Tamayo-Nuñez Jessica, de la Mora Javier, Padilla-Vaca Felipe, Vargas-Maya Naurú Idalia, Rangel-Serrano Ángeles, Anaya-Velázquez Fernando, Páramo-Pérez Itzel, Reyes-Martínez Juana Elizabeth, España-Sánchez Beatríz Liliana, Franco Bernardo
Departamento de Biologia, Division de Ciencias Naturales y Exactas, Universidad de Guanajuato, Noria Alta s/n, Guanajuato, Gto. 36050, Mexico.
Instituto de Fisiologia Celular, Universidad Nacional Autonoma de Mexico, Circuito Exterior S/N, Mexico City, 04510, Mexico.
Protein Pept Lett. 2020;27(1):74-84. doi: 10.2174/0929866526666190806145740.
Marine sessile organisms display a color palette that is the result of the expression of fluorescent and non-fluorescent proteins. Fluorescent proteins have uncovered transcriptional regulation, subcellular localization of proteins, and the fate of cells during development. Chromoproteins have received less attention until recent years as bioreporters. Here, we studied the properties of aeBlue, a a 25.91 kDa protein from the anemone Actinia equina.
To assess the properties of aeBlue chromoprotein under different physicochemical conditions.
In this article, during the purification of aeBlue we uncovered that it suffered a color shift when frozen. We studied the color shift by different temperature incubation and physicochemical conditions and light spectroscopy. To assess the possible structural changes in the protein, circular dichroism analysis, size exclusion chromatography and native PAGE was performed.
We uncover that aeBlue chromoprotein, when expressed from a synthetic construct in Escherichia coli, showed a temperature dependent color shift. Protein purified at 4 °C by metal affinity chromatography exhibited a pinkish color and shifts back at higher temperatures to its intense blue color. Circular dichroism analysis revealed that the structure in the pink form of the protein has reduced secondary structure at 4 °C, but at 35 °C and higher, the structure shifts to a native conformation and Far UV- vis CD spectra revealed the shift in an aromatic residue of the chromophore. Also, the chromophore retains its properties in a wide range of conditions (pH, denaturants, reducing and oxidants agents). Quaternary structure is also maintained as a tetrameric conformation as shown by native gel and size exclusion chromatography.
Our results suggest that the chromophore position in aeBlue is shifted from its native position rendering the pink color and the process to return it to its native blue conformation is temperature dependent.
海洋固着生物呈现出的色彩组合是荧光蛋白和非荧光蛋白表达的结果。荧光蛋白已揭示了转录调控、蛋白质的亚细胞定位以及发育过程中细胞的命运。直到近年来，作为生物报告分子，色素蛋白受到的关注较少。在此，我们研究了来自海葵（Actinia equina）的一种25.91 kDa的aeBlue蛋白的特性。
评估aeBlue色素蛋白在不同物理化学条件下的特性。
在本文中，我们在纯化aeBlue的过程中发现，它在冷冻时会发生颜色变化。我们通过不同温度孵育、物理化学条件和光谱学研究了这种颜色变化。为了评估蛋白质可能的结构变化，进行了圆二色性分析、尺寸排阻色谱和非变性聚丙烯酰胺凝胶电泳。
我们发现，当aeBlue色素蛋白在大肠杆菌中由合成构建体表达时，呈现出温度依赖性颜色变化。通过金属亲和色谱在4℃纯化的蛋白质呈粉红色，在较高温度下会变回深蓝色。圆二色性分析表明，该蛋白质粉红色形式的结构在4℃时二级结构减少，但在35℃及更高温度下，结构转变为天然构象，远紫外可见圆二色光谱显示发色团的芳香族残基发生了变化。此外，发色团在广泛的条件（pH、变性剂、还原剂和氧化剂）下都能保持其特性。如非变性凝胶和尺寸排阻色谱所示，四级结构也保持为四聚体构象。
我们的结果表明，aeBlue中发色团的位置从其天然位置发生了偏移，从而产生粉红色，而使其恢复到天然蓝色构象的过程取决于温度。
