Structural basis of allostery and mechanical properties of F-actin

 描述(申请人提供)：我们将采用多学科的实验方法，包括分子细胞生物学、生物化学、生物物理方法、计算分析和高分辨率冷冻电子显微镜(Cryo-EM)来提供不同功能状态下的肌动蛋白细丝(F-肌动蛋白)的结构。在我们3-5？的目标分辨率下，α-螺旋和β-链将被分解，大的侧链将可见，从而允许准确地放置多肽主干和许多侧链。低温电子断层摄影术将通过提供不使用螺旋对称性的地图来补充这些努力，从而允许可视化细丝中的原型可变性。我们将充分利用低温电磁领域的最新技术进步，特别是直接探测器成像设备、相位板技术、基于最大似然法的数据处理和强大的数据收集设备(Titan Krios)。因此，通过直接测定F-肌动蛋白前体在肌丝内以及与肌动蛋白结合蛋白(ABPs)的结构和界面，以及通过检测肌动蛋白细丝上的原型构象的变化，我们将解决F-肌动蛋白功能的三个关键方面：(I)解释ABP与F-肌动蛋白结合亲和力的核苷酸依赖性；(Ii)解释ABP与F-肌动蛋白结合的协同性；以及(Iii)确定F-肌动蛋白僵硬调节的结构基础。

 DESCRIPTION (provided by applicant): We will employ a multidisciplinary experimental approach that includes molecular cell biology, biochemistry, biophysical approaches, computational analysis, and high-resolution cryo electron microscopy (cryo-EM) to provide structures of actin filaments (F-actin) in different functional states. At our target resolution of3-5Å, α- helices and β-strands will be resolved and large side chains will be visible allowing accurate placement of the polypeptide backbone and of many side chains. Cryo electron tomography will supplement these efforts by providing maps without the application of helical symmetry allowing visualization of protomer variability in the filament. We will take full advantage of recent technical advances in the cryo-EM field, specifically direct detector imaging devices, phase plate technology, maximum likelihood based data processing, and robust data collection equipment (Titan Krios). Thus, by direct determination of the structures and interfaces of F-actin protomers within the filament and with actin-binding proteins (ABPs) and by examining the variability of protomer conformations along actin filaments, we will address three key aspects of F-actin function: (i) to explain the nucleotide-dependence of ABP binding affinity to F-actin; (ii) to explain the cooperativity of ABP binding in F-actin; and (iii) to define the structural basis of F-actin stiffness regulation.