Site-Specific Labelling of Multidomain Proteins by Amber Codon Suppression

Abstract

17Amber codon suppression is a powerful tool to site-specifically modify proteins to 18 generate novel biophysical probes. Yet, its application on large and complex 19 multidomain proteins is challenging, leading to difficulties during structural and 20 conformational characterization using spectroscopic methods. The animal fatty acid 21 synthase type I is a 540 kDa homodimer displaying large conformational variability. 22As the key enzyme of de novo fatty acid synthesis, it attracts interest in the fields of 23 obesity, diabetes and cancer treatment. Substrates and intermediates remain 24 covalently bound to the enzyme during biosynthesis and are shuttled to all catalytic 25 domains by the acyl carrier protein domain. Thus, conformational variability of animal 26 FAS is an essential aspect for fatty acid biosynthesis. We investigate this 27 multidomain protein as a model system for probing amber codon suppression by 28 genetic encoding of non-canonical amino acids. The systematic approach relies on a 29 microplate-based reporter assay of low complexity, that was used for quick screening 30 of suppression conditions. Furthermore, the applicability of the reporter assay is 31 demonstrated by successful upscaling to both full-length constructs and increased 32 expression scale. The obtained fluorescent probes of murine FAS type I could be 33 subjected readily to a conformational analysis using single-molecule fluorescence 34 resonance energy transfer. 35All rights reserved. No reuse allowed without permission.(which was not peer-reviewed) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.

The architectures of iterative type I PKS and FAS

“…These fragments have successfully been employed in screening small-molecule FAS inhibitors as potential leads against cancer and metabolic disorders. mFAS continues to serve as a test-bed for functional studies of ACP-containing multienzymes, as exemplified by attempts towards analysing mFAS dynamics at the single molecule level via fluorescence methods 136 or high-speed atomic force microscopy 41 .…”