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.
Sci Rep. 2018 Oct 5;8(1):14864. doi: 10.1038/s41598-018-33115-5.
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.
获得关于大型蛋白质动态行为的信息通常受到限制，因为光谱方法需要在特定位置附着生物物理探针。一种强大的新兴工具可以解决这个问题，那就是琥珀密码子抑制。迄今为止，尚未有报道将其应用于 MDa 大小的大型复杂多域蛋白质。在此，我们通过遗传密码扩展系统地研究了在同源二聚体脂肪酸合酶 I 中引入不同非天然氨基酸的可行性，随后进行荧光标记。我们的方法依赖于使用 GFP 融合蛋白的基于微孔板的低复杂度报告基因测定，快速筛选出足够的抑制条件。一旦确定，这些发现被成功地用于扩大表达规模和蛋白质大小至全长构建体。这些脂肪酸合酶的荧光标记样品进行了初步的生物物理实验，包括 HPLC 分析、活性测定和荧光光谱分析。成功地将这些探针引入脂肪酸合酶等分子机器中，可能为理解构象可变性铺平道路，这是所有催化功能有效相互作用所必需的主要内在特性，并为其工程化提供了可能。
