Land and Water, CSIRO, Clunies Ross Street, Canberra, ACT 2601, Australia.
Manufacturing, CSIRO, Research Way, Clayton, VIC 3168, Australia.
Acta Crystallogr D Struct Biol. 2022 May 1;78(Pt 5):599-612. doi: 10.1107/S2059798322002625. Epub 2022 Apr 8.
Anthozoan chromoproteins are highly pigmented, diversely coloured and readily produced in recombinant expression systems. While they are a versatile and powerful building block in synthetic biology for applications such as biosensor development, they are not widely used in comparison to the related fluorescent proteins, partly due to a lack of structural characterization to aid protein engineering. Here, high-resolution X-ray crystal structures of four open-source chromoproteins, gfasPurple, amilCP, spisPink and eforRed, are presented. These proteins are dimers in solution, and mutation at the conserved dimer interface leads to loss of visible colour development in gfasPurple. The chromophores are trans and noncoplanar in gfasPurple, amilCP and spisPink, while that in eforRed is cis and noncoplanar, and also emits fluorescence. Like other characterized chromoproteins, gfasPurple, amilCP and eforRed contain an sp-hybridized N-acylimine in the peptide bond preceding the chromophore, while spisPink is unusual and demonstrates a true sp-hybridized trans-peptide bond at this position. It was found that point mutations at the chromophore-binding site in gfasPurple that substitute similar amino acids to those in amilCP and spisPink generate similar colours. These features and observations have implications for the utility of these chromoproteins in protein engineering and synthetic biology applications.
腔肠动物色蛋白高度色素化，颜色多样，易于在重组表达系统中产生。虽然它们在合成生物学中是一种多功能且强大的构建模块，可用于生物传感器开发等应用，但与相关的荧光蛋白相比，它们的应用并不广泛，部分原因是缺乏结构特征来辅助蛋白质工程。在这里，介绍了四种开源色蛋白 gfasPurple、amilCP、spisPink 和 eforRed 的高分辨率 X 射线晶体结构。这些蛋白质在溶液中为二聚体，在保守的二聚体界面处发生突变会导致 gfasPurple 失去可见的颜色发展。gfasPurple、amilCP 和 spisPink 中的生色团为反式且非共面，而 eforRed 中的生色团为顺式且非共面，并且还发出荧光。与其他经过表征的色蛋白一样，gfasPurple、amilCP 和 eforRed 在色团前的肽键中含有 sp 杂化的 N-酰亚胺，而 spisPink 则不同，在该位置显示出真正的 sp 杂化反式肽键。研究发现，gfasPurple 中色团结合位点的点突变取代了与 amilCP 和 spisPink 中相似的氨基酸，会产生相似的颜色。这些特征和观察结果对这些色蛋白在蛋白质工程和合成生物学应用中的实用性具有重要意义。
