Sortase-mediated segmental labeling: A method for segmental assignment of intrinsically disordered regions in proteins

Abstract

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.

Nine-residue low-complexity disordered peptide as a model system, an NMR/CD study

“…This sizable internal linker, spanning 192 residues, has a distinct charge-partitioning pattern: the 144-residue N-terminal basic segment with the isoelectric point is at 11.5 and the 48-residue C-terminal acidic one with the isoelectric point 4.1. [14] The region has been shown to have a novel capacity to bind/bundle actin filaments. [15] Notably, the closest match to the basic RC9 oligopeptide is basic (SRSMSFSP) and it is positioned within the basic part of the IDR in the villin 4 linker.…”

From Immunogenic Peptides to Intrinsically Disordered Proteins

“…The recent advances in cryo‐electron microscopy, giving high‐resolution structures of large macromolecules, are also not applicable, since the disordered regions are too heterogeneous to be resolved. NMR approaches using inteins or sortase to perform segmental isotope labeling are highly promising for characterization of full‐length proteins containing both structured and disordered regions [48–50] …”

“…NMR approaches using inteins or sortase to perform segmental isotope labeling are highly promising for characterization of full-length proteins containing both structured and disordered regions. [48][49][50]…”

Chemical tools for study and modulation of biomolecular phase transitions

“…In the context of IDPs and LLPS, there are several important considerations that need to be kept in mind. 93,94 First, to carry out the ligation reaction, sortase needs to be folded, which precludes the use of high concentrations of denaturants. Second, the reaction requires the presence of Ca 2+ ions in the buffer, which may be incompatible with LLPS studies for some proteins.…”