CONFORMATIONS OF LIGHT-SENSITIVE PROTEINS

这个子项目是许多研究子项目中利用 资源由NIH/NCRR资助的中心拨款提供。子项目和 调查员(PI)可能从NIH的另一个来源获得了主要资金， 并因此可以在其他清晰的条目中表示。列出的机构是 该中心不一定是调查人员的机构。 天然蛋白质光传感器是在生物系统中进行光学控制的很有前途的工具。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LOV2光电开关，在照明时可以选择性地结合DNA。我们证明了这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所介导的。在光和暗条件下对该蛋白质进行的SAXS测量得到了意想不到的结果。即使共享的螺旋在光线中展开，整体形状也几乎没有变化。建模要求，由于疏水表面上的弱相互作用，在明暗两种状态下，结构域相互关联。手稿正在准备中。 尽管大多数折叠中间体都能逃过检测，但它们的特性对于阐明折叠机制至关重要。在这里，我们概述了一种强大的策略来填充部分展开的中间体：用带电残基(例如，Leu？Glu-)取代埋藏的脂肪残基，以破坏蛋白质的特定区域并展开。我们将这一策略应用于泛素(Ub)，可逆地捕获其中5链展开的折叠中间体(N-5)。在温和的酸性条件下，当电荷中和时，中间体重新折叠成天然的结构。我们使用SAXS比较了野生型Ub和中间型Ub的全局维度。在pH 7.8时，测得两种蛋白质的回转半径分别为13.2°0.2和13.8°0.1。这一差异与原生态的Rg和N-5中间体的模型之间的0.5？差异相匹配。这一通用策略可以与其他方法相结合，并在蛋白质折叠和其他需要捕获高能态的反应的研究中有广泛的应用。

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.