Using Singular Value Decomposition to Characterize Protein–Protein Interactions by In‐cell NMR Spectroscopy |
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| Authors: | Subhabrata Majumder Christopher M. DeMott Dr. David S. Burz Dr. Alexander Shekhtman |
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| Affiliation: | Department of Chemistry, University at Albany, State University of New York, 1400 Washington Ave., Albany, NY 12222 (USA) |
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| Abstract: | Distinct differences between how model proteins interact in‐cell and in vitro suggest that the cytosol might have a profound effect in modulating protein–protein and/or protein–ligand interactions that are not observed in vitro. Analyses of in‐cell NMR spectra of target proteins interacting with physiological partners are further complicated by low signal‐to‐noise ratios, and the long overexpression times used in protein–protein interaction studies may lead to changes in the in‐cell spectra over the course of the experiment. To unambiguously resolve the principal binding mode between two interacting species against the dynamic cellular background, we analyzed in‐cell spectral data of a target protein over the time course of overexpression of its interacting partner by using single‐value decomposition (SVD). SVD differentiates between concentration‐dependent and concentration‐independent events and identifies the principal binding mode between the two species. The analysis implicates a set of amino acids involved in the specific interaction that differs from previous NMR analyses but is in good agreement with crystallographic data. |
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| Keywords: | NMR spectroscopy protein‐protein interactions single‐cell measurements single‐value decomposition statistical analysis |
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