HIGH THROUGHPUT CHARACTERIZATION OF THE YEAST METALLOME

描述（由申请人提供）：已知金属离子在生物系统中发挥各种关键作用，提供结构、催化和信号传导功能，并且据信所有蛋白质中约有三分之一是金属蛋白。然而，从来没有一个全面的调查的分布和功能的金属离子在一个有机体，即完整的“金属组”。该项目的总体目标是使用酿酒酵母作为初始测试生物体来表征金属离子在生物学中的作用。本项目的第一步是通过开发高通量表达和纯化谷胱甘肽-S-转移酶- ORF（开放阅读框）文库中所有融合蛋白的方法来开发金属组筛选方法。啤酒。金属：将使用用于高灵敏度金属分析的ICP-MS和作为用于高通量非破坏性金属分析的主要工具的X射线荧光的组合来确定这些融合蛋白的蛋白质比率，并且这些工具将用于定义S。酿酒金属体对于选定的金属蛋白，将使用X射线吸收光谱法确定金属离子的局部环境（氧化态和连接）。然后，将使用为初步筛选开发的方法在金属组范围内调查生长条件变化的后果。将使用两个模型系统：在有限Zn条件下的生长作为金属离子浓度对金属组中金属分布的影响的模型;以及在Pb（II）存在下的生长作为非天然金属离子对金属组的影响的模型。

DESCRIPTION (provided by applicant): Metal ions are known to play a variety of critical roles in biological systems, providing structural, catalytic, and signaling functions, and approximately one-third of all proteins are believed to be metalloproteins. However, there has never been a comprehensive survey of the distribution and function of metal ions in an organism, that is, of the complete "metallome". The overall goal of the project is to characterize the role of metal ions in biology, using Saccharomyces cerevisiae as an initial test organism. The first step in this project will be to develop methods for metallome screening by developing methodology for high throughput expression and purification of all of the fusion proteins in the glutathione-S-transferase - ORF (open reading frame) library from S. cerevisiae. The metal: protein ratios for these fusion proteins will be determined using a combination of ICP-MS for high-sensitivity metal analysis and x-ray fluorescence as the primary tool for high-throughput non-destructive metal analysis and these tools will be used to define the S. cerevisiae metallome. For selected metalloproteins, the local environment of the metal ion (oxidation state and ligation) will be determined using x-ray absorption spectroscopy. The methodology developed for the initial screening will then be used to investigate, on a metallome-wide scale, the consequences of variations in growth conditions. Two model systems will be used: growth under conditions of limited Zn as a model for the effect of metal ion concentration on metal distribution in the metallome; and growth in the presence of Pb(ll) as a model for the effect of non-natural metal ions on the metallome.