aeBlue Chromoprotein Color is Temperature Dependent

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.

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Combination of circular dichroism spectroscopy and size-exclusion chromatography coupled with HDX-MS for studying global conformational structures of peptides in solution

Talanta ◽

10.1016/j.talanta.2018.09.077 ◽

2019 ◽

Vol 194 ◽

pp. 177-182 ◽

Cited By ~ 4

Author(s):

Nicole M. Schiavone ◽

Gregory F. Pirrone ◽

Erik D. Guetschow ◽

Ian Mangion ◽

Alexey A. Makarov

Keyword(s):

Circular Dichroism ◽

Size Exclusion Chromatography ◽

Circular Dichroism Spectroscopy ◽

Size Exclusion ◽

Exclusion Chromatography ◽

Hdx Ms ◽

Circular Dichroïsm

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Investigation of thermoreversible polymer networks by temperature dependent size exclusion chromatography

Polymer Chemistry ◽

10.1039/c7py01262d ◽

2017 ◽

Vol 8 (43) ◽

pp. 6598-6605 ◽

Cited By ~ 5

Author(s):

Josef Brandt ◽

Johannes Lenz ◽

Kai Pahnke ◽

Friedrich Georg Schmidt ◽

Christopher Barner-Kowollik ◽

...

Keyword(s):

Size Exclusion Chromatography ◽

Polymer Networks ◽

Diels Alder ◽

Size Exclusion ◽

Temperature Dependent ◽

Novel Approach ◽

Exclusion Chromatography

We introduce a novel approach for studying thermoreversible Diels–Alder networks by Temperature Dependent SEC.

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Variable-Temperature Size Exclusion Chromatography for the Study of the Structural Changes in G-Quadruplex

ISRN Biochemistry ◽

10.1155/2013/631875 ◽

2013 ◽

Vol 2013 ◽

pp. 1-7 ◽

Cited By ~ 1

Author(s):

Sanae Benabou ◽

Ramon Eritja ◽

Raimundo Gargallo

Keyword(s):

Size Exclusion Chromatography ◽

Promoter Region ◽

Structural Changes ◽

Variable Temperature ◽

Size Exclusion ◽

G Quadruplex ◽

Conformational Equilibria ◽

Exclusion Chromatography ◽

In The Wild ◽

Circular Dichroïsm

The conformational equilibria of a guanine-rich sequence found at the promoter region of the human c-kit oncogene are studied by means of circular dichroism spectroscopy (CD) and variable-temperature size exclusion chromatography (SEC). It is shown that the wild sequence ckit21 exists as a mixture of monomeric and multimeric G-quadruplexes. Appropriate mutation of several bases in the wild sequence produces the shift from parallel to antiparallel G-quadruplex, as well as the disappearance of multimeric species. The shift from the antiparallel to the parallel conformation induced by temperature is reflected in both CD and SEC profiles.

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Conformation and stability of the Streptococcus pyogenes pSM19035-encoded site-specific β recombinase, and identification of a folding intermediate

Biological Chemistry ◽

10.1515/bc.2006.068 ◽

2006 ◽

Vol 387 (5) ◽

pp. 525-533 ◽

Cited By ~ 2

Author(s):

Anshul Bhardwaj ◽

Karin Welfle ◽

Rolf Misselwitz ◽

Silvia Ayora ◽

Juan C. Alonso ◽

...

Keyword(s):

Circular Dichroism ◽

Ionic Strength ◽

Analytical Ultracentrifugation ◽

Size Exclusion ◽

Sedimentation Equilibrium ◽

Folding Intermediate ◽

Guanidinium Chloride ◽

Site Specific ◽

Exclusion Chromatography ◽

Circular Dichroïsm

Abstract Solution properties of β recombinase were studied by circular dichroism and fluorescence spectroscopy, size exclusion chromatography, analytical ultracentrifugation, denaturant-induced unfolding and thermal unfolding experiments. In high ionic strength buffer (1 M NaCl) β recombinase forms mainly dimers, and strongly tends to aggregate at ionic strength lower than 0.3 M NaCl. Urea and guanidinium chloride denaturants unfold β recombinase in a two-step process. The unfolding curves have bends at approximately 5 M and 2.2 M in urea and guanidinium chloride-containing buffers. Assuming a three-state unfolding model (N2→2I→2U), the total free energy change from 1 mol of native dimers to 2 mol of unfolded monomers amounts to ΔG tot=17.9 kcal/mol, with ΔG N2→2I=4.2 kcal/mol for the first transition and ΔG I→U=6.9 kcal/mol for the second transition. Using sedimentation-equilibrium analytical ultracentrifugation, the presence of β recombinase monomers was indicated at 5 M urea, and the urea dependence of the circular dichroism at 222 nm strongly suggests that folded monomers represent the unfolding intermediate.

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