MADMAX: Precise Measurement of Conformational Changes in Proteins

描述(由申请人提供)：该项目涉及开发一种新的方法，即使用中角X射线溶液散射的多波长异常衍射(MADMAX)，用于精确测量蛋白质和其他水溶液中的大分子的原子间距离。它将使现有技术无法研究的结构变化和分子内运动的表征成为可能。蛋白质是动态的分子，其活动对所有生物过程都有贡献。几乎所有的蛋白质功能都需要分子内运动，无论是施加机械力、化学转化还是分子移位。MadMax将有可能对溶液中蛋白质的这些运动进行精确的原子水平测量。MADMAX应该普遍适用于溶液中的大分子，并能够在包括变构相互作用、蛋白质-配体相互作用、通道门控、结构域移动和蛋白质折叠在内的广泛现象中阐明蛋白质的作用。它应该能够测量原子间矢量长度的变化，精度高达~1°。它可以被改装成时间分辨率的研究--至少达到毫秒级的分辨率。该项目的目标是：(I)展示对具有良好特性的蛋白质在溶液散射中的异常差异的准确测量。(Ii)从原子坐标集预测异常差异。(3)确定该方法的适用范围：该方法适用于多大的蛋白质？检测需要多少个标签？标签位置的混乱程度可以容忍多少？(4)展示使用MadMax精确测量由于配体结合而引起的结构变化。(V)演示利用MadMax研究膜蛋白的结构变化。(Vi)演示使用时间分辨的MadMax来研究蛋白质构象变化。充分利用细菌表达系统和蛋白质化学合成的能力引入标记，可能进行的实验范围几乎是无限的。其全方位能力的发展将使MadMax成为观察水溶液中蛋白质结构和功能的一种重要的新方法。这个项目将导致开发一种新的工具，用于精确测量蛋白质内部的原子间距离和它们在作用过程中经历的运动。先进疗法的发展依赖于对这些分子作用的深入理解，因为它们构成了几乎所有生物功能的基础。将这一方法应用于涉及发病机制的分子系统，将对制定诊断和治疗广泛的人类疾病的策略做出重大贡献。

DESCRIPTION (provided by applicant): This project involves development of a new method, Multi-wavelength Anomalous Diffraction using Medium Angle X-ray solution scattering (MADMAX), for the precise measurement of interatomic distances within proteins and other macromolecules in aqueous solution. It will make possible the characterization of structural changes and intra-molecular movements that cannot be studied by existing techniques. Proteins are dynamic molecules whose activities contribute to all biological processes. Virtually all protein function requires intra- molecular movement, whether for the application of mechanical force, chemical transformation or molecular translocation. MADMAX will make possible accurate atomic-level measurement of these movements within proteins in solution. MADMAX should be generally applicable to macromolecules in solution and capable of elucidating protein action during a broad range of phenomena including allosteric interactions; protein-ligand interactions; channel gating; domain movements; and protein folding. It should be capable of measuring changes in the length of interatomic vectors with an accuracy of up to ~1 ¿. It can be adapted for time- resolved studies - to at least millisecond resolution. The goals of this project are: (i) Demonstrate the accurate measurement of anomalous differences in solution scattering from well characterized proteins. (ii) Predict the anomalous differences from atomic coordinate sets. (iii) Determine the practical range of the method: How large a protein is this method applicable to? How many labels are required for detection? How much disorder in the label position can be tolerated? (iv) Demonstrate the use of MADMAX for precise measurement of structural changes due to ligand binding. (v) Demonstrate the use of MADMAX for the study of structural changes in membrane proteins. (vi) Demonstrate the use of time-resolved MADMAX for study of protein conformational changes. Taking full advantage of the capabilities of bacterial expression systems and chemical synthesis of proteins for the introduction of labels, the range of experiments that will be possible is almost limitless. Development of its full range of capabilities will establish MADMAX as an important new approach to the observation of protein structure and function in aqueous solution. This project will result in the development of a novel tool for the precise measurement of interatomic distances within proteins and the motions that they undergo during action. Development of advanced therapies depends on a deep understanding of these molecular actions because they form the basis of virtually all biological functions. Application of this approach to molecular systems involved in pathogenesis will contribute significantly to the development of strategies for the diagnosis and treatment of a broad range of human diseases.