aeBlue Chromoprotein Color is Temperature Dependent

Author(s): Jessica Tamayo-Nuñez, Javier de la Mora, Felipe Padilla-Vaca, Naurú Idalia Vargas-Maya, Ángeles Rangel-Serrano, Fernando Anaya-Velázquez, Itzel Páramo-Pérez, Juana Elizabeth Reyes-Martínez, Beatríz Liliana España-Sánchez, Bernardo Franco*

Journal Name: Protein & Peptide Letters

Volume 27 , Issue 1 , 2020

Become EABM
Become Reviewer

Graphical 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.

Methods: 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.

Keywords: Aeblue chromoprotein, color shift, protein secondary structure analysis, chromophore, cold chain reporter, bioreporters.

Alieva, N.O.; Konzen, K.A.; Field, S.F.; Meleshkevitch, E.A.; Hunt, M.E.; Beltran-Ramirez, V.; Miller, D.J.; Wiedenmann, J.; Salih, A.; Matz, M.V. Diversity and evolution of coral fluorescent proteins. PLoS One, 2008, 3(7)e2680
[] [PMID: 18648549]
Dove, S.G.; Hoegh-Guldberg, O.; Ranganathan, S. Major color patterns of reef-building corals are due to a family of GFP-like proteins. Coral Reefs, 2001, 19, 197-204.
Prescott, M.; Ling, M.; Beddoe, T.; Oakley, A.J.; Dove, S.; Hoegh-Guldberg, O.; Devenish, R.J.; Rossjohn, J. The 2.2 A crystal structure of a pocilloporin pigment reveals a nonplanar chromophore conformation. Structure, 2003, 11(3), 275-284.
[] [PMID: 12623015]
Levy, O.; Appelbaum, L.; Leggat, W.; Gothlif, Y.; Hayward, D.C.; Miller, D.J.; Hoegh-Guldberg, O. Light-responsive cryptochromes from a simple multicellular animal, the coral Acropora millepora. Science, 2007, 318(5849), 467-470.
[] [PMID: 17947585]
Zawada, D.G.; Mazel, C.H. Fluorescence-based classification of Caribbean coral reef organisms and substrates. PLoS One, 2014, 9(1)e84570
[] [PMID: 24482676]
Battad, J.M.; Wilmann, P.G.; Olsen, S.; Byres, E.; Smith, S.C.; Dove, S.G.; Turcic, K.N.; Devenish, R.J.; Rossjohn, J.; Prescott, M. A structural basis for the pH-dependent increase in fluorescence efficiency of chromoproteins. J. Mol. Biol., 2007, 368(4), 998-1010.
[] [PMID: 17376484]
Liljeruhm, J.; Funk, S.K.; Tietscher, S.; Edlund, A.D.; Jamal, S.; Wistrand-Yuen, P. Dyrhage, K.; Gynnå, A.; Ivermark, K.; Lövgren, J.; Törnblom, V.; Virtanen, A.; Lundin, ER.; Wistrand-Yuen, E.; Forster, A.C. Engineering a palette of eukaryotic chromoproteins for bacterial synthetic biology. J. Biol. Eng., 2018, 12, 8.
[] [PMID: 29760772]
Tafoya-Ramírez, M.D.; Padilla-Vaca, F.; Ramírez-Saldaña, A.P.; Mora-Garduño, J.D.; Rangel-Serrano, A.; Vargas-Maya, N.I.; Herrera-Gutiérrez, L.J.; Franco, B. Replacing standard reporters from molecular cloning plasmids with Chromoproteins for positive clone selection. Molecules, 2018, 23(6), 31.
Chiang, C.Y.; Lee, C.C.; Lo, S.Y.; Wang, A.H.; Tsai, H.J. Chromophore deprotonation state alters the optical properties of blue chromoproteins. PLoS One, 2015, 10(7), 28.
[] [PMID: 26218063]
Shkrob, M.A.; Yanushevich, Y.G.; Chudakov, D.M.; Gurskaya, N.G.; Labas, Y.A.; Poponov, S.Y.; Mudrik, N.N.; Lukyanov, S.; Lukyanov, K.A. Far-red fluorescent proteins evolved from a blue chromoprotein from Actinia equina. Biochem. J., 2005, 392(Pt 3), 649-654.
[] [PMID: 16164420]
Simeon, S.; Shoombuatong, W.; Anuwongcharoen, N.; Preeyanon, L.; Prachayasittikul, V.; Wikberg, J.E.; Nantasenamat, C. osFP: a web server for predicting the oligomeric states of fluorescent proteins. J. Cheminform., 2016, 8, 72.
[] [PMID: 28053671]
Kelley, L.A.; Mezulis, S.; Yates, C.M.; Wass, M.N.; Sternberg, M.J. The Phyre2 web portal for protein modeling, prediction and analysis. Nat. Protoc., 2015, 10(6), 845-858.
[] [PMID: 25950237]
Johansson, M.U.; Zoete, V.; Michielin, O.; Guex, N. Defining and searching for structural motifs using DeepView/Swiss-PdbViewer. BMC Bioinformatics, 2012, 13, 173.
[] [PMID: 22823337]
Bowie, J.U.; Lüthy, R.; Eisenberg, D. A method to identify protein sequences that fold into a known three-dimensional structure. Science, 1991, 253(5016), 164-170.
[] [PMID: 1853201]
Lüthy, R.; Bowie, J.U.; Eisenberg, D. Assessment of protein models with three-dimensional profiles. Nature, 1992, 356(6364), 83-85.
[] [PMID: 1538787]
DeLano, W. The PyMOL Molecular Graphics System, Version 1.8; Shrodinger LLC: New York, NY, USA, 2015.
Zhang, Y.; Skolnick, J. TM-align: a protein structure alignment algorithm based on the TM-score. Nucleic Acids Res., 2005, 33(7), 2302-2309.
[] [PMID: 15849316]
Subach, F.V.; Subach, O.M.; Gundorov, I.S.; Morozova, K.S.; Piatkevich, K.D.; Cuervo, A.M.; Verkhusha, V.V. Monomeric fluorescent timers that change color from blue to red report on cellular trafficking. Nat. Chem. Biol., 2009, 5(2), 118-126.
[] [PMID: 19136976]
Dedecker, P.; De Schryver, F.C. Hofkens, J. Fluorescent proteins: shine on, you crazy diamond. J. Am. Chem. Soc., 2013, 135(7), 2387-2402.
[] [PMID: 23317378]
Chudakov, D.M.; Matz, M.V.; Lukyanov, S.; Lukyanov, K.A. Fluorescent proteins and their applications in imaging living cells and tissues. Physiol. Rev., 2010, 90(3), 1103-1163.
[] [PMID: 20664080]
Krasowska, J.; Olasek, M.; Bzowska, A.; Clark, P.L.; Wielgus-Kutrowska, B. The comparison of aggregation and folding of enhanced green fluorescent protein (EGFP) by spectroscopic studies. Spectroscopy (Springf.), 2010, 24, 343-348.
Delgado-Galván, C.J.; Padilla-Vaca, F.; Montiel, F.B.R.; Rangel-Serrano, Á.; Paramo-Pérez, I.; Anaya-Velázquez, F.; Franco, B. Red fluorescent protein (DsRFP) optimization for Entamoeba histolytica expression. Exp. Parasitol., 2018, 187, 86-92.
[] [PMID: 29476758]
Pletnev, S.; Subach, F.V.; Dauter, Z.; Wlodawer, A.; Verkhusha, V.V. Understanding blue-to-red conversion in monomeric fluorescent timers and hydrolytic degradation of their chromophores. J. Am. Chem. Soc., 2010, 132(7), 2243-2253.
[] [PMID: 20121102]
Cao, E.; Chen, Y.; Cui, Z.; Foster, P.R. Effect of freezing and thawing rates on denaturation of proteins in aqueous solutions. Biotechnol. Bioeng., 2003, 82(6), 684-690.
[] [PMID: 12673768]
Wicksten, M.K. Why are there bright colors in sessile marine invertebrates? Bull. Mar. Sci., 1989, 45, 519-530.
Leutenegger, A.; Kredel, S.; Gundel, S.; D’Angelo, C.; Salih, A.; Wiedenmann, J. Analysis of fluorescent and non-fluorescent sea anemones from the Mediterranean Sea during a bleaching event. J. Exp. Mar. Biol. Ecol., 2007, 353, 221-234.
Rodriguez-Lanetty, M.; Harii, S.; Hoegh-Guldberg, O. Early molecular responses of coral larvae to hyperthermal stress. Mol. Ecol., 2009, 18(24), 5101-5114.
[] [PMID: 19900172]
Seneca, F.O.; Forêt, S.; Ball, E.E.; Smith-Keune, C.; Miller, D.J.; van Oppen, M.J. Patterns of gene expression in a scleractinian coral undergoing natural bleaching. Mar. Biotechnol. (NY), 2010, 12(5), 594-604.
[] [PMID: 20041338]
Ivleva, I.V. Elements of energetic balance in sea anemones. Trans. Sevastopol Biol. Sta. Acad. Sci. USSR, 1964, 25, 410-428.
Chomskya, O.; Kamenira, Y.; Hyamsa, M.; Dubinskya, Z.; Chadwick-Furman, N.E. Effects of temperature on growth rate and body size in the Mediterranean Sea anemone Actinia equina. J. Exp. Mar. Biol. Ecol., 2004, 313, 63-73.
Wilmann, P.G.; Petersen, J.; Devenish, R.J.; Prescott, M.; Rossjohn, J. Variations on the GFP chromophore: A polypeptide fragmentation within the chromophore revealed in the 2.1-A crystal structure of a nonfluorescent chromoprotein from Anemonia sulcata. J. Biol. Chem., 2005, 280(4), 2401-2404.
[] [PMID: 15542608]
Turcic, K.; Pettikiriarachchi, A.; Battad, J.; Wilmann, P.G.; Rossjohn, J.; Dove, S.G.; Devenish, R.J.; Prescott, M. Amino acid substitutions around the chromophore of the chromoprotein Rtms5 influence polypeptide cleavage. Biochem. Biophys. Res. Commun., 2006, 340(4), 1139-1143.
[] [PMID: 16414348]

Rights & PermissionsPrintExport Cite as

Article Details

Year: 2020
Page: [74 - 84]
Pages: 11
DOI: 10.2174/0929866526666190806145740
Price: $65

Article Metrics

PDF: 27