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 overla...

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Autores principales:	Kristina V Boyko, Erin A Rosenkranz, Derrick M Smith, Heather L Miears, Melissa Oueld Es Cheikh, Micah Z Lund, Jeffery C Young, Patrick N Reardon, Mark Okon, Serge L Smirnov, John M Antos
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/1d443f071c4e45259e7777728beab55b
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spelling	oai:doaj.org-article:1d443f071c4e45259e7777728beab55b2021-12-02T20:16:31ZSortase-mediated segmental labeling: A method for segmental assignment of intrinsically disordered regions in proteins.1932-620310.1371/journal.pone.0258531https://doaj.org/article/1d443f071c4e45259e7777728beab55b2021-01-01T00:00:00Zhttps://doi.org/10.1371/journal.pone.0258531https://doaj.org/toc/1932-6203A 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.Kristina V BoykoErin A RosenkranzDerrick M SmithHeather L MiearsMelissa Oueld Es CheikhMicah Z LundJeffery C YoungPatrick N ReardonMark OkonSerge L SmirnovJohn M AntosPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 16, Iss 10, p e0258531 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Kristina V Boyko Erin A Rosenkranz Derrick M Smith Heather L Miears Melissa Oueld Es Cheikh Micah Z Lund Jeffery C Young Patrick N Reardon Mark Okon Serge L Smirnov John M Antos Sortase-mediated segmental labeling: A method for segmental assignment of intrinsically disordered regions in proteins.
description	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.
format	article
author	Kristina V Boyko Erin A Rosenkranz Derrick M Smith Heather L Miears Melissa Oueld Es Cheikh Micah Z Lund Jeffery C Young Patrick N Reardon Mark Okon Serge L Smirnov John M Antos
author_facet	Kristina V Boyko Erin A Rosenkranz Derrick M Smith Heather L Miears Melissa Oueld Es Cheikh Micah Z Lund Jeffery C Young Patrick N Reardon Mark Okon Serge L Smirnov John M Antos
author_sort	Kristina V Boyko
title	Sortase-mediated segmental labeling: A method for segmental assignment of intrinsically disordered regions in proteins.
title_short	Sortase-mediated segmental labeling: A method for segmental assignment of intrinsically disordered regions in proteins.
title_full	Sortase-mediated segmental labeling: A method for segmental assignment of intrinsically disordered regions in proteins.
title_fullStr	Sortase-mediated segmental labeling: A method for segmental assignment of intrinsically disordered regions in proteins.
title_full_unstemmed	Sortase-mediated segmental labeling: A method for segmental assignment of intrinsically disordered regions in proteins.
title_sort	sortase-mediated segmental labeling: a method for segmental assignment of intrinsically disordered regions in proteins.
publisher	Public Library of Science (PLoS)
publishDate	2021
url	https://doaj.org/article/1d443f071c4e45259e7777728beab55b
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Sortase-mediated segmental labeling: A method for segmental assignment of intrinsically disordered regions in proteins.

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