Mechanism of fluorescence and conformational changes of the sarcoplasmic calcium binding protein of the sand worm Nereis diversicolor upon Ca2+ or Mg2+ binding

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 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.

FIGURE 1

MOLSCRIPT representation of the x-ray structure of NSCP Ca²⁺. The white spheres represent the calcium ions.

FIGURE 2

Typical phase measurement of NSCP W4F/W170F/R25D/D58R apo and graphical tests incorporated in the data analysis (spreading and autocorrelation analysis of the residuals). Fit to four exponentials.

FIGURE 3

Normalized decay-associated spectra (DAS) of Wt, W57F/W170F, and W4F/W57F in the Ca²⁺, Mg²⁺, and apo states. Spectra are from 300 nm to 450 nm. ▴, longest lifetime; □, second longest lifetime; •, middle lifetime; ⋄, smallest lifetime; –, measured emission spectra; and …, sum of the decay-associated spectra.

FIGURE 4

Normalized decay-associated spectra (DAS) of W4F/W170F, W4F/W170F/R25D, and W4F/W170F/R25D/D58R in the Ca²⁺, Mg²⁺, and apo states. Spectra are from 300 nm to 450 nm. ▴, longest lifetime; □, second longest lifetime; •, middle lifetime; ⋄, smallest lifetime; –, measured emission spectra; and …, sum of the decay-associated spectra.

FIGURE 5

Fluorescence titration with EGTA of NSCP Wt containing 0.2 mM Ca²⁺. Fluorescence is measured at 338 nm. Continuous line is the best fit using Eq. 6.

FIGURE 6

Normalized fluorescence titration with EGTA of NSCP mutants containing 0.2 mM Ca²⁺. Fluorescence is measured at 338 nm for W170 and at 317 nm for W57. Continuous line is the best fit using Eq. 5.