Alexander Shekhtman, Professor, The RNA Institute, SUNY at Albany, will give a talk on "In-cell NMR - what is inside."

ABSTRACT

High-resolution structural studies of proteins and protein complexes in a native prokaryotic and eukaryotic environment present a challenge to structural biology. In-cell NMR can characterize atomic resolution structures but requires high concentrations of labeled proteins to be delivered into intact cells. Most exogenous delivery techniques are either limited to specific cell types or are too destructive to preserve cellular physiology. We demonstrate the feasibility of microfluidics transfection or volume exchange for the convective transfer, VECT, as a means to deliver labeled targets to HeLa cells. VECT delivery maintains cell viability thereby providing a route for long-term eukaryotic in-cell NMR experiments. The high viability allows changes in the metabolic state due to internal and external stimuli, and stresses to be monitored. Protein-protein interactions, PPIs, underlie most cellular processes, but many PPIs depend on a particular metabolic state that can only be observed in live, actively metabolizing cells. Real-time in-cell NMR spectroscopy, RT-NMR,  utilizes a bioreactor to maintain cells in an active metabolic state and unmasks functional protein-protein interactions that are absent without active metabolism. The combination of a bioreactor and VECT technology will facilitate the study of protein interactions in the most relevant physiological states.