Comprehensive analysis of the roles of 'black' and 'gray' clusters in structure and function of rat β-parvalbumin.

A1 Journal article (refereed)

Internal Authors/Editors

Publication Details

List of Authors: Permyakov SE, Vologzhannikova AA, Khorn PA, Shevelyova MP, Kazakov AS, Emelyanenko VI, Denesyuk AI, Denessiouk K, Uversky VN, Permyakov EA.
Publisher: Elsevier
Publication year: 2018
Journal: Cell Calcium
Volume number: 75
Start page: 64
End page: 78


Recently we found two highly conserved structural motifs in the proteins
of the EF-hand calcium binding protein family. These motifs provide a
supporting scaffold for the Ca2+ binding loops and contribute
to the hydrophobic core of the EF-hand domain. Each structural motif
forms a cluster of three amino acids called cluster I ('black' cluster)
and cluster II ('grey' cluster). Cluster I is much more conserved and
mostly incorporates aromatic amino acids. In contrast, cluster II
includes a mix of aromatic, hydrophobic, and polar amino acids. The
'black' and 'gray' clusters in rat β-parvalbumin consist of F48, A100,
F103 and G61, L64, M87, respectively. In the present work, we
sequentially substituted these amino acids residues by Ala, except
Ala100, which was substituted by Val. Physical properties of the mutants
were studied by circular dichroism, scanning calorimetry, dynamic light
scattering, chemical crosslinking, and fluorescent probe methods. The
Ca2+ and Mg2+ binding affinities of these mutants
were evaluated by intrinsic fluorescence and equilibrium dialysis
methods. In spite of a rather complicated pattern of contributions of
separate amino acid residues of the 'black' and 'gray' clusters into
maintenance of rat β-parvalbumin structural and functional status, the
alanine substitutions in the cluster I cause noticeably more pronounced
changes in various structural parameters of proteins, such as
hydrodynamic radius of apo-form, thermal stability of Ca2+/Mg2+-loaded forms, and total energy of Ca2+
binding in comparison with the changes caused by amino acid
substitutions in the cluster II. These findings were further supported
by the outputs of computational analysis of the effects of these
mutations on the intrinsic disorder predisposition of rat β-parvalbumin,
which also indicated that local intrinsic disorder propensities and the
overall levels of predicted disorder were strongly affected by
mutations in the cluster I, whereas mutations in cluster II had less
pronounced effects. These results demonstrate that amino acids of the
cluster I provide more essential contribution to the maintenance of
structuraland functional properties of the protein in comparison with
the residues of the cluster II.


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