白球头或亚纹理1（S1）的晶体结构显示，由几乎完整的N末端催化和C端杠杆臂制成的明确分子，该分子在间间肽中由于局部变形而改变其相对位置。该系统通过扩增催化域活动位点的小结构变化，在该结构中，ATP被水解为大杠杆臂运动。阐明积极位点结构在杠杆臂运动中变化的传播和扩增的机制是该提案的主要目的是通过构建显式模型来实现。该模型涉及单个残基参与者进行能量转导的参与者，因此可以实际分析特定残基修饰或突变的效果。在AIM 1中，从策略性位置的外部探针和固有的ATP敏感色氨酸检测到转导期间的肌球蛋白构象。在AIM 2中，生化和分子生物学方法探测结构化表面环在肌动蛋白相互作用中的作用。 AIM 3是在收缩周期中构建代表骨骼肌肌球蛋白构象的原子结构，从蛋白质数据库挖掘的晶体学结构与AIM 1和2数据的结构含义相结合，使用能量传输模型。该模型同时满足了晶体学，光谱和生物化学/分子生物学的多个独立要求，可以为肌球蛋白转导中间结构提供最佳解决方案。此外，随着新数据的可用，该模型将要进行累积改进。 AIM 4是实施可靠的可靠例程，以将肽结构转换为光谱信号，该过程的准确性对于拟议的模型构建至关重要。频谱翻译程序的结构对生物物理学中的结构传感光谱具有更广泛的影响。