Substrate targeting mechanism of a DesCEND pathway

摘要 该补助金R 01 GM 135592的补充申请建议购买一台自动化高- 生产量结晶筛选系统，以支持研究目标，重点是调查基板 新型DesCEND途径中SCF遍在蛋白连接酶F-box亚基FBXO 31的靶向机制。 通过在系统水平和分子水平上识别和表征FBXO 31的底物，我们 提出阐明FBXO 31-降解决定子对的互补结构决定因素， 底物特异性和决定蛋白质识别和命运。为了实现我们的目标，我们必须拥有 筛选获得高分辨率蛋白质所必需的广泛结晶条件的能力， 衬底复杂结构。该提案要求提供资金，以取代10岁的Gryphon-100 ul Flex 同一制造商生产的类似但最新版本的仪器。除了拥有良好的声誉外， 性能质量，拟议的系统涵盖了广泛的筛选应用，并满足 自动化要求，需要开发一个精简的方法，结晶在高通量 时尚.我们得到了分子生物学和生物物理学系的机构支持。的 仪器将确保继续进行母公司中提出的生物化学，生物物理和结构研究 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.