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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

Alain Sillen ^*, Stefan Verheyden ^*, Lotte Delfosse ^*, Tania Braem ^*, Johan Robben , Guido Volckaert  and Yves Engelborghs ^*

^* Laboratory of Biomolecular Dynamics; and Laboratory of Gene Technology; Catholic University of Leuven, Leuven, Belgium

Correspondence: Address reprint requests to Yves Engelborghs, Katholieke Universiteit Leuven, Celestijnenlaan 200D, B-3001 Leuven, Belgium. Tel.: 321-632-7160, Fax: 321-632-7982; E-mail: yves.engelborghs{at}fys.kuleuven.ac.be.

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.