Three segment ligation of a 104 kDa multi-domain protein by SrtA and OaAEP1.

Content Provider	Europe PMC
Author	Azatian, Stephan B. Canny, Marella D. Latham, Michael P.
Abstract	NMR spectroscopy is an excellent tool for studying protein structure and dynamics which provides a deeper understanding of biological function. As the size of the biomolecule of interest increases, it can become advantageous to dilute the number of observed signals in the NMR spectrum to decrease spectral overlap and increase resolution. One way to limit the number of resonances in the NMR data is by selectively labeling a smaller domain within the larger macromolecule, a process called segmental isotopic labeling. Many examples of segmental isotopic labeling have been described where two segments of a protein are ligated together by chemical or enzymatic means, but there are far fewer descriptions of a three or more segment ligation reaction. Herein, we describe an enzymatic segmental labeling scheme that combines the widely used Sortase A and more recently described OaAEP1 for a two site ligation strategy. In preparation to study proposed long-range allostery in the 104 kDa DNA damage repair protein Rad50, we ligated side-chain methyl group labeled Zn Hook domain between two long segments of otherwise unlabeled P.furiosus Rad50. Enzymatic activity data demonstrated that the scars resulting from the ligation reactions did not affect Rad50 function within the Mre11-Rad50 DNA double strand break repair complex. Finally, methyl-based NMR spectroscopy confirmed the formation of the full-length ligated protein. Our strategy highlights the strengths of OaAEP1 for segmental labeling, namely faster reaction times and a smaller recognition sequence, and provides a straightforward template for using these two enzymes in multisite segmental labeling reactions.
Related Links	https://europepmc.org/backend/ptpmcrender.fcgi?accid=PMC10149453&blobtype=pdf
ISSN	09252738
Journal	Journal of Biomolecular Nmr [J Biomol NMR]
Volume Number	77
DOI	10.1007/s10858-022-00409-w
PubMed Central reference number	PMC10149453
Issue Number	1-2
PubMed reference number	36539644
e-ISSN	15735001
Language	English
Publisher	Springer Netherlands
Publisher Date	2022-12-21
Publisher Place	Dordrecht
Access Restriction	Open
Rights License	Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. © The Author(s) 2022
Subject Keyword	Segmental labeling Protein ligation Side-chain methyl group labeling Sortase A Asparaginyl endopeptidase
Content Type	Text
Resource Type	Article
Subject	Spectroscopy Biochemistry