CONFORMATIONS OF LIGHT-SENSITIVE PROTEINS

光敏蛋白的构象

• 批准号: 8168623
• 负责人: Tobin R Sosnick
• 金额: $ 0.54万
• 依托单位: ILLINOIS INSTITUTE OF TECHNOLOGY
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-01-01 至 2010-12-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8168623
• 关键词: Charge; Computer Retrieval of Information on Scientific Projects Database; Coupled; DNA Binding; Detection; Dimensions; Engineering; Face; Funding; Grant; Institution; Light; Link; Manuscripts; Measurement; Measures; Mediating; Methods; Modeling; Molecular Conformation; Optics; Preparation; Protein Region; Proteins; Radial; Reaction; Reporting; Research; Research Personnel; Resources; Shapes; Signal Transduction; Source; Structure; Ubiquitin; United States National Institutes of Health; biological systems; design; detector; protein folding; tool

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Natural protein photosensors are promising tools for engineering optical control into biological systems. We are just beginning to understand how these genetically encoded light detectors can be easily coupled to arbitrary effectors. We have reported a designed LOV2 photoswitch that selectively binds DNA when illuminated. We demonstrated that signal transduction between the two domains is mediated by the local unfolding of a shared, linking helix. SAXS measurements on the protein in the light and dark yield unexpected results. The overall shape barely changes even though the shared helix unfolds in the light. Modeling mandates that the domains associate due to weak interactions on the hydrophobic face in both the light and dark states. Manuscript is in preparation. Although most folding intermediates escape detection, their characterization is crucial to the elucidation of folding mechanisms. Here we outline a powerful strategy to populate partially unfolded intermediates: A buried aliphatic residue is substituted with a charged residue (e.g., Leu¿Glu-) to destabilize and unfold a specific region of the protein. We apply this strategy to Ubiquitin (Ub), reversibly trapping a folding intermediate in which the ¿5 strand is unfolded (N-¿5). The intermediate refolds to a native-like structure upon charge neutralization under mildly acidic conditions. We compare the global dimensions of wild-type Ub to the intermediate using SAXS. At pH 7.8, the measured radius of gyration (Rg) is 13.2 ¿ 0.2 and 13.8 ¿ 0.1 ¿ for the two proteins, respectively. This difference matches the 0.5 ¿ difference between Rg's of the native state and the model for the N-¿5 intermediate. This general strategy may be combined with other methods and have broad applications in the study of protein folding and other reactions that require trapping of high-energy states.

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目和 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 天然蛋白质光传感器是将光学控制工程化到生物系统中的有前途的工具。我们才刚刚开始了解这些基因编码的光探测器是如何容易地与任意效应器耦合的。我们已经报道了一种设计的LOV 2光开关，它在光照下选择性地结合DNA。我们证明了两个结构域之间的信号转导是由共享的连接螺旋的局部展开介导的。SAXS在光和暗下对蛋白质的测量产生了意想不到的结果。即使共享的螺旋在光线下展开，整体形状也几乎没有变化。建模任务，域相关联，由于弱相互作用的疏水面在光明和黑暗的状态。曼彻斯特正在筹备中。 虽然大多数折叠中间体逃脱检测，其特性是至关重要的折叠机制的阐明。在这里，我们概述了一个强大的策略，以填充部分未折叠的中间体：一个掩埋的脂肪族残基被取代的带电残基（例如，Leu <$Glu-）使蛋白质的特定区域不稳定并展开。我们将这种策略应用于泛素（Ub），可逆地捕获折叠中间体，其中<$5链未折叠（N-<$5）。在弱酸性条件下电荷中和后，中间体重新折叠成天然样结构。我们使用SAXS比较了野生型Ub与中间体的全局尺寸。在pH 7.8时，两种蛋白质的回转半径（Rg）分别为13.2。 该差异与天然状态的Rg和N-差异相匹配。这种一般策略可以与其他方法相结合，并在研究蛋白质折叠和其他需要捕获高能态的反应中具有广泛的应用。