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The Protein Journal

, 28:1 | Cite as

An Anionic Porphyrin Binds β-Lactoglobulin A at a Superficial Site Rich in Lysine Residues

  • Ivan Silva
  • Samuel Sansone
  • Lorenzo Brancaleon
Article

Abstract

Binding of small ligands to globular proteins remains a major research topic in biophysics. We have studied the binding of several photoactive dyes to β-lactoglobulin (BLG), as a model to investigate the photoinduced effects of porphyrins on proteins. A combination of optical spectroscopies (fluorescence, circular dichroism) and molecular docking simulations were used to estimate the pH-dependence of the binding parameters and the docking location for meso-tetrakis(sulfonatophenyl)-porphyrin (TPPS). We have observed that the binding of TPPS is not modulated by the pH-mediated conformational transition of the protein (i.e., Tanford transition). Binding of TPPS appears to occur with some degree of negative cooperativity. Moreover, TPPS remains bound even upon partial denaturation of the protein. These results are consistent with a superficial binding site at a location removed from the aperture of the interior β-barrel. Binding occurs through electrostatic interactions between the negative SO3 groups of TPPS and positively charged Lys and Arg residues. This is the first study that explores the interaction of an anionic porphyrin with BLGA in a pH range that spans across the Tanford transition. Establishing the location of the binding site will enable us to explain the photoinduced conformational effects mediated by TPPS on BLG.

Keywords

Lactoglobulin Porphyrin Fluorescence spectroscopy Binding Photodynamic therapy (PDT) 

Abbreviations

PPIX

Protoporphyrin IX

β-lg

β-Lactoglobulin

GI

Gastrointestinal tract

DMSO

Dimethylsulfoxide

KI

Potassium iodide

S–V

Stern–Volmer

Trp

Tryptophan

TPPS4

Meso-tetrakis(sulfonatophenyl)-porphine

ANS

1-Anilinonaphthalene-8-sulfonate

FRET

Fluorescence resonance energy transfer

CD

Circular dichroism

Notes

Acknowledgments

The research was supported by the 2006 Faculty Research Award of the University of Texas at San Antonio (to L.B.) and by the AFRL/HE grant # FA8650-07-1-6850 (to L.B.). The author would also like to thank Dr. Markandeswar Panda for the use of the CD spectrometer.

Supplementary material

10930_2008_9158_MOESM1_ESM.doc (146 kb)
(DOC 146 kb)

References

  1. 1.
    Kuwata K, Hoshino M, Forge V, Era S, Batt CA, Goto Y (1999) Prot Sci 8:2541–2545Google Scholar
  2. 2.
    Oliveira KMG, Valente-Mesquita VL, Botelho MM, Sawyer L, Ferreira ST, Polikarpov I (2001) Eur J Biochem 268:477–484Google Scholar
  3. 3.
    Tian F, Johnson K, Lesar AE, Moseley H, Ferguson J, Samuel IDW, Mazzini A, Brancaleon L (2005) Biochim Biophys Acta 1760:38–46Google Scholar
  4. 4.
    Fernandez NF, Sansone S, Mazzini A, Brancaleon L (2008) J Phys Chem B 112:7592–7600CrossRefGoogle Scholar
  5. 5.
    Kubelka J, Hofrichter J, Eaton WA (2004) Curr Opin Struct Biol 14:76–88CrossRefGoogle Scholar
  6. 6.
    Lee CT Jr, Smith KA, Hatton TA (2005) Biochemistry 44:524–536CrossRefGoogle Scholar
  7. 7.
    Dougherty TJ, Gomer CJ, Henderson BW, Jori G, Kessel D, Korbelik M, Moan J, Peng Q (1998) J Nat Canc Inst 90:889–905CrossRefGoogle Scholar
  8. 8.
    Pope AJ, Bown SG (1991) Br J Urol 68:1–9CrossRefGoogle Scholar
  9. 9.
    Castelli M, Reiners JJ, Kessel D (2004) Cell Death Differ 11:906–914CrossRefGoogle Scholar
  10. 10.
    Tsaytler PA, O’Flaherty MC, Sakharov DV, Krijgsveld J, Egmond MR (2008) J Proteome Res 7:3868–3878CrossRefGoogle Scholar
  11. 11.
    Usuda J, Chiu SM, Murphy ES, Lam M, Nieminen AL, Oleinick NL (2003) J Biol Chem 278:2021–2029CrossRefGoogle Scholar
  12. 12.
    Kessel D (2002) Photochem Photobiol Sci 1:837–840CrossRefGoogle Scholar
  13. 13.
    Andrade SM, Costa SMB (2002) Biophys J 82:1607–1619CrossRefGoogle Scholar
  14. 14.
    Qin BY, Bewley MC, Creamer LK, Baker HM, Baker EN, Jameson GB (1998) Biochemistry 37:14014–14023CrossRefGoogle Scholar
  15. 15.
    Tanford C, Bunville LG, Nozaki Y (1959) J Am Chem Soc 81:4032–4036CrossRefGoogle Scholar
  16. 16.
    Maiti NC, Ravikanth M, Mazumdar S, Periasamy N (1995) J Phys Chem 99:17192–17197CrossRefGoogle Scholar
  17. 17.
    Tian F, Johnson EM, Zamarripa M, Sansone S, Brancaleon L (2007) Biomacromol 8:3767–3778CrossRefGoogle Scholar
  18. 18.
    D’Alfonso L, Collini M, Baldini G (2002) Biochemistry 41:326–333CrossRefGoogle Scholar
  19. 19.
    Harvey BJ, Bell E, Brancaleon L (2007) J Phys Chem B 111:2610–2620CrossRefGoogle Scholar
  20. 20.
    Bilsel O, Buchler JW, Hammerschmitt P, Rodriguez J, Holten D (1991) Chem Phys Lett 182:415–421CrossRefGoogle Scholar
  21. 21.
    Lang K, Mosinger J, Wagnerova DM (2004) Coord Chem Rev 248:321–350CrossRefGoogle Scholar
  22. 22.
    Folgolari F, Ragona L, Zetta L, Romagnoli S, De Kruif KG, Molinari H (1998) FEBS Lett 436:149–154CrossRefGoogle Scholar
  23. 23.
    Fukamizo T, Juffer AH, Voge HJ, Honda Y, Tremblay H, Boucher I, Neugebauer WA, Brzezinski R (2000) J Biol Chem 275:25633–25640CrossRefGoogle Scholar
  24. 24.
    Coval ML (1970) J Biol Chem 245:6335–6336Google Scholar
  25. 25.
    Weiss JN (1997) FASEB J 11:835–841Google Scholar
  26. 26.
    Lakowicz JR (2006) Principles of Fluorescence spectroscopy, 3rd edn. Springer, New YorkGoogle Scholar
  27. 27.
    Tatikolov AS, Costa SM (2004) Photochem Photobiol 80:250–256CrossRefGoogle Scholar
  28. 28.
    Lehrer SS (1971) Biochemistry 10:3254–3263CrossRefGoogle Scholar
  29. 29.
    Cho Y, Batt CA, Sawyer L (1994) J Biol Chem 269:11102–11107Google Scholar
  30. 30.
    Wu P, Brand L (1994) Anal Biochem 218:1–13CrossRefGoogle Scholar
  31. 31.
    Chuang TJ, Eisenthal KB (1972) J Chem Phys 57:5094–5097CrossRefGoogle Scholar
  32. 32.
    Galani D, Apenten RKO (1999) Food Res Internat 32:93–100CrossRefGoogle Scholar
  33. 33.
    Eftink MR (1994) Biophys J 66:482–501CrossRefGoogle Scholar
  34. 34.
    Pettersen EF, Goddard TD, Huang CC, Couch GS, Greenblatt DM, Meng EC, Ferrin TE (2004) J Comput Chem 25:1605–1612CrossRefGoogle Scholar
  35. 35.
    Collini M, D’Alfonso L, Baldini G (2000) Prot Sci 9:1968–1974CrossRefGoogle Scholar
  36. 36.
    Horowitz P, Prasad V, Luduena RF (1984) J Biol Chem 259:14647–14650Google Scholar
  37. 37.
    Tulp A, Verwoerd D, Hard AA (1997) Electrophoresis 18:767–773CrossRefGoogle Scholar
  38. 38.
    Hamada D, Dobson CM (2002) Protein Sci 11:2417–2426CrossRefGoogle Scholar
  39. 39.
    Capron I, Nicolai T, Durand D (1999) Food Hydrocoll 13:1–5CrossRefGoogle Scholar
  40. 40.
    Gottschalk M, Nilsson H, Roos H, Halle B (2003) Protein Sci 12:2404–2411CrossRefGoogle Scholar
  41. 41.
    Brownlow S, Morais Cabral JH, Cooper R, Flower DR, Yewdall SJ, Polikarpov I, North ACT, Sawyer L (1997) Structure 5:481–495CrossRefGoogle Scholar
  42. 42.
    Fessas D, Iametti S, Schiraldi A, Bonomi F (2001) Eur J Biochem 268:5439–5448CrossRefGoogle Scholar
  43. 43.
    Kelbauskas L, Bagdonas S, Dietel W, Rotomskis R (2003) J Luminesc 101:253–262CrossRefGoogle Scholar
  44. 44.
    Jameson GB, Adams JJ, Creamer LK (2002) Int Dairy J 12:319–329CrossRefGoogle Scholar
  45. 45.
    Busti P, Scarpeci S, Gatti C, DeLorenzi N (2002) Food Res Internat 35:871–877CrossRefGoogle Scholar
  46. 46.
    Dahlquist FW (1978) Methods Enzymol 48:270–299CrossRefGoogle Scholar
  47. 47.
    Tominaga TT, Yushimanov VE, Borissevitch IE, Imasato H, Tabak M (1997) J Inorg Biochem 65:235–244CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2008

Authors and Affiliations

  • Ivan Silva
    • 1
  • Samuel Sansone
    • 1
  • Lorenzo Brancaleon
    • 1
  1. 1.Department of Physics and AstronomyUniversity of Texas at San AntonioSan AntonioUSA

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