PT  - JOURNAL ARTICLE
AU  - Christina S. Heil
AU  - Alexander Rittner
AU  - Bjarne Goebel
AU  - Daniel Beyer
AU  - Martin Grininger
TI  - Site-Specific Labelling of Multidomain Proteins by Amber Codon Suppression
AID  - 10.1101/282525
DP  - 2018 Jan 01
TA  - bioRxiv
PG  - 282525
4099  - http://biorxiv.org/content/early/2018/03/14/282525.short
4100  - http://biorxiv.org/content/early/2018/03/14/282525.full
AB  - 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.