Site-specific labelling of multidomain proteins by amber codon suppression

  • The access to information on the dynamic behaviour of large proteins is usually hindered as spectroscopic methods require the site-specific attachment of biophysical probes. A powerful emerging tool to tackle this issue is amber codon suppression. Till date, its application on large and complex multidomain proteins of MDa size has not been reported. Herein, we systematically investigate the feasibility to introduce different non-canonical amino acids into a 540 kDa homodimeric fatty acid synthase type I by genetic code expansion with subsequent fluorescent labelling. Our approach relies on a microplate-based reporter assay of low complexity using a GFP fusion protein to quickly screen for sufficient suppression conditions. Once identified, these findings were successfully utilized to upscale both the expression scale and the protein size to full-length constructs. These fluorescently labelled samples of fatty acid synthase were subjected to initial biophysical experiments, including HPLC analysis, activity assays and fluorescence spectroscopy. Successful introduction of such probes into a molecular machine such as fatty acid synthases may pave the way to understand the conformational variability, which is a primary intrinsic property required for efficient interplay of all catalytic functionalities, and to engineer them.

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Author:Christina S. Heil, Alexander Rittner, Bjarne Goebel, Daniel Beyer, Martin GriningerORCiDGND
URN:urn:nbn:de:hebis:30:3-476744
DOI:https://doi.org/10.1038/s41598-018-33115-5
ISSN:2045-2322
Pubmed Id:https://pubmed.ncbi.nlm.nih.gov/30291265
Parent Title (English):Scientific reports
Publisher:Macmillan Publishers Limited, part of Springer Nature
Place of publication:[London]
Document Type:Article
Language:English
Year of Completion:2018
Date of first Publication:2018/10/05
Publishing Institution:Universitätsbibliothek Johann Christian Senckenberg
Release Date:2018/10/09
Tag:Biochemistry; Proteins
Volume:8
Issue:1, Art. 14864
Page Number:15
First Page:1
Last Page:15
Note:
Rights and permissions: 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 license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license 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 license, visit http://creativecommons.org/licenses/by/4.0/.
HeBIS-PPN:438464389
Institutes:Biochemie, Chemie und Pharmazie / Biochemie und Chemie
Biochemie, Chemie und Pharmazie / Pharmazie
Exzellenzcluster / Exzellenzcluster Makromolekulare Komplexe
Dewey Decimal Classification:5 Naturwissenschaften und Mathematik / 57 Biowissenschaften; Biologie / 570 Biowissenschaften; Biologie
Sammlungen:Universitätspublikationen
Licence (German):License LogoCreative Commons - Namensnennung 4.0