ABSTRACT
STINT-NMR is an in-cell technique[6], [7] for examining the structural changes in a target protein resulting from protein-protein interactions [5], [8]. Protein overexpression is rst induced in uniformly labeled medium [U, 15N-] to produce a target protein containing NMR-active nuclei. e cells are washed and resuspended in non-labeling medium to induce overexpression of the interactor protein(s). We then use 15N-edited heteronuclear single quantum coherence (1H{15N}-HSQC) NMR experiments to monitor changes in the chemical shifts of target backbone amide nuclei as the concentration of the interactor(s) increases. In cases where the target binds to a high molecular weight interactor protein, we monitor changes in the NMR spectrum of the free target since only this species gives rise to visible peaks. Depending on the chemical exchange rate between the free and bound states of the target, aected NMR peaks, corresponding to backbone amides, can either shift, broaden their line shape or disappear completely, thereby delineating the intermolecular interaction surface between the target and the interactor(s). By changing the isotopic composition on induction, we selectively label the target while the interactor proteins remain cryptic, thus reducing NMR spectral complexity. e resulting NMR data provide a complete titration of the interaction and identify the amino acids that comprise the interaction surface of a target protein. It is important to note that in-cell titrations lack the precision of binding isotherms that are obtained in vitro because of the variable levels of protein expression that are inherent when using living cell, and thus, are largely qualitative. However, in each case, the same structural endpoint is attained. Tight temporal control over protein expression allows us to perform in-cell biochemistry, such as phosphorylation, by expressing a kinase domain capable of post-translationally modifying the interactor protein, o an inducible plasmid (Fig. 1ab).
