Real Time (RT) In-cell NMR technology to study protein interactions in live cells Alexander Shekhtman1 1Department of Chemistry, University at Albany, State University of New York, Albany, NY 12222 ABSTRACT: Since intracellular changes induced by stimuli can take hours to fully manifest into detectable signals, one of the overarching goals of modern biology is to understand temporal protein structure-function relationships at atomic resolution within the complexity of a live cell. In-cell solution NMR spectroscopy is an important step towards this goal but is limited by the long data acquisition times and the static nature of in-cell NMR experiments that provide snapshots rather than continuous monitoring of time dependent changes in protein structure. In addition, weak quinary interactions between the protein of interest and intracellular components, particularly RNAs, which are omnipresent in live cells, result in a dramatic increase in the apparent in-cell molecular weight and render all but a few proteins invisible by standard in-cell NMR approaches. There are currently no structural biology tools to characterize time dependent protein interactions in live cells at atomic resolution even though these interactions affect protein physicochemical properties, protein-protein, protein-ligand, and protein-drug binding. We showed that the combination of protein deuteration and NMR experiments using optimized transverse relaxation allowed us to obtain in-cell NMR spectra of previously invisible proteins. The goal of this project is to develop real time (RT) in-cell NMR technology to characterize protein interactions in situ over a long (more than a day) period of time at atomic resolution inside live prokaryotic and eukaryotic cells. We will apply this technology to study how exogenous and endogenous challenges to cells result in specific temporal changes in protein structure. We will build the infrastructure to make this new technology available to the scientific community. We expect that the technology will be critical to bridge the gap between in vitro and in-cell protein biochemistry, which is an absolute requirement to understand cell biology and to develop effective therapeutics against protein targets.
. Real time(RT) in-cell NMR spectroscopy will provide time dependent atomic resolution information on protein interactions inside live prokaryotic and eukaryotic cells. This technology bridges the gap between atomic resolution structural biology techniques to study protein interactions in vitro and low- resolution microscopy techniques to study protein interactions in vivo. RT in-cell NMR will prove to be invaluable for identifying new biologically active (drug) compounds against established therapeutic targets.
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