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Ciliate Euplotes octocarinatus centrin (EoCen) is an EF-hand calcium-binding protein closely related to the prototypical calcium sensor protein calmodulin. The first amino acid of the Ca2+-binding loops found in the EF-hand calcium-binding proteins is a highly conserved aspartic acid residue. The D37K mutant was produced to elucidate the metal binding role of the first aspartic acid of the EF-loop I of EoCen. Aromatic-sensitized Tb3+ fluorescence results indicated that the metal binding ability of loop I was lost due to the D37K mutation. Based on fluorescence titration curves of Lu2-D37K, the conditional binding constants of the EoCen loop II were quantitatively found to be KII = (1.61 ± 0.04) x 105 L mol-1 and KII = (3.52 ± 0.08) x 10² L-1 mol with Tb3+ and Ca2+, respectively. Using 2-p-toluidinylnaphthalene-6-sulfonate as a hydrophobic probe, exposure of the hydrophobic surface upon metal binding was found to be significantly reduced for the metal ion-saturated EoCen D37K mutant. [ABSTRACT FROM AUTHOR]

Interactions between model target peptide melittin (ME) and Euplotes octocarinatus centrin (EoCen) were investigated by fluorescence spectra, circular dichroism (CD) spectra and native polyacrylamide gel electrophoresis (PAGE). In 0.1 mol/L N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (Hepes) and 150 mmol/L NaCI at pH 7.4, EoCen and isolated short C-terminal domain of EoCen (SC-EoCen) form 1:1 peptide: protein complexes. However, no detectable signal changes can be observed while isolated N-terminal domain of EoCen (N-EoCen) or isolated long C-terminal domain of EoCen (LC-EoCen) was added into solution of ME. The interaction between EoCen and ME is specified exclusively for the short C-terminal domain of EoCen. On the basis of fluorescence titration curves, the conditional binding constants of ME with EoCen and SC-EoCen were calculated to be logKME-EoCen = 6.81±0.33 and logKME-EoCen = 6.51±0.45, respectively. [ABSTRACT FROM AUTHOR]