NSF/MCB-BSF: Dynamic protein autoinhibition as a mechanism for rapid DNA recognition

在这个NSF-BSF项目中，研究小组将寻求一种分子机制，使蛋白质在复杂的细胞样条件下有效地找到它们的结合靶标。为了使蛋白质发挥其生物学功能，蛋白质必须找到目标分子并与之结合。与目标分子相互作用的蛋白质结合表面（功能域）也可以与同一蛋白质的其他部分相互作用，从而阻止蛋白质发挥其功能。这个过程被称为“自身抑制”，已经在各种蛋白质中发现，包括基因调节蛋白和酶。目前的项目将阐明自抑制如何影响蛋白质在众多相似分子中寻找目标的效率。研究小组测试了一个假设，即自抑制可以降低蛋白质被困在非目标上的风险，从而加速与目标的结合。这个国际项目由美国国家科学基金会和英国科学基金会共同支持，涉及到对高中生的教育推广，向学生展示跨学科合作的重要性和全球科学的普遍性。该项目可以为各种生物技术目的提供一种创新的方法来设计蛋白质的分子特性。这个项目是理论驱动的，以协同的方式整合了实验和计算生物物理学的方法，以阐明自抑制的基本原理。研究小组将研究dna结合蛋白在大量无功能高亲和力配体（称为诱饵）存在的情况下，在其靶标搜索过程中的自抑制作用。在寻找过程中，dna结合蛋白会遇到许多诱饵，这些诱饵会严重阻碍与靶标的结合。研究小组将研究动态自抑制作为减少诱饵不利影响的潜在机制。经过动态构象平衡的自抑制将在自然和人工系统中进行研究，其中抑制片段和配体（诱饵或靶标）竞争dna结合域的带正电的分子表面。研究团队将追求两个具体目标：1)描述分子内与分子间相互作用的竞争；2)阐明动态自抑制在目标搜索动力学中的作用。该研究将利用核磁共振（NMR）方法，停止流动荧光方法和计算方法。该项目预计将描述是什么决定了自抑制和非抑制状态之间的平衡，以及平衡如何影响目标搜索动力学。该项目还将探索动态自抑制在蛋白质工程中的应用，以改善dna结合蛋白的动力学特性。本项目由生物科学理事会分子和细胞生物科学部分子生物物理学和遗传机制组支持。该奖项反映了美国国家科学基金会的法定使命，并通过使用基金会的知识价值和更广泛的影响审查标准进行评估，被认为值得支持。

In this NSF-BSF project, the research team will seek a molecular mechanism whereby proteins efficiently find their binding target in complex, cell-like conditions. For a protein to perform its biological function, the protein must find and bind to a target molecule. The binding surface on the protein (the functional domain), that interacts with the target molecule, can also interact with other parts of that same protein, preventing the protein from performing its function. This process is called ‘autoinhibition’ has been found in various proteins, including gene-regulatory proteins and enzymes. The current project will elucidate how autoinhibition impacts the efficiency of proteins in their search their targets among numerous similar molecules. The research team tests a hypothesis that autoinhibition can reduce the risk for the proteins to get trapped at nontargets and thereby accelerate the binding to the targets. This international project, supported by both NSF and BSF, involves educational outreach for high school students that demonstrates for students the importance of interdisciplinary collaboration and the universal nature of science across the globe. The project can provide an innovative way to engineer the molecular properties of proteins for various biotechnological purposes. This project is theory-driven and integrates approaches of experimental and computational biophysics in a synergistic fashion to shed light on the fundamental principles of autoinhibition. The research team will study the role of autoinhibition of DNA-binding proteins in their target search process in the presence of numerous nonfunctional high-affinity ligands, which are referred to as decoys. During the search process, DNA-binding proteins encounter with numerous decoys, which can severely impede association with targets. The research team will examine the dynamic autoinhibition as a potential mechanism to diminish this adverse impact of decoys. The autoinhibition that undergoes dynamic conformational equilibrium will be studied for natural and artificial systems where an inhibitory segment and ligands (decoy or target) compete for the positively charged molecular surface of a DNA-binding domain. The research team will pursue two specific objectives: 1) to delineate competition of intra- vs. inter-molecular interactions; and 2) to elucidate the role of dynamic autoinhibition in target search kinetics. The research will utilize nuclear magnetic resonance (NMR) methods, stopped-flow fluorescence methods, and computational approaches. The project is expected to delineate what dictate the balance between the autoinhibited and uninhibited states and how the balance influences the target search kinetics. The project will also explore applications of the dynamic autoinhibition to protein engineering for improving the kinetic properties of DNA-binding proteins.This project is supported by the Molecular Biophysics and Genetic Mechanism Clusters of the Division of Molecular and Cellular Biosciences in Biological Sciences Directorate.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.