Mechanism of fluorescence and conformational changes of the sarcoplasmic calcium binding protein of the sand worm Nereis diversicolor upon Ca2+ or Mg2+ binding.
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 Ca(2+) or Mg(2+). 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 Ca(2+) or Mg(2+). 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.
Sillen
Alain
A
Laboratory of Biomolecular Dynamics, Catholic University of Leuven, Belgium.
Verheyden
Stefan
S
Delfosse
Lotte
L
Braem
Tania
T
Robben
Johan
J
Volckaert
Guido
G
Engelborghs
Yves
Y
D000634:Aminoquinolines / Q000737:chemistry; D000818:Animals; D016880:Anisotropy; D000845:Annelida; D001120:Arginine / Q000737:chemistry; D001483:Base Sequence; D001665:Binding Sites; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000737:chemistry*; D004305:Dose-Response Relationship, Drug; D004533:Egtazic Acid / Q000494:pharmacology; D004926:Escherichia coli / Q000378:metabolism; D007700:Kinetics; D008274:Magnesium / Q000737:chemistry; D008958:Models, Molecular; D015233:Models, Statistical; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry; D014466:Ultraviolet Rays
D000634:Aminoquinolines; D002135:Calcium-Binding Proteins; D004533:Egtazic Acid; D014364:Tryptophan; D001120:Arginine; D008274:Magnesium; C035739:Quin2; D002118:Calcium