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An insight into pH-induced changes in FAD conformational structure by means of time-resolved fluorescence and circular dichroism

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Abstract

Optical properties of flavin adenine dinucleotide (FAD) moiety are widely used nowadays for biotechnological applications. Given the fundamental role played by FAD, additional structural information about this enzymatic cofactor can be extremely useful in order to obtain a greater insight into its functional role in proteins. For this purpose, we have investigated FAD behaviour in aqueous solutions at different pH values by a novel approach based on the combined use of time-resolved fluorescence and circular dichroism spectroscopies. The results showed that pH strongly affects time-resolved fluorescence emission and the analysis allowed us to detect a three-component decay for FAD in aqueous solution with pH-depending lifetimes and relative amplitudes. Circular dichroism data were analyzed by a multi-Gaussian fitting procedure and the trends of properly chosen parameters confirmed pH-depending changes. The comparison between the results obtained by these two optical techniques allowed us to improve the significance of the outcome of circular dichroism. This combined approach may provide a useful tool for biotechnological investigation.

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References

  • Berova N, Nakanishi K, Woody RW (2000) Circular dichroism: principles and applications, 2nd edn. Wiley, New York

    Google Scholar 

  • Bloomfield VA, Crothers DM, Tinoco I Jr (2000) Nucleic acids: structures, properties, and functions. University Science Books, Sausalito

    Google Scholar 

  • Bruen D, Delaney C, Florea L, Diamond D (2017) Glucose sensing for diabetes monitoring: recent developments. Sensors 17:1866. https://doi.org/10.3390/s17081866

    Article  CAS  Google Scholar 

  • Chosrowjan H, Taniguchi S, Mataga N, Tanaka F, Visser AJWG (2003) The stacked flavin adenine nucleotide conformation in water is fluorescent on picoseconds timescale. Chem Phys Lett 378:354–358

    Article  CAS  Google Scholar 

  • D’Anna JA, Tollin G (1972) Studies of flavin–protein interaction in flavoproteins using protein fluorescence and circular dichroism. Biochemistry 11:1073–1080

    Article  PubMed  Google Scholar 

  • Delfino I, Esposito R, Portaccio M, Lepore M (2017) Dynamical and structural properties of flavin adenine dinucleotide in aqueous solutions and bound to free and sol-gel immobilized glucose oxidase. J Sol Gel Sci Technol 82(1):239–252

    Article  CAS  Google Scholar 

  • Dijkman WP, de Gonzalo G, Mattevi A, Fraaije MW (2013) Flavoprotein oxidases: classification and applications. Appl Microbiol Biotechnol. 97:5177–5188

    Article  CAS  PubMed  Google Scholar 

  • Drossler P, Holzer W, Penzkofer A, Hegemann P (2002) pH dependence of the absorption and emission behaviour of riboflavin in aqueous solution. Chem Phys 282:429–439

    Article  CAS  Google Scholar 

  • Dumitraşcu L, Stănciuc N, Bahrim GE, Ciumac A, Aprodu I (2016) pH and heat-dependent behaviour of glucose oxidase down to single molecule level by combined fluorescence spectroscopy and molecular modeling. J Sci Food Agric 96:1906–1914

    Article  CAS  PubMed  Google Scholar 

  • Edmondson DE, Tollin G (1971) Circular dichroism studies of the flavin chromophore and of the relation between redox properties and flavin environment in oxidases and dehydrogenases. Biochemistry 10:113–124

    Article  CAS  PubMed  Google Scholar 

  • Esposito R, Della Ventura B, De Nicola S, Altucci C, Velotta R, Mita DG, Lepore M (2011) Glucose sensing by time-resolved fluorescence of sol-gel immobilized glucose oxidase. Sensors 11:3483–3497

    Article  CAS  PubMed  Google Scholar 

  • Esposito R, Altucci C, Velotta R (2013) Analysis of simulated fluorescence intensities decays by a new maximum entropy method algorithm. J Fluoresci 23(1):203–211

    Article  CAS  Google Scholar 

  • Esposito R, Delfino I, Lepore M (2013) Time-resolved flavin adenine dinucleotide fluorescence study of the interaction between immobilized glucose oxidase and glucose. J Fluoresci 23(5):947–955

    Article  CAS  Google Scholar 

  • Esposito R, Mensitieri G, De Nicola S (2015) Improved maximum entropy method for the analysis of fluorescence spectroscopy data: evaluating zero-time shift and assessing its effect on the determination of fluorescence lifetimes. Analyst 140:8138–8147

    Article  CAS  PubMed  Google Scholar 

  • Fujiwara A, Mizutani Y (2008) Photoinduced electron transfer in glucose oxidase: a picosecond time-resolved ultraviolet resonance Raman study. J Raman Spectrosc 39:1600–1605

    Article  CAS  Google Scholar 

  • Galbán J, Sanz-Vicente I, Navarro J, de Marcos S (2016) The intrinsic fluorescence of FAD and its application in analytical chemistry: a review. Methods Appl Fluores 4:042005

    Article  CAS  Google Scholar 

  • Grininger M, Seiler F, Zeth K, Oesterhelt D (2006) Dodecin sequesters FAD in closed conformation from the aqueous solution. J Mol Biol 364:561–566

    Article  CAS  PubMed  Google Scholar 

  • Gu Q, Cheng C, Gonela R, Suryanarayanan S, Anabathula S, Dai K, Haynie DT (2006) DNA nanowire fabrication. Nanotechnology 17:R14–R25

    Article  CAS  Google Scholar 

  • Hartnett AM, Ingersoll CM, Baker GA, Bright FV (1999) Kinetics and thermodynamics of free flavins and the flavins-based redox active site within glucose oxidase dissolved in solution or sequestered within a sol–gel derived glass. Anal Chem 71:1215–1224

    Article  CAS  PubMed  Google Scholar 

  • Islam SDM, Susdorf T, Penzkofer A, Hegemann P (2003) Fluorescence quenching of flavin adenine dinucleotide in aqueous solution by pH dependent isomerisation and photo-induced electron transfer. Chem Phys 295:137–149

    Article  CAS  Google Scholar 

  • Islam MS, Honma M, Nakabayashi T, Kinjo M, Ohta N (2013) pH dependence of the fluorescence lifetime of FAD in solution and in cells. Int J Mol Sci 14:1952–1963. https://doi.org/10.3390/ijms14011952

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Joosten V, van Berkel WJ (2007) Flavoenzymes. Curr Opin Chem Biol 11:195–202

    Article  CAS  PubMed  Google Scholar 

  • Kelly SM, Price NC (2000) The use of circular dichroism in the investigation of protein structure and function. Curr Protein Pept Sci 1:349–384

    Article  CAS  PubMed  Google Scholar 

  • Lakowicz JR (2006) Principles of fluorescence spectroscopy, 2nd edn. Kluwer Academic, Plenum

    Book  Google Scholar 

  • Lepore M, Portaccio M, De Tommasi E, De Luca P, Bencivenga U, Maiuri P, Mita DG (2004) Glucose concentration determination by means of fluorescence emission spectra of soluble and insoluble glucose oxidase: some useful indications for optical fibre-based sensors. J Mol Catal B Enzym 31:151–158

    Article  CAS  Google Scholar 

  • Mataga N, Chosrowjan H, Shibata Y, Tanaka F, Nishina Y, Shiga K (2000) Dynamics and mechanisms of ultrafast fluorescence quenching reactions of flavin chromophores in protein nanospace. J Phys Chem 104:10667–10677

    Article  CAS  Google Scholar 

  • McShane MJ (2002) Potential for glucose monitoring with nanoengineered fluorescent biosensors. Diabetes Technol Therap 4:533–538

    Article  CAS  Google Scholar 

  • Miles DW, Urry DW (1968) Reciprocal relations and proximity of bases in flavin–adenin dinucleotide. Biochemistry 7:2791–2799

    Article  CAS  PubMed  Google Scholar 

  • Miles AJ, Wallace BA (2016) Circular dicrhroism of membrane proteins. Chem Soc Rev 45:4859–4872. https://doi.org/10.1039/c5cs00084j

    Article  CAS  PubMed  Google Scholar 

  • Molano-Arevalo JC, Hernandez DR, Gonzalez WG et al (2014) Flavin adenine dinucleotide structural motifs: from solution to gas phase. Anal Chem 86:10223–10230

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Nakabayashi T, Islam MS, Ohta N (2010) Fluorescence decay dynamics of flavin adenine dinucleotide in a mixture of alcohol and water in the femtosecond and nanosecond time range. J Phys Chem B 114:15254–15260

    Article  CAS  PubMed  Google Scholar 

  • Radoszkowicz L, Huppert D, Nachliel E, Gutman M (2009) Sampling the conformation space of FAD in water–methanol mixtures through molecular dynamics and fluorescence measurements. J Phys Chem A 114:1017–1022

    Article  CAS  Google Scholar 

  • Radoszkowicz L, Presiado I, Erez Y, Nachliel E, Huppert D, Gutman M (2011) Time-resolved emission of flavin adenine dinucleotide in water and water–methanol mixtures. Phys Chem Chem Phys 13:12058–12066

    Article  CAS  PubMed  Google Scholar 

  • Ranjbar B, Gill P (2009) Circular dichroism techniques: biomolecular and nanostructural analyses—a review. Chem Biol Drug Des 74:101–120

    Article  CAS  PubMed  Google Scholar 

  • Raszka M, Kaplan NO (1974) Intramolecular hydrogen bonding in flavin adenine dinucleotide. Proc Natl Acad Sci USA 71:4546–4550

    Article  CAS  PubMed  Google Scholar 

  • Robinson PK (2015) Enzymes: principles and biotechnological applications. Essays Biochem 59:1–41

    Article  PubMed  PubMed Central  Google Scholar 

  • Sengupta A, Khade RV, Hazra P (2011) pH dependent dynamic behavior of flavin mononucleotide (FMN) and flavin adenine dinucleotide (FAD) in femtosecond to nanosecond time scale. J. Photochem Photobiol A 221:105–112

    Article  CAS  Google Scholar 

  • Sengupta A, Singh RK, Gavvala K, Koninti RK, Mukherjee A, Hazra P (2014) Urea induced unfolding dynamics of flavin adenine dinucleotide (FAD): spectroscopic and molecular dynamics simulation studies from femto-second to nanosecond regime. J Phys Chem B 118:1881–1890

    Article  CAS  PubMed  Google Scholar 

  • Shao Q, Wu P, Xu X, Zhang H, Cai C (2012) Insight into the effects of graphene oxide sheets on the conformation and activity of glucose oxidase: towards developing a nanomaterial-based protein conformation assay. Phys Chem Chem Phys 14:9076–9085

    Article  CAS  PubMed  Google Scholar 

  • Stanley R, MacFarlane A (2000) Ultrafast excited state dynamics of oxidized flavins: direct observations of quenching by purines. J Phys Chem A 104:6899–6906

    Article  CAS  Google Scholar 

  • Tinoco I Jr (1962) Theoretical aspects of optical activity part two: polymers. Adv Chem Phys 4:113–160

    Google Scholar 

  • Tollin G (1968) Magnetic circular dichroism and circular dichroism of riboflavin and its analogs. Biochemistry 7:1720–1727

    Article  CAS  PubMed  Google Scholar 

  • van den Berg P, Feenstra K, Mark A, Berendsen H, Visser A (2002) Dynamic conformations of flavin adenine dinucleotide: simulated molecular dynamics of the flavin cofactor related to the time-resolved fluorescence characteristics. J Phys Chem B 106:8858–8869

    Article  CAS  Google Scholar 

  • Villar-Guerra R, Trent JO, Chaires JB (2018) G-quadruplex secondary structure obtained from circular dichroism spectroscopy. Angew Chem Int Ed 57:7171–7175

    Article  CAS  Google Scholar 

  • Walsh CT, Wencewicz TA (2013) Flavoenzymes: versatile catalysts in biosynthetic pathways. Nat Prod Rep 30:175–200

    Article  CAS  PubMed  Google Scholar 

  • Weber G (1950) Fluorescence of riboflavin and flavin–adenine dinucleotide. Biochem J 47:114–121

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Yildiz HB, Kiralp S, Toppare Yağci Y (2005) Immobilization of glucose oxidase in conducting graft copolymers and determination of glucose amount in orange juices with enzyme electrodes. Int J Biol Macromol 37:174–178

    Article  CAS  PubMed  Google Scholar 

  • Zhong D, Zewail AH (2001) Femtosecond dynamics of flavoproteins: charge separation and recombination in riboflavine (vitamin B2)-binding protein and in glucose oxidase enzyme. Proc Natl Acad Sci USA 98:11867–11872

    Article  CAS  PubMed  Google Scholar 

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Authors and Affiliations

  1. Dipartimento di Ingegneria Chimica, dei Materiali e della Produzione industriale, Università Federico II, P.le Tecchio 80, 80125, Naples, Italy

    Rosario Esposito

  2. Dipartimento di Scienze Ecologiche e Biologiche, Università della Tuscia, Largo dell’Università snc, 01100, Viterbo, Italy

    Ines Delfino

  3. Dipartimento di Medicina Sperimentale, Università della Campania “Luigi Vanvitelli”, Via S.M. Costantinopoli 16, 80134, Naples, Italy

    Marianna Portaccio & Maria Lepore

  4. Dipartimento di Medicina di Precisione, Università della Campania “Luigi Vanvitelli”, Via L. De Crecchio 7, 80138, Naples, Italy

    Clara Iannuzzi

Authors
  1. Rosario Esposito
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  2. Ines Delfino
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  3. Marianna Portaccio
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  4. Clara Iannuzzi
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  5. Maria Lepore
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Correspondence to Ines Delfino.

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Esposito, R., Delfino, I., Portaccio, M. et al. An insight into pH-induced changes in FAD conformational structure by means of time-resolved fluorescence and circular dichroism. Eur Biophys J 48, 395–403 (2019). https://doi.org/10.1007/s00249-019-01369-0

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  • DOI: https://doi.org/10.1007/s00249-019-01369-0

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