Protein-Protein Interactions in Natural Product Biosynthesis

项目摘要。 该计划研究蛋白质·蛋白质相互作用在载体蛋白依赖性生物合成中的作用， 包括脂肪酸合成酶、聚酮化合物合成酶和非核糖体肽合成酶途径。的 与这些途径相关的天然产物可用作治疗剂，包括抗生素、抗真菌剂和 抗癌剂。指导这些途径的生物合成以生产未经试验的 生物活性化合物对于新的和改进的疗法是至关重要的。在过去的几年里，我们证明了 载体蛋白和伴侣蛋白结构域之间的蛋白质相互作用如何指导反应性， 引导持续性。此外，我们开发了一套化学生物学工具来稳定和询问这些 通过创新的交联剂开发和结构生物学，以原子分辨率进行相互作用。我们现在 我建议用新的笼状交联剂来扩展这些工具，这些交联剂旨在捕获瞬时伴侣蛋白。 有了这个全面的交联剂库，我们将评估载体蛋白与伴侣的相互作用。 包括酮还原酶、烯酰还原酶和来自脂肪酸和聚酮化合物的硫酯酶的蛋白质 来自聚酮脱氢酶的脱氢酶、酮合酶/链长因子和酰基转移酶，和 缩合结构域、卤化酶和来自非核糖体肽合成酶的氧化酶。一旦被捕， 这些交联的物质将通过溶液相NMR、动力学和热力学分析、X射线衍射和透射电镜来研究。 晶体学和分子动力学模拟，以更全面地阐明机制和特异性 由载体蛋白质-底物/中间体/产物相互作用赋予。最后，我们将使用收集的数据， 该计划作为一个信息平台，使新的天然产物杂交的合成生物生产。 为了提高探针制备的规模和纯度，加快我们的生产， 探针和交联剂合成。探针和交联剂纯化是我们的方法中最大的瓶颈之一。 发现管道，并要求这种仪器将加强这些努力的严谨性。

Project Summary. This program investigates the role of protein·protein interactions in carrier protein-dependent biosynthesis, including fatty acid synthase, polyketide synthase, and non-ribosomal peptide synthetase pathways. The natural products associated with these pathways serve as therapeutics including antibiotics, antifungals, and anticancer agents. The ability to direct the biosynthesis of these pathways for the production of untried bioactive compounds is of critical importance for new and improved therapies. In prior years, we demonstrated how protein'protein interactions between carrier proteins and partner protein domains direct reactivity and guide processivity. Moreover, we developed a suite of chemical biology tools to stabilize and interrogate these interactions at atomic resolution through innovative crosslinker development and structural biology. We now propose to expand these tools with new, caged crosslinkers designed to capture transient partner proteins. With this comprehensive library of crosslinkers, we will evaluate carrier protein interactions with partner proteins that include ketoreductases, enoyl reductases, and thioesterases from fatty acid and polyketide synthases, ketosynthase/chain-length factors and acyltransferases from polyketide synthases, and condensation domains, halogenases, and oxidases from non-ribosomal peptide synthetases. Once captured, these crosslinked species will be studied by solution-phase NMR, kinetic and thermodynamic assays, X-ray crystallography, and molecular dynamics simulations to more fully elucidate mechanism and specificity conferred by carrier protein-substrate/intermediate/product interactions. Finally, we will use data collected in this program as an informatic platform to enable synthetic biological production of new natural product hybrids. This equiment supplement is requested to improve the scale and purity of prepared probes and expedite our probe and crosslinker synthesis. Probe and crosslinker purification is one of the largest bottlenecks within our discovery pipeline, and request for this instrumentation will enhance the rigor of these efforts.