MUTIDIMENSIONAL DIFFUSION EXPERIMENTS IN A CELL-FREE SYSTEM

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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