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起着各种关键作用，提供结构，催化和信号功能，并且大约三分之一的蛋白质被认为是金属蛋白。然而，对金属离子在生物体内的分布和功能，即完整的“金属组”，还没有一个全面的调查。该项目的总体目标是表征金属离子在生物学中的作用，使用酿酒酵母作为初始测试生物。该项目的第一步将是通过开发高通量表达和纯化酿酒酵母谷胱甘肽- 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。这些融合蛋白的金属：蛋白质比例将使用ICP-MS（高灵敏度金属分析）和x射线荧光（高通量非破坏性金属分析的主要工具）的组合来确定，这些工具将用于定义酿酒酵母的金属组。对于选定的金属蛋白，金属离子的局部环境（氧化态和结扎）将使用x射线吸收光谱来确定。为初步筛选开发的方法随后将用于在金属组范围内调查生长条件变化的后果。将使用两种模型系统：在有限Zn条件下的生长作为金属离子浓度对金属在金属组中分布影响的模型；和Pb（ll）存在下的生长，作为非天然金属离子对金属组影响的模型。

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.