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

项目摘要 G-四链体（G4）是四链核酸基序，其涉及多种不同的生物学功能。 生物学功能和疾病，并已成为药物设计的有吸引力的目标。协调 碱金属离子（例如，K+，Na+）转化为鸟嘌呤C6=O羰基是它们组装和稳定的关键因素， 因此，可能会影响与蛋白质的相互作用，这些蛋白质识别并重塑G4，以进行调节 功能协调发展的尽管已经对数千个G4进行了结构表征， 它们的体外折叠/解折叠行为，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（2D IR）光谱是局部静电、振动和辐射的强有力的探测器。 耦合和飞秒-皮秒波动。与特定地点标签一起，它已被应用于 许多蛋白质，以发现往往隐藏在高分辨率的结合相互作用的微妙变化 技术.我们最近开发了用于G4 s结构分析的2D IR方法，并表明同位素 编辑可以检测金属位置占用、可塑性和动力学的变化。结合2D IR， 有用的方法来探测金属配位，折叠/展开，和蛋白质结合在G4之间的相互作用。 该提案扩展了两个具体目标中的既定方法。在第一个目标中，我们使用2D IR， 同位素编辑，以测试天然状态动力学和C6=O键频率与 G4的全局稳定性、顺序组装和拆卸机制以及自然态金属交换 rates.在第二个目标中，我们研究了G4与来自蛋白质的肽的相互作用， 稳定或解旋G4，并检验肽（蛋白质）结合偏向金属稳定的G4核心的假设 根据功能，沿展开或折叠的方向。如果这项研究取得成功，它将提供基本的 深入了解G4的物理化学性质，并可能为如何靶向G4提供线索：蛋白质复合物 药物和成像探针。该项目还为研究生提供了一个跨学科的培训环境 和本科生，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.