Improved spectral resolution of [(13)C,(1)H]-HSQC spectra of aromatic amino acid residues in proteins produced by cell-free synthesis from inexpensive (13)C-labelled precursors

Cell-free protein synthesis using eCells allows production of amino acids from inexpensive (13)C-labelled precursors. We show that the metabolic pathway converting pyruvate, glucose and erythrose into aromatic amino acids is maintained in eCells. Judicious choice of (13)C-labelled starting material...

Descripción completa

Detalles Bibliográficos
Autores principales: Van Raad, Damian, Huber, Thomas, Otting, Gottfried
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Springer Netherlands 2023
Materias:
Article
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10406723/
https://www.ncbi.nlm.nih.gov/pubmed/37338652
http://dx.doi.org/10.1007/s10858-023-00420-9
_version_ 1785085808083992576
author Van Raad, Damian
Huber, Thomas
Otting, Gottfried
author_facet Van Raad, Damian
Huber, Thomas
Otting, Gottfried
author_sort Van Raad, Damian
collection PubMed
description Cell-free protein synthesis using eCells allows production of amino acids from inexpensive (13)C-labelled precursors. We show that the metabolic pathway converting pyruvate, glucose and erythrose into aromatic amino acids is maintained in eCells. Judicious choice of (13)C-labelled starting material leads to proteins, where the sidechains of aromatic amino acids display [(13)C,(1)H]-HSQC cross-peaks free of one-bond (13)C–(13)C couplings. Selective (13)C-labelling of tyrosine and phenylalanine residues is achieved simply by using different compositions of the reaction buffers. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10858-023-00420-9.
format Online
Article
Text
id pubmed-10406723
institution National Center for Biotechnology Information
language English
publishDate 2023
publisher Springer Netherlands
record_format MEDLINE/PubMed
spelling pubmed-104067232023-08-09 Improved spectral resolution of [(13)C,(1)H]-HSQC spectra of aromatic amino acid residues in proteins produced by cell-free synthesis from inexpensive (13)C-labelled precursors Van Raad, Damian Huber, Thomas Otting, Gottfried J Biomol NMR Article Cell-free protein synthesis using eCells allows production of amino acids from inexpensive (13)C-labelled precursors. We show that the metabolic pathway converting pyruvate, glucose and erythrose into aromatic amino acids is maintained in eCells. Judicious choice of (13)C-labelled starting material leads to proteins, where the sidechains of aromatic amino acids display [(13)C,(1)H]-HSQC cross-peaks free of one-bond (13)C–(13)C couplings. Selective (13)C-labelling of tyrosine and phenylalanine residues is achieved simply by using different compositions of the reaction buffers. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s10858-023-00420-9. Springer Netherlands 2023-06-20 2023 /pmc/articles/PMC10406723/ /pubmed/37338652 http://dx.doi.org/10.1007/s10858-023-00420-9 Text en © The Author(s) 2023 https://creativecommons.org/licenses/by/4.0/Open Access This 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/ (https://creativecommons.org/licenses/by/4.0/) .
spellingShingle Article
Van Raad, Damian
Huber, Thomas
Otting, Gottfried
Improved spectral resolution of [(13)C,(1)H]-HSQC spectra of aromatic amino acid residues in proteins produced by cell-free synthesis from inexpensive (13)C-labelled precursors
title Improved spectral resolution of [(13)C,(1)H]-HSQC spectra of aromatic amino acid residues in proteins produced by cell-free synthesis from inexpensive (13)C-labelled precursors
title_full Improved spectral resolution of [(13)C,(1)H]-HSQC spectra of aromatic amino acid residues in proteins produced by cell-free synthesis from inexpensive (13)C-labelled precursors
title_fullStr Improved spectral resolution of [(13)C,(1)H]-HSQC spectra of aromatic amino acid residues in proteins produced by cell-free synthesis from inexpensive (13)C-labelled precursors
title_full_unstemmed Improved spectral resolution of [(13)C,(1)H]-HSQC spectra of aromatic amino acid residues in proteins produced by cell-free synthesis from inexpensive (13)C-labelled precursors
title_short Improved spectral resolution of [(13)C,(1)H]-HSQC spectra of aromatic amino acid residues in proteins produced by cell-free synthesis from inexpensive (13)C-labelled precursors
title_sort improved spectral resolution of [(13)c,(1)h]-hsqc spectra of aromatic amino acid residues in proteins produced by cell-free synthesis from inexpensive (13)c-labelled precursors
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC10406723/
https://www.ncbi.nlm.nih.gov/pubmed/37338652
http://dx.doi.org/10.1007/s10858-023-00420-9
work_keys_str_mv AT vanraaddamian improvedspectralresolutionof13c1hhsqcspectraofaromaticaminoacidresiduesinproteinsproducedbycellfreesynthesisfrominexpensive13clabelledprecursors
AT huberthomas improvedspectralresolutionof13c1hhsqcspectraofaromaticaminoacidresiduesinproteinsproducedbycellfreesynthesisfrominexpensive13clabelledprecursors
AT ottinggottfried improvedspectralresolutionof13c1hhsqcspectraofaromaticaminoacidresiduesinproteinsproducedbycellfreesynthesisfrominexpensive13clabelledprecursors

Ejemplares similares