MUTIDIMENSIONAL DIFFUSION EXPERIMENTS IN A CELL-FREE SYSTEM

这个子项目是利用由NIH/NCRR资助的中心拨款提供的资源的许多研究子项目之一。子项目和调查员(PI)可能从另一个NIH来源获得了主要资金，因此可能会出现在其他CRISE条目中。列出的机构是针对中心的，而不一定是针对调查员的机构。扩散有序光谱(DOSY)是一种根据小分子在溶液中扩散系数的差异来区分复杂混合物中小分子的有效工具，该方法通常应用于相对较小的分子(奎宁、樟烯、香叶醇、碳水化合物等)的混合物。目前还没有一种方法来区分大分子多肽和蛋白质与小分子。我们开发了DOSY-HSQC脉冲序列，用于直接从光谱上分离蛋白质引起的共振和小分子引起的共振。该方法可用于复杂生物介质的光谱分析，包括蛋白质水解物、活细胞和含有过表达的同位素标记蛋白质的无细胞体系

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Diffusion-ordered spectroscopy (DOSY) is an effective tool to spectroscopically discriminate small molecules within complex mixtures based on differences in their diffusion coefficient in solution.This method has typically been applied to mixtures of relatively small molecules (quinine, camphene, geraniol, carbohydrates, etc.). No method is currently available to discriminate large polypeptides and proteins from smaller molecules.We have developed the DOSY-HSQC pulse sequence for the straightforward spectroscopic separation of resonances due to proteins from those due to small molecules.This method can be applied to the spectroscopic analysis of complex biological media, including proteolytic mixtures, living cells, and cell-free systems containing overexpressed isotopically labeled proteins