Journal
BIOPHYSICAL JOURNAL
Volume 115, Issue 9, Pages 1690-1695Publisher
CELL PRESS
DOI: 10.1016/j.bpj.2018.08.049
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Funding
- Interdisciplinary Quantitative Biology program at the BioFrontiers Institute, University of Colorado, Boulder (National Science Foundation) [1144807]
- Boettcher Foundation
- University of Colorado Innovative Seed Grant
- National Institutes of Health-National Institute of General Medical Sciences [R35 GM119755]
- National Institutes of Health [RR011969, RR16649]
- National Science Foundation [DBI-0230966, 960241]
- University of Colorado Department of Physics
- BioFrontiers Institute
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In-cell NMR spectroscopy is a powerful tool to determine the properties of proteins and nucleic acids within living cells. In-cell NMR can give site-specific measurements of interactions, modifications, and dynamics as well as their modulation by the cellular environment. In-cell NMR requires selective incorporation of heavy isotopes into a protein of interest, either through the introduction of exogenously produced protein to a cell's interior or the selective overexpression of a protein. We developed conditions to allow the use of Saccharomyces cerevisiae, which was chosen because of its genetic tractability, as a eukaryotic expression system for in-cell NMR. We demonstrate this technique using a fragment of S. cerevisiae Nsp1, an FG Nup. FG Nups are intrinsically disordered proteins containing phenylalanine (F)-glycine (G) repeats and form the selective barrier within the nuclear pore complex. Yeast FG Nups have previously been shown to be maintained in a highly dynamic state within living bacteria as measured by in-cell NMR. Interactions thought to stabilize this dynamic state are also present in the protein's native organism, although site specificity of interaction is different between the two cytosols.
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