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.