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. https://dx.doi.org/10.1016/S0006-3495(03)74616-5
In: Biophysical Journal. Cell Press: New York. ISSN 0006-3495; e-ISSN 0006-3495, more
|  |
| 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. |
|
