Abstract
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.
Highlights
While the overall architecture of type I PKSs has not yet been firmly elucidated[10,11], high resolution data of FASs in different structural arrangements are available[6,12]
The results demonstrate that non-canonical amino acids (ncAAs) labelling is a promising new tool that can be used to characterize the dynamics of large megasynthases and may help in better understanding the molecular mechanism of such biosynthetic machineries
Out of a growing repertoire of ncAAs that are introduced by amber codon suppression, we limited our screening to eight different ncAAs with functional groups that can be used for bioconjugation with click chemistry or oxime formation (Fig. 2A; for syntheses of respective ncAAs, see Supplementary Methods)[19]
Summary
While the overall architecture of type I PKSs has not yet been firmly elucidated[10,11], high resolution data of FASs in different structural arrangements are available[6,12]. More detailed insights into the conformational versatility of animal FAS were given by a recent negative stain electron microscopy (EM) study on rat FAS, high-speed atomic force microscopy on insect FAS and by computational modelling with porcine FAS data[7,8,15]. These studies revealed large conformational changes within the enzyme with complete relative rotational and swinging freedom between the condensing and processing wing (Fig. 1C). Our method of choice was the genetic encoding of non-canonical amino acids (ncAAs) through the amber codon suppression technology[19,20] Such ncAAs carry orthogonal functional groups, which can be used to site- attach spectroscopic labels by post-translational modification. The results demonstrate that ncAA labelling is a promising new tool that can be used to characterize the dynamics of large megasynthases and may help in better understanding the molecular mechanism of such biosynthetic machineries
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