Examining G-quadruplex metal site heterogeneity and the influence of peptide binding using 2D IR spectroscopy

项目总结 G-四链体(G4S)是四链核酸基序，与一系列不同的 生物功能和疾病，并已成为药物设计的有吸引力的目标。协调的关系 碱金属离子(例如，K、Na)到鸟嘌呤C6=O羰基是它们组装和稳定的关键因素， 因此，可能会影响与识别和重塑G4S的蛋白质的相互作用，以执行调节 功能。尽管数以千计的G4已经在结构上得到了表征，人们对它的理解也很广泛 关于它们的体外折叠/去折叠行为，总体上缺乏关于它们与 蛋白质导致在理解金属结合和蛋白质相互作用是否/如何耦合方面存在重大差距。 超快二维红外光谱是研究局部静电、振动的一种强有力的探针。 耦合和飞秒-皮秒波动。与现场特定标签一起，它已被应用于 许多蛋白质发现结合相互作用中的细微变化，这些变化往往是高分辨率所隐藏的 技巧。我们最近发展了用于G4S结构分析的2D IR方法，并证明了同位素 编辑可以检测金属站点占有率、可塑性和动力学方面的变化。结合起来，2D IR提供了 探索G4S中金属配位、折叠/去折叠和蛋白质结合之间相互作用的有用方法。 这项提案在两个具体目标中对既定方法进行了扩展。在第一个目标中，我们使用2D IR和 同位素编辑以测试自然状态动力学和C6=O键频率与 G4的全局稳定性、顺序组装和拆卸机制以及原生态金属交换 费率。在第二个目标中，我们研究了G4S与来自以下蛋白质的多肽的相互作用 稳定或解除G4S，并测试多肽(蛋白质)结合偏向金属稳定的G4核心的假设 根据功能在展开或折叠的方向上。如果这项研究成功，它将为 深入了解G4S的物理化学性质，并可能为如何靶向G4：蛋白质复合体提供线索 使用药物和成像探头。该项目还为毕业生提供了一个跨学科的培训环境 以及本科生，这将为他们未来的研究生涯做好准备。

PROJECT SUMMARY G-quadruplexes (G4s) are four-stranded nucleic acid motifs that have been implicated in a diverse array of biological functions and diseases and have emerged as attractive targets for drug design. The coordination of alkali metal ions (e.g., K+, Na+) to guanine C6=O carbonyls is a critical factor in their assembly and stabilization, and thus, is likely to influence interactions with proteins that recognize and remodel G4s to perform regulatory functions. Although thousands of G4s have been structurally characterized and there is extensive understanding of their in vitro folding/unfolding behaviors, the overall lack of molecular-level data about their interactions with proteins results in a major gap in understanding if/how metal binding and protein interactions are coupled. Ultrafast two-dimensional infrared (2D IR) spectroscopy is a powerful probe of local electrostatics, vibrational coupling, and femtosecond-picosecond fluctuations. Together with site specific labeling, it has been applied to many proteins to discover subtle variations in binding interactions that are often hidden from high-resolution techniques. We have recently developed 2D IR methods for structural analysis of G4s and showed that isotope editing can detect variations in metal site occupancy, plasticity, and dynamics. Combined, 2D IR provides a useful method to probe the interplay between metal coordination, folding/unfolding, and protein binding in G4s. This proposal expands on established methods in two Specific Aims. In the first Aim, we use 2D IR and isotope editing to test the hypothesis that native state dynamics and C6=O bond frequencies correlate with the global stability of a G4, sequential assembly and disassembly mechanisms, and native state metal exchange rates. In the second Aim, we examine the interactions of G4s with peptides derived from proteins that either stabilize or unwind G4s and test the hypothesis that peptide (protein) binding biases the metal stabilized G4 core in the direction of unfolding or folding depending on function. If this study is successful, it will provide fundamental insight into the physicochemical properties of G4s and may provide clues for how to target G4:protein complexes with drugs and imaging probes. The project also provides an interdisciplinary training environment for graduate and undergraduate students that will prepare them for future careers in research.