4.6 Article

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

期刊

PLOS ONE
卷 16, 期 10, 页码 -

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PUBLIC LIBRARY SCIENCE
DOI: 10.1371/journal.pone.0258531

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资金

  1. National Science Foundation [CHE-2004237, REU-1757629, REU-1359330]
  2. Western Washington University
  3. Denice (Ambrose) Hougen Chemistry Undergraduate Fellowship
  4. Karen and Joseph Morse Research Fellowship
  5. NSF [MRI-1532269]
  6. Canada Foundation for Innovation
  7. British Columbia Knowledge Development Fund
  8. Natural Sciences and Engineering Research Council of Canada

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This study demonstrates the use of SML for segmental isotopic labeling of IDR-containing samples, reducing complexity of NMR spectra. Through segmental assignment, different segments within the IDR can be identified and studied for their characteristics and responses. This method shows promise in selectively labeling and probing target segments within larger IDR contexts.
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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