MUTIDIMENSIONAL DIFFUSION EXPERIMENTS IN A CELL-FREE SYSTEM

本子项目是利用由NIH/NCRR资助的中心赠款提供的资源的众多研究子项目之一。子项目和研究者（PI）可能已经从另一个NIH来源获得了主要资金，因此可以在其他CRISP条目中表示。列出的机构是中心的，不一定是研究者的机构。扩散有序光谱（DOSY）是一种基于溶液中小分子扩散系数差异来鉴别复杂混合物中的小分子的有效工具。该方法通常应用于相对小分子的混合物（奎宁、莰烯、香叶醇、碳水化合物等）。目前还没有一种方法可以区分大分子多肽和小分子蛋白质。我们已经开发了DOSY-HSQC脉冲序列，用于直接光谱分离由蛋白质引起的共振和由小分子引起的共振。该方法可应用于复杂生物介质的光谱分析，包括蛋白水解混合物、活细胞和含有过表达同位素标记蛋白质的无细胞系统

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