Title: Mechanism of fluorescence and conformational changes of the sarcoplasmic calcium binding protein of the sand worm Nereis diversicolor upon Ca2+ or Mg2+ binding
Authors: Sillen, A ×
Verheyden, Stefan
Delfosse, L
Braem, T
Robben, Johan
Volckaert, Guido
Engelborghs, Yves #
Issue Date: Jan-2003
Publisher: Biophysical society
Series Title: Biophysical Journal vol:85 issue:3 pages:1882-1893
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.
ISSN: 0006-3495
Publication status: published
KU Leuven publication type: IT
Appears in Collections:Division of Gene Technology (-)
Biochemistry, Molecular and Structural Biology Section
× corresponding author
# (joint) last author

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