Substrate targeting mechanism of a DesCEND pathway

摘要 这项对拨款的补充申请R01GM135592建议收购自动化高- 生产能力结晶筛选系统支持专注于研究底物的研究目标 干细胞因子泛素连接酶F-box亚单位FBXO31在一种新的下行途径中的靶向机制。 通过在系统水平和分子水平上鉴定和表征FBXO31底物，我们 建议阐明FBXO31-degron对的互补结构决定因素 底物专一性决定蛋白质的识别和命运。为了实现我们的目标，我们必须拥有 能够筛选获得高分辨率蛋白质所必需的各种结晶条件- 衬底复杂结构。这项提案要求提供资金来更换一辆有10年历史的鹰头狮-100ul Flex 使用来自同一制造商的类似但最新版本的仪器。除了拥有声誉良好的 性能质量，建议的系统涵盖广泛的筛选应用程序，并满足 在高通量中开发简化结晶方法所需的自动化要求 时尚。我们得到了分子生物学和生物物理系的机构支持。这个 仪器将确保母公司建议的生化、生物物理和结构研究继续进行 获奖。

Abstract This request for a supplement to the grant R01GM135592 proposes the acquisition of an automated high- throughput crystallization screening system to support the research aims that focus on investigating the substrate targeting mechanisms of the F-box subunit FBXO31 of the SCF ubiquitin ligase in a novel DesCEND pathway. By identifying and characterizing substrates of FBXO31 at both a systems level and the molecular level, we proposed to elucidate the complementary structural determinants of the FBXO31–degron pair that confer substrate specificity and dictate protein recognition and fate. To achieve our goals, it is vital that we have the ability to screen wide ranges of crystallization conditions that are essential for obtaining high resolution protein- substrate complex structures. This proposal requests funds to replace a 10-year old Gryphon-100ul Flex instrument with a similar but up-to-date version from the same manufacturer. In addition to having reputable performance quality, the proposed system covers a broad range of screening applications and meets the automation requirements needed to develop a streamlined approach for crystallization in a high-throughput fashion. We have obtained institutional support from the Department of Molecular Biology and Biophysics. The instrument will ensure the continued biochemical, biophysical and structural studies proposed in the parent award.