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Fluorescence of a Histidine-Modified Enhanced Green Fluorescent Protein (EGFP) Effectively Quenched by Copper(II) Ions. Part II. Molecular Determinants

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Abstract

The histidine-modified EGFP was characterized as a sensing element that preferentially binds nanomolar concentrations of Cu2+ in a reversible manner (Kd = 15 nM). This research aims to determine the causes of nanomolar-affinity of this mutant by investigating significant structural and energetic alterations of the chromophore in the presence of different copper ion concentrations. In order to reveal the unknown parts of the quenching mechanism we have elaborated a specific approach that combines theoretical and experimental techniques. The theoretical experiment included the modeling of potential distortions of the chromophores and the corresponding changes in energy using quantum mechanical calculations. Differences between the modeled energy profiles of planar and distorted conformations represented the energies of activation for the chromophore distortions. We found that some values of the experimental activation energies, which were derived from fluorescence lifetime decay analysis (ex: 470 nm, em: 507 nm), were consistent with the theoretical ones. Thus, it has been revealed similarity between the theoretical activation energy (50 kJmol−1) for 40° phenolate-ring distortion and the experimental activation energy (52.17 kJmol−1) required for histidine-modified EGFP saturation with copper. This chromophore conformation was further investigated and it has been found that the large decrease in fluorescence emission is attributed to the significant charge transfer over the molecule which triggers proton transfer thereby neutralizing the cromophore.

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References

  1. Hsiu-Chuan LV, Chien MT, Tseng YY, Ou KL (2006) Assessment of heavy metal bioavailability in contaminated sediments and soils using green fluorescent protein-based bacterial biosensors. Environ Pollut 142(1):17–23

    Article  Google Scholar 

  2. Hartter DE, Barnea A (1988) Brain tissue accumulates 67copper by two ligand-dependent saturable processes. A high affinity, low capacity and a low affinity, high capacity process. J Biol Chem 263:799–805

    CAS  PubMed  Google Scholar 

  3. Lovell MA, Robertson JD, Teesdale WJ, Campbell JL, Markesbery WR (1998) Copper, iron and zinc in Alzheimer's disease senile plaques. J Neurol Sci 158(1):47–52

    Article  CAS  PubMed  Google Scholar 

  4. Desai V, Kaler S (2008) Role of copper in human neurological disorders. Am J Clin Nutr 88(3):855S–858S

    CAS  PubMed  Google Scholar 

  5. Huang GG, Yang J (2003) Selective detection of copper ions in aqueous solution based on an evanescent wave infrared absorption spectroscopic method. Anal Chem 75(10):2262–2269

    Article  CAS  PubMed  Google Scholar 

  6. Lin M, Cho M, Choe WS, Son Y, Lee Y (2009) Electrochemical detection of copper ion using a modified copolythiophene electrode. Electrochim Acta 54(27):7012–7017

    Article  CAS  Google Scholar 

  7. Yang M, Yu Y, Shen F, Dierks K, Fang W, Li Q (2010) Detection of copper ion with laser-induced fluorescence in a capillary electrophoresis microchip. Anal Lett 43(18):2883–2891

    Article  CAS  Google Scholar 

  8. Isarankura-Na-Ayudhya C, Tantimongcolwat T, Galla HJ, Prachayasittikul V (2010) Fluorescent protein-based optical biosensor for copper Ion quantitation. Biol Trace Elem Res 134(3):352–363

    Article  CAS  PubMed  Google Scholar 

  9. Kneen M, Farinas J, Li Y, Verkman AS (1998) Green fluorescent protein as a noninvasive intracellular pH indicator. Biophys J 74(3):1591–1599

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  10. Szabó (P) M, Petres J, Szilágyi L, Miklóssy I, Ábrahám B, Lányi S (2012) Possible application of metal sensitive red fluorescent proteins in environmental monitoring. Environ Eng Manag J 11(1):193–198

    Google Scholar 

  11. Richmond TA, Takahashi TT, Shimkhada R, Bernsdorf J (2000) Engineered metal binding sites on green fluorescence protein. Biochem Biophys Res Commun 268(2):462–465

    Article  CAS  PubMed  Google Scholar 

  12. Palfi M, Kovács E, Miklóssy I, Szilágyi L, Ábrahám B (2009) Engineered green fluorescent protein as a potential metal sensor. Studia Universitatis Babes-Bolyai Chemia, Special Issue: 35–45

  13. Bálint EÉ, Petres J, Szabó M, Orbán CK, Szilágyi L, Ábrahám B (2013) Fluorescence of a histidine-modified enhanced Green Fluorescent Protein (EGFP) effectively quenched by copper(II) ions. J Fluoresc 23(2):273–281

    Article  PubMed  Google Scholar 

  14. Baker M (2011) Microscopy: bright light, better labels. Nature 478:137–142

    Article  CAS  PubMed  Google Scholar 

  15. Chen MC, Lambert CR, Urgitis JD, Zimmer M (2001) Photoisomerization of green fluorescent protein and the dimensions of the chromophore cavity. Chem Phys 270:157–164

    Article  CAS  Google Scholar 

  16. West CW, Hudson AS, Cobb SL, Verlet JRR (2013) Communication: autodetachment versus internal conversion from the S1 state of the isolated GFP chromophore anion. J Chem Phys 139:071104–1–071104–4

    Google Scholar 

  17. Follenius-Wund A, Bourotte M, Schmitt M, Iyice F, Lami H, Bourguignon JJ, Haiech J, Pigault C (2003) Fluorescent derivatives of the GFP chromophore give a new insight into the GFP fluorescence process. Biophys J 85(3):1839–1850

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  18. Kummer AD, Wiehler J, Rehaber H, Kompa C, Steipe B, Michel-Beyerle ME (2000) Effects of threonine 203 replacements on excited-state dynamics and fluorescence properties of the Green Fluorescent Protein (GFP). J Phys Chem B 104(19):4791–4798

    Article  CAS  Google Scholar 

  19. Jung G, Wiehler J, Zumbusch A (2005) The photophysics of green fluorescent protein: influence of the key amino acids at positions 65, 203, and 222. Biophys J 88(3):1932–1947

    Article  CAS  PubMed Central  PubMed  Google Scholar 

  20. Cheng CW, Huang GJ, Hsu HY, Prabhakar C, Lee YP, Diau EWG, Yang JS (2013) Effects of hydrogen bonding on internal conversion of GFP-like chromophores. II. The meta-amino systems. J Phys Chem B 117:2705–2716

    Article  CAS  PubMed  Google Scholar 

  21. Paul BK, Guchhait N (2013) Looking at the Green Fluorescent Protein (GFP) chromophore from a different perspective: a computational insight. Spectrochim Acta A Mol Biomol Spectrosc 103:295–303

    Article  CAS  PubMed  Google Scholar 

  22. Van Den Zegel M, Boens N, Daems D, DeSchryver FC (1986) Possibilities and limitations of the timecorrelated single photon counting technique: a comparative study of correction methods for the wavelength dependence of the instrument response function. Chem Phys 101:311–335

    Article  Google Scholar 

  23. Granovsky AA, Firefly version 8.0.0, http://classic.chem.msu.su/gran/firefly/index.html

  24. Marques MA, López X, Varsano D, Castro A, Rubio A (2003) Time-dependent density-functional approach for biological chromophores: the case of the green fluorescent protein. Phys Rev Lett 90(25):258101-1–258101-4

    Article  Google Scholar 

  25. Fang C, Frontiera RR, Tran R, Mathies RA (2009) Mapping GFP structure evolution during proton transfer with femtosecond Raman spectroscopy. Nature 462:200–204

    Article  CAS  PubMed  Google Scholar 

  26. Lossau H, Kummer A, Heinecke R, Pöllinger-Dammer F, Kompa C, Bieser G, Jonsson T, Silva CM, Yang MM, Youvan DC, Michel-Beyerle ME (1996) Time-resolved spectroscopy of wild-type and mutant green fluorescent proteins reveals excited state deprotonation consistent with fluorophore–protein interactions. Chem Phys 213:1–16

    Article  CAS  Google Scholar 

  27. Voityuk AA, Kummer AD, Michel-Beyerle ME, Rösch N (2001) Absorption spectra of the GFP chromophore in solution: comparision of theoretical and experimental results. Chem Phys 269:83–91

    Article  CAS  Google Scholar 

  28. Polyakov IV, Grigorenko BL, Epifanovsky EM, Krylov AI, Nemukhin AV (2010) Potential energy landscape of the electronic states of the GFP chromophore in different protonation forms: electronic transition energies and conical intersections. J Chem Theory Comput 6:2377–2387

    Article  CAS  Google Scholar 

  29. Philips GN Jr (1996) Structure and dynamics of green fluorescent proteins. Curr Opin Struct Biol 7(6):821–827

    Article  Google Scholar 

  30. Epifanovsky E, Polyakov I, Grigorenko B, Nemukhin A, Krylov AI (2009) Quantum chemical benchmark studies of the electronic properties of the green fluorescent protein chromophore. 1. Electronically excited and ionized states of the anionic chromophore in the gas phase. J Chem Theory Comput 5:1895–1906

    Article  CAS  Google Scholar 

  31. Laino T, Nifosí R, Tozzini V (2004) Relationship between structure and optical properties in green fluorescent proteins: a quantum mechanical study of the chromophore environment. Chem Phys 298:17–28

    Article  CAS  Google Scholar 

  32. Lakowicz JR (2006) Principles of fluorescence spectroscopy, 3rd edn. Springer Science+Business Media, New York

    Book  Google Scholar 

  33. Hötzer B, Ivanov R, Altmeier S, Kappl R, Jung G (2011) Determination of copper(II) Ion concentration by lifetime measurements of green fluorescent protein. J Fluoresc 21:2143–2153

    Article  PubMed  Google Scholar 

  34. Huang GJ, Cheng CW, Hsu HY, Prabhakar C, Lee YP, Diau EWG, Yang JS (2013) Effects of hydrogen bonding on internal conversion of GFP-like chromophores. I. The para-amino systems. J Phys Chem B 117:2695–2704

    Article  CAS  PubMed  Google Scholar 

  35. Hasegawa JY, Fujimoto K, Swerts B, Miyahara T, Nakatsuji H (2007) Excited states of GFP chromophore and active site studied by the SAC-CI method: effect of protein-environment and mutations. J Comput Chem 28:2443–2452

    Article  CAS  PubMed  Google Scholar 

  36. Lill MA, Helms V (2001) Proton shuttle in green fluorescent protein studied by dynamic simulations. PNAS 99(5):2778–2781

  37. Arpino JAJ, Rizkallah PJ, Jones DD (2012) Crystal structure of enhanced green fluorescent protein to 1.35 a° resolution reveals alternative conformations for Glu222. PLos One 7(10):e4132/1-8

    Article  Google Scholar 

  38. Sniegowski JA, Phail ME, Wachter RM (2005) Maturation efficiency, trypsin sensitivity, and optical properties of Arg96, Glu222, and Gly67 variants of green fluorescent protein. Biochem Biophys Res Commun 332:657–663

    Article  CAS  PubMed  Google Scholar 

  39. Voityuk AA, Michel-Beyerle ME, Rösch N (1998) Quantum chemical modeling of structure and absorption spectra of the chromophore in green fluorescent proteins. Chem Phys 231:13–25

    Article  CAS  Google Scholar 

Download references

Acknowledgments

The work has been funded by the Sectorial Operational Programme Human Resources Development 2007–2013 of the Romanian Ministry of Labour, Family and Social Protection through the Financial Agreement POSDRU/88/1.5/S/60203 and by the University of Sapientia, Department of Bioengineering.

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Authors and Affiliations

  1. Department of Inorganic Substances Technology and Environment, Protection, “Politechnica” University of Bucharest, Polizu street No. 1-7, 011061, Bucharest, Romania

    Judit (Petres) Péterffy

  2. Department of Bioengineering, Sapientia Hungarian University of Transylvania, Libertatii square No.1, 530104, Miercurea Ciuc, Romania

    Mária Szabó, László Szilágyi, Szabolcs Lányi & Beáta Ábrahám

  3. Institute of Biology, Department of Biochemistry, Eötvös Loránd University, Pázmány P. s. 1C, 1117, Budapest, Hungary

    László Szilágyi

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  1. Judit (Petres) Péterffy
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  2. Mária Szabó
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Correspondence to Judit (Petres) Péterffy.

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Péterffy, J.(., Szabó, M., Szilágyi, L. et al. Fluorescence of a Histidine-Modified Enhanced Green Fluorescent Protein (EGFP) Effectively Quenched by Copper(II) Ions. Part II. Molecular Determinants. J Fluoresc 25, 871–883 (2015). https://doi.org/10.1007/s10895-015-1567-4

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  • DOI: https://doi.org/10.1007/s10895-015-1567-4

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