Structure and Localization of the Flavonoid Multienzyme Complex

生物大分子之间的相互作用几乎构成了每个细胞过程的基础，包括将相互协作的酶组织成大分子复合体。多酶系统提供了获得高的局部底物浓度，调节分支通路之间的竞争，协调相互依赖的通路的活动，并将有毒或挥发性中间产物隔离在细胞内的手段。然而，人们对任何酶复合体的结构或指导单个成分相互作用的分子机制知之甚少。该项目将建立在Winkel实验室先前工作的基础上，以表征拟南芥类黄酮酶复合体的结构和功能，包括使用小角中子散射来开发该系统的三维模型。将测试几种用于研究交互界面的新技术，以努力确认在Planta中提出的交互作用，并确定可能仲裁复合体组装的因素。最近发现的类黄酮酶的差异定位的决定因素也将被表征。这些研究将有助于全面理解大分子相互作用是如何发生的，具有必要的特异性和灵活性，以有效地调节拥挤和动态的细胞内部的生物过程。这些知识对于代谢工程在植物农艺和营养改良中的有效应用以及在微生物、植物和动物中生产有价值的代谢物是必不可少的。该项目将为本科生、研究生和博士后提供当代分子遗传学、生物化学和计算生物学的培训基地。这项工作的更广泛的影响还包括一项具体的努力，即研究本科生的研究经验对参与的学生及其导师的结果和影响。大约35名本科生在温克尔实验室进行了十多年的研究；这为以严格和广泛适用的方式研究这些经验提供了理想的方案。将评估本科生在获得研究经验后所追求的学术和职业机会，研究经验在多大程度上准备和/或鼓励本科生追求学术和职业机会，以及本科生的研究经验对导师的影响程度。

Interactions between biological macromolecules underlie virtually every cellular process, including the organization of cooperating enzymes into macromolecular complexes. Multienzyme systems provide the means to attain high local substrate concentrations, regulate competition among branch pathways, coordinate the activities of interdependent pathways, and sequester toxic or volatile intermediates within the cell. Yet remarkably little is known about the architecture of any enzyme complex or the molecular mechanisms that direct the interaction of the individual components. The project will build on previous work in the Winkel laboratory to characterize the structure and function of the flavonoid enzyme complex in Arabidopsis thaliana, including the use of small angle neutron scattering to develop a three-dimensional model of this system. Several new technologies for studying interaction interfaces will be tested in an effort to confirm proposed interactions in planta, and to identify factors that may arbitrate assembly of the complex. The determinants of the recently discovered differential localization of flavonoid enzymes will also be characterized. These studies will contribute to a general understanding of how macromolecular interactions occur with the requisite specificity and flexibility to effectively mediate biological processes in the crowded and dynamic cell interior. This knowledge is essential for the effective application of metabolic engineering to the agronomic and nutritional improvement of plants and for the bioproduction of valuable metabolites in microorganisms, plants, and animals.The project will provide a training ground in contemporary molecular genetics, biochemistry, and computational biology for undergraduates, graduate students, and a postdoctoral fellow. The broader impact of this work also involves a specific effort to study the outcomes and impacts of research experiences for undergraduates on the participating students and their mentors. Some 35 undergraduate students have conducted research in the Winkel laboratory over more than a decade; this provides an ideal scenario for studying these experiences in a rigorous and broadly applicable way. An assessment will be made of the academic and career opportunities that undergraduates pursue following their research experience, the extent to which the research experience prepare and/or encourage undergraduates to pursue academic and career opportunities, and the extent to which research experiences for undergraduates impacted the mentors.