Sortase-mediated
ligation is a powerful method for generating site-specifically
modified proteins. However, this process is limited by the inherent
reversibility of the ligation reaction. To address this, here we report
the continued development and optimization of an experimentally facile
strategy for blocking reaction reversibility. This approach, which
we have termed metal-assisted sortase-mediated ligation (MA-SML),
relies on the use of a solution additive (Ni2+) and a C-terminal
tag (LPXTGGHH5) that is widely used for converting protein
targets into sortase substrates. In a series of model systems utilizing
a 1:1 molar ratio of sortase substrate and glycine amine nucleophile,
we find that MA-SML consistently improves the extent of ligation.
This enables the modification of proteins with fluorophores, PEG,
and a bioorthogonal cyclooctyne moiety without the need to use precious
reagents in excess. Overall, these results demonstrate the potential
of MA-SML as a general strategy for improving reaction efficiency
in a broad range of sortase-based protein engineering applications.
A significant number of proteins possess sizable intrinsically disordered regions (IDRs). Due to the dynamic nature of IDRs, NMR spectroscopy is often the tool of choice for characterizing these segments. However, the application of NMR to IDRs is often hindered by their instability, spectral overlap and resonance assignment difficulties. Notably, these challenges increase considerably with the size of the IDR. In response to these issues, here we report the use of sortase-mediated ligation (SML) for segmental isotopic labeling of IDR-containing samples. Specifically, we have developed a ligation strategy involving a key segment of the large IDR and adjacent folded headpiece domain comprising the C-terminus of A. thaliana villin 4 (AtVLN4). This procedure significantly reduces the complexity of NMR spectra and enables group identification of signals arising from the labeled IDR fragment, a process we refer to as segmental assignment. The validity of our segmental assignment approach is corroborated by backbone residue-specific assignment of the IDR using a minimal set of standard heteronuclear NMR methods. Using segmental assignment, we further demonstrate that the IDR region adjacent to the headpiece exhibits nonuniform spectral alterations in response to temperature. Subsequent residue-specific characterization revealed two segments within the IDR that responded to temperature in markedly different ways. Overall, this study represents an important step toward the selective labeling and probing of target segments within much larger IDR contexts. Additionally, the approach described offers significant savings in NMR recording time, a valuable advantage for the study of unstable IDRs, their binding interfaces, and functional mechanisms.
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