aeBlue Chromoprotein Color is Temperature Dependent

Abstract

Background: 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. Objective: To assess the properties of aeBlue chromoprotein under different physicochemical conditions. Method: 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. Results: 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. Conclusion: 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.

Biodesign of Frugal CRISPR Kits for Equitable and Accessible Education

“…2d ). On the contrary, aeBlue CRISPRi showed a smaller fold change (2.5-fold), likely due to the suboptimal measurement using absorbance 24 . Yet, visually from parafilm or tube images taken by smartphone, we observed strong pigmentation without CRISPRi and almost no color with CRISPRi ( Fig.…”

“…We further linked CRISPRi-based Streptococcus pyogenes dCas9 activity to a pigmentation readout via the control of chromoproteins, which are pigmented proteins that do not require special light wavelengths or filters to visualize. We chose three chromoproteins with visually distinct colors that resemble the RGB color mode (red, yellow/green, and blue): eforRed 20 , fwYellow 21 , and aeBlue 22 . We designed single guide RNAs (sgRNAs) that specifically target the protein-coding sequence of each chromoprotein to achieve efficient and specific transcriptional repression.…”

“…2c , see Methods ). We chose to measure absorbance of aeBlue because, unlike eforRed or fwYellow, there is not a suitable fluorescence excitation/emission wavelength that has been found for this protein 22 .…”

Chromogenic Escherichia coli reporter strain for screening DNA damaging agents

“…Once diluted by diffusion, the second aspect is the activation of the reporter strain, that canbe evidenced in the surrounding cell death zone and the 'on/off' response can provide information for fast decision making in a similar fashion as described by Zhang et al ( 2013 ) or generate more portable and complementary tools for in-field detection (Roggo and van der Meer 2017 ). The choice of AmilCP is based on a previous study that showed that AmilCP is stable at a wide range of pH values and exposure to urea and an oxidant (Tamayo-Nuñez et al 2020 ), thus making it suitable as a reporter under harsh conditions, such as contaminated water and soil samples (Jiang et al 2017 ).…”