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Mechanism of fluorescence and conformational changes of the sarcoplasmic calcium binding protein of the sand worm Nereis diversicolor upon Ca2+ or Mg2+ binding
Sillen, A.; Verheyden, S.; Delfosse, L.; Braem, T.; Robben, J.; Volckaert, G.; Engelborghs, Y. (2003). Mechanism of fluorescence and conformational changes of the sarcoplasmic calcium binding protein of the sand worm Nereis diversicolor upon Ca2+ or Mg2+ binding. Biophys. J. 85(3): 1882-1893. dx.doi.org/10.1016/S0006-3495(03)74616-5
In: Biophysical journal. Published for the Biophysical Society by the Rockefeller University Press: New York,. ISSN 0006-3495, more
Peer reviewed article  

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Keyword
    Marine

Authors  Top 
  • Sillen, A.
  • Verheyden, S.
  • Delfosse, L.
  • Braem, T.
  • Robben, J.
  • Volckaert, G.
  • Engelborghs, Y.

Abstract
    The calcium-binding protein isolated from the sarcoplasm of the muscles of the sand worm Nereis diversicolor has four EF-hands and three active binding sites for Ca2+ or Mg2+. Nereis diversicolor sarcoplasmic calcium-binding protein contains three tryptophan residues at positions 4, 57, and 170, respectively. The Wt protein shows a very limited fluorescence increase upon binding of Ca2+ or Mg2+. Single-tryptophan-containing mutants were produced and purified. The fluorescence titrations of these mutants show a limited decrease of the affinity for calcium, but no alterations of the cooperativity. Upon adding calcium, Trp170 shows a strong fluorescence increase, Trp57 an extensive fluorescence decrease, and Trp4 shows no fluorescence change. Therefore mutant W4F/W170F is ideally suited to analyze the fluorescence titrations and to study the binding mechanism. Mutations of the calcium ligands at the z-position in the three binding sites show no effect at site I and a total loss of cooperativity at sites III and IV. The quenching of Trp57 upon calcium binding is dependent on the presence of arginine R25, but this residue is not just a simple dynamic quencher. The role of the salt bridge R25-D58 is also investigated.

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