Recently we found two highly conserved structural motifs in the members of the EF-hand protein family, which provide a supporting scaffold for their Ca2+ binding loops. Each structural motif is formed by a cluster of three amino acids. These clusters were called 'black' cluster (cluster I) and 'gray' cluster (cluster II). In the present work, we studied the relationship between the location of the 'black' and 'gray' structural clusters in parvalbumins and the location of the amino acid sequence regions with a tendency for intrinsic disorder. This analysis revealed that in parvalbumins, the residues in the vicinity of the conserved structural clusters constitute parts of the conserved motifs enriched in the disorder-promoting residues. Therefore, the clusters found in parvalbumins are characterized not only by the presence of conserved amino acid residues, but also by the conserved distribution of the intrinsic disorder predisposition within their sequences, suggesting the presence of conserved structural dynamics in the apo-forms of parvalbumins, where the black cluster appears to have greater mobility than the gray cluster.

Two highly conserved structural motifs observed in members of the EF-hand family of calcium binding proteins. The motifs provide a supporting scaffold for the Ca2+ binding loops and contribute to the hydrophobic core of the EF-hand domain. Each structural motif represents a cluster of three amino acids called cluster I ('black' cluster) and cluster II ('grey' cluster). Cluster I is more conserved and mostly incorporates aromatic amino acids. In contrast, cluster II is noticeably less conserved and includes a mix of aromatic, hydrophobic, and polar amino acids of different sizes. In the human calcium binding S100 P protein, these 'black' and 'gray' clusters include residues F15, F71, and F74 and L33, L58, and K30, respectively. To evaluate the effects of these clusters on structure and functionality of human S100 P, we have performed Ala scanning. The resulting mutants were studied by a multiparametric approach that included circular dichroism, scanning calorimetry, dynamic light scattering, chemical crosslinking, and fluorescent probes. Spectrofluorimetric Ca2+-titration of wild type S100 P showed that S100 P dimer has 1-2 strong calcium binding sites (K1 = 4 × 106 M-1) and two cooperative low affinity (K2 = 4 × 104 M-1) binding sites. Similarly, the S100 P mutants possess two types of calcium binding sites. This analysis revealed that the alanine substitutions in the clusters I and II caused comparable changes in the S100 P functional properties. However, analysis of heat- or GuHCl-induced unfolding of these proteins showed that the alanine substitutions in the cluster I caused notably more pronounced decrease in the protein stability compared to the changes caused by alanine substitutions in the cluster II. Opposite to literature data, the F15 A substitution did not cause the S100 P dimer dissociation, indicating that F15 is not crucial for dimer stability. Overall, similar to parvalbumins, the S100 P cluster I is more important for protein conformational stability than the cluster II.