Abstract 1387 Enzyme-Mediated Segmental Labeling Allows NMR Characterization of Large Disordered Proteins

The actin-binding protein dematin has a small villin-type headpiece domain and a large disordered region. The folded headpiece has been extensively studied, but possible conformations of the intrinsically disordered region (IDR) remain unknown. It is hypothesized that the N-terminal region of the IDR binds transiently to the headpiece domain. NMR analysis of the fully labeled protein is hindered by its size and the resulting degree of spectral overlap. To overcome this, we expressed the 15N-labeled headpiece domain independently and ligated it to an unlabeled IDR fragment. We collected NMR data on this headpiece-IDR construct and the labeled headpiece in isolation, then compared the two to determine the effect of the IDR fragment on headpiece dynamics. We found differences in peak position and intensity consistent with altered backbone dynamics between the two samples, suggesting that the IDR fragment does interact with the headpiece domain. Furthermore, we believe this is a promising technique for the characterization of other large disordered proteins. This work was supported by the WWU Department of Chemistry and the National Science Foundation REU Program under grant NSF CHE-2243968.