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Site-Specific Labelling of Multidomain Proteins by Amber Codon Suppression

View ORCID ProfileChristina S. Heil, View ORCID ProfileAlexander Rittner, Bjarne Goebel, Daniel Beyer, View ORCID ProfileMartin Grininger
doi: https://doi.org/10.1101/282525
Christina S. Heil
Institute of Organic Chemistry and Chemical Biology, Buchmann Institute for Molecular Life Sciences, Cluster of Excellence for Macromolecular Complexes, Goethe University Frankfurt, Max-von-Laue-Str. 15, 60438 Frankfurt am Main, Germany
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Alexander Rittner
Institute of Organic Chemistry and Chemical Biology, Buchmann Institute for Molecular Life Sciences, Cluster of Excellence for Macromolecular Complexes, Goethe University Frankfurt, Max-von-Laue-Str. 15, 60438 Frankfurt am Main, Germany
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Bjarne Goebel
Institute of Organic Chemistry and Chemical Biology, Buchmann Institute for Molecular Life Sciences, Cluster of Excellence for Macromolecular Complexes, Goethe University Frankfurt, Max-von-Laue-Str. 15, 60438 Frankfurt am Main, Germany
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Daniel Beyer
Institute of Organic Chemistry and Chemical Biology, Buchmann Institute for Molecular Life Sciences, Cluster of Excellence for Macromolecular Complexes, Goethe University Frankfurt, Max-von-Laue-Str. 15, 60438 Frankfurt am Main, Germany
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Martin Grininger
Institute of Organic Chemistry and Chemical Biology, Buchmann Institute for Molecular Life Sciences, Cluster of Excellence for Macromolecular Complexes, Goethe University Frankfurt, Max-von-Laue-Str. 15, 60438 Frankfurt am Main, Germany
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  • For correspondence: grininger@chemie.uni-frankfurt.de
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Abstract

Amber codon suppression is a powerful tool to site-specifically modify proteins to generate novel biophysical probes. Yet, its application on large and complex multidomain proteins is challenging, leading to difficulties during structural and conformational characterization using spectroscopic methods. The animal fatty acid synthase type I is a 540 kDa homodimer displaying large conformational variability. As the key enzyme of de novo fatty acid synthesis, it attracts interest in the fields of obesity, diabetes and cancer treatment. Substrates and intermediates remain covalently bound to the enzyme during biosynthesis and are shuttled to all catalytic domains by the acyl carrier protein domain. Thus, conformational variability of animal FAS is an essential aspect for fatty acid biosynthesis. We investigate this multidomain protein as a model system for probing amber codon suppression by genetic encoding of non-canonical amino acids. The systematic approach relies on a microplate-based reporter assay of low complexity, that was used for quick screening of suppression conditions. Furthermore, the applicability of the reporter assay is demonstrated by successful upscaling to both full-length constructs and increased expression scale. The obtained fluorescent probes of murine FAS type I could be subjected readily to a conformational analysis using single-molecule fluorescence resonance energy transfer.

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Posted March 14, 2018.
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Site-Specific Labelling of Multidomain Proteins by Amber Codon Suppression
Christina S. Heil, Alexander Rittner, Bjarne Goebel, Daniel Beyer, Martin Grininger
bioRxiv 282525; doi: https://doi.org/10.1101/282525
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Site-Specific Labelling of Multidomain Proteins by Amber Codon Suppression
Christina S. Heil, Alexander Rittner, Bjarne Goebel, Daniel Beyer, Martin Grininger
bioRxiv 282525; doi: https://doi.org/10.1101/282525

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