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Magic‐angle spinning (MAS) NMR coupled with dynamic nuclear polarization (DNP) has the possibility to increase the sensitivity of MAS NMR by several orders of magnitude. While DNP enables many experiments that are sensitivity limited, such as those on dilute samples or those that measure long‐range distances, interpretation of DNP NMR spectra is often limited by broad lines and chemical shift degeneracy. Segmental isotopic labeling using split intein technology can provide an opportunity to overcome this issue. Isotopic labeling of only a segment of a protein that is otherwise unlabeled reduces the chemical shift degeneracy. In this article, we describe the current state of the art for producing segmentally isotopically labeled proteins using split inteins. We discuss some of the potential applications of segmental isotopic labeling, particularly those that exploit the increased experimental sensitivity of DNP‐enhanced MAS NMR spectroscopy
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Sortase-mediated segmental labeling: A method for segmental assignment of intrinsically disordered regions in proteins
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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- PAR ID:
- 10312349
- Editor(s):
- van der Wel, Patrick
- Date Published:
- Journal Name:
- PLOS ONE
- Volume:
- 16
- Issue:
- 10
- ISSN:
- 1932-6203
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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- Free Publicly Accessible Full Text
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Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1371/journal.pone.0258531
