Cyanylated cysteine as vibrational probe of binding-induced structural changes in

描述（由申请人提供）：需要新的实验来观察和理解最动态和无序蛋白质的结构。蛋白质的振动光谱可以提供具有非常快的固有时间分辨率的特定信息，这可以导致整个结构分布的快照，而不是时间加权平均值。这里提出了几种策略，以进一步发展的C=N伸缩振动的氰基化的侧链的半胱氨酸作为位点特异性探针，可以实现在动态和无序的蛋白质的任意大小。C=N伸缩振动在水性生物分子红外光谱的透明且不拥挤的区域中吸收，并且它可以通过翻译后修饰在游离半胱氨酸侧链处引入。两个系统，其中动态蛋白质形成结构上未表征的结合结构的目标研究使用单网站氰基化半胱氨酸标签。第一种是副粘病毒Hendra和Nipah，其内在无序的NTAIL蛋白以未表征的几何形状与其P蛋白结合。第二个天然系统是钙调素，一种普遍存在的钙调节蛋白，它结合600多种不同的靶序列，具有不寻常的结合灵活性和通常未知的几何形状。将通过诱变/修饰方案产生这些蛋白质中的每一种的含有单个氰基化半胱氨酸的变体，以映射感兴趣的蛋白质与可能的结合配偶体之间的残基特异性结构形成和接触形成。将使用等温滴定量热法独立评价变体蛋白中结合的热力学。新的实验方法也将被用来放置在一个更可靠的技术基础氰基化半胱氨酸的一般应用。这些实验包括通过拉曼光谱观察C=N伸缩带，将C=N放置在半塌陷、无序但结合的结构内以检查探针对水排斥的依赖性，以及将C=N放置在球状蛋白质的疏水内部。拟议的工作将解决两个特别困难的表征问题，在重要的结合结构，并在同一时间，设置了更广泛的应用振动光谱氰化半胱氨酸在许多其他可能的蛋白质系统的阶段。 公共卫生相关性：缺乏明确结构或具有动态结构和许多可能的生理作用的蛋白质的缺陷和功能障碍通常与系统性人类疾病有关。一种新的实验方案将用于这项工作，揭示原子级的信息结合事件在无序和动态的蛋白质，使用标准的分子生物学技术和分析实验室设备。这种方法的普遍性将使人们有可能更详细地了解与无序和动态蛋白质相关的疾病的分子基础。

DESCRIPTION (provided by applicant): New experiments are needed to view and to understand the structure of the most dynamic and disordered proteins. Vibrational spectra of proteins can provide this specific information with very fast intrinsic time resolution, which can lead to a snapshot of the entire structural distribution rather than a time-weighted average. Several strategies are proposed here to further develop the C=N stretching vibration of the cyanylated side chain of cysteine as a site-specific probe which can be implemented in dynamic and disordered proteins of arbitrary size. The C=N stretching vibration absorbs in a transparent and uncrowded region of the aqueous biomolecular infrared spectrum, and it can be introduced via post-translational modification at free cysteine side chains. Two systems in which dynamic proteins form structurally uncharacterized bound structures are targeted for study using single site cyanylated cysteine labels. The first is the paramyxoviruses Hendra and Nipah, whose intrinsically disordered NTAIL proteins bind to their P proteins in uncharacterized geometries. The second natural system is calmodulin, a ubiquitous calcium regulatory protein which binds to over 600 different target sequences, with an unusual flexibility in binding and often unknown geometries. Single cyanylated-cysteine-containing variants of each of these proteins will be generated via a mutagenesis/modification scheme in order to map both residue-specific structure formation and contact formation between the protein of interest and possible binding partners. Thermodynamics of binding in the variant proteins will be independently evaluated using isothermal titration calorimetry. New experimental approaches will also be used to place the general application of cyanylated cysteine on a more sure technical footing. These experiments include observation of the C=N stretching band by Raman spectroscopy, placement of C=N inside a semi-collapsed, disordered-yet-bound structure to examine the probe's dependence on water exclusion, and placement of C=N inside the hydrophobic interior of a globular protein. The proposed work will solve two particularly difficult characterization problems in important bound structures, and at the same time, set the stage for wider application of vibrational spectroscopy cyanylated cysteine in many other possible protein systems. PUBLIC HEALTH RELEVANCE: Defects and dysfunction in proteins without well-defined structures, or with dynamic structures and many possible physiological roles, are often associated with systemic human diseases. A novel experimental protocol will be used in this work to reveal atom-level information on binding events in disordered and dynamic proteins, using standard molecular biology techniques and analytical laboratory equipment. The generality of this approach will make it possible to understand, in greater detail, the molecular basis of diseases associated with disordered and dynamic proteins.