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

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.     

Parvalbumin as a metal-dependent antioxidant

Parvalbumin (PA) is a Ca²⁺-binding protein of vertebrates massively expressed in tissues with high oxygen uptake and respectively elevated level of reactive oxygen species (ROS). To characterize antioxidant properties of PA, antioxidant capacity (AOC) of intact rat α-PA has been explored. ORAC, TEAC and hydrogen peroxide AOC assays evidence conformation-dependent oxidation of the PA. AOC value for the apo-PA 4-11-fold exceeds that for the Ca²⁺-loaded protein. Despite folded conformation of apo-PA, it has AOC equivalent to that of the proteolized protein. The most populated under resting conditions PA form, Mg²⁺-bound PA, has AOC similar to that of apo-PA. ROS-induced changes in absorption spectrum of PA evidence an oxidation of PA's phenylalanines in the ORAC assay. Sensitivity of PA oxidation to its conformation enabled characterization of its metal affinity and pH-dependent behavior: a transition with pK_a of 7.6 has been revealed for the Ca²⁺-loaded PA. Since total AOC of PA under in vivo conditions may reach the level of reduced glutathione, we propose that PA might modulate intracellular redox equilibria and/or signaling in a calcium-dependent manner. We speculate that the oxidation-mediated damage of some of PA-GABAergic interneurons observed in schizophrenia is due to a decline in total AOC of the reduced glutathione–PA pair.     

Parvalbumins distribution and physical state inside the muscle cell

Single skinned muscle fibres (frog) have been submitted to double Ouchterlony immunodiffusion assays with antibodies directed against the two species of frog parvalbumin. The antigenic material which diffuses out of each fibre contains the two parvalbumins. Their presence in each cell is thus demonstrated. The amount of parvalbumins having diffused out of the fibre has been quantified. It corresponds to the parvalbumin content of the cell. This implies that these proteins are freely soluble in the muscle sarcoplasm.     

Binding of calcium by fragment 38-108 of pike parvalbumin

Cyanogen bromide splitting of pike parvalbumin III was carried out and fragment 38-108 containing two calcium-binding domains was isolated. The obtained fragment binds calcium rather well (Kd = 1.6.10(-5) M), possesses a secondary structure, its CD spectrum changes after removal of calcium.     

In search for globally disordered apo-parvalbumins: Case of parvalbumin β-1 from coho salmon

Parvalbumin (PA) is a classical EF-hand calcium-binding protein of muscle, neuronal, and other tissues, and a major fish allergen. Although certain apo-PAs lack tertiary structure, functional implications of that feature and its structural prerequisites remain unclear. In a search for unstable PAs, we probed conformational stability of parvalbumin β-1 from coho salmon (csPA), a cold water fish species, using circular dichroism, scanning calorimetry, hydrophobic probe fluorescence, limited proteolysis, chemical crosslinking and dynamic light scattering techniques. Apo-csPA is shown to be mainly monomeric protein with markedly disorganized secondary structure and lack of rigid tertiary structure. Examination of per-residue propensity for intrinsic disorder in the PA groups with either folded or unfolded apo-form using the average PONDR^® VSL2P profiles revealed that the N-terminal region that includes α-helix A, AB-loop and N-terminal half of α-helix B is predicted to be less ordered in PAs with disordered apo-state. Application of the structural criteria developed for discrimination of disordered PAs indicate that the latter comprise about 16–19% of all PAs. We show that structural instability of apo-β-PA serves as a hallmark of elevated calcium affinity of the protein. Therefore, the successful predictions of unstable apo-PAs might facilitate search for PAs with maximal calcium affinity and possibly serving as calcium sensors.     

Some aspects of studies of thermal transitions in proteins by means of their intrinsic fluorescence

The changes in intrinsic fluorescence parameters induced by thermal transitions in proteins are developed on the background of the common thermal fluorescence quenching due to an activation of collisions between the excited chromophores and neighbouring quenching groups. Two methods of separation of the thermai quenching and conformational change contributions to the temperature dependence of the fluorescence parameters are presented. One is based on the use of the linearity of the plots of the reciprocal fluorescence quantum yield, l/q, vs. the t/η ratio (T. temperature; η, solvent viscosity) for native proteins containing a single fluorescing chromophore (T.L. Bushueva, E.P. Busel and E.A. Burstein, Biochim. Biophys. Acta 534 (1978) 141). The other method is based on a consideration of the phase plots for the tryptophan fluorescence of proteins (fluorescence intensity at a fixed wavelength vs. intensity at any other fixed wavelength). The methods have been used for a study of the thermal transitions in Mg²⁺-loaded whiting parvalbumin (tryptophan fluorescence), Mg²⁺-loaded pike parvalbumins pI 4.2 (tyrosine fluorescence) and pI 5.0 (phenylalanine fluorescence), and Ca²⁺-loaded bovine α-lactalbumin (tryptophan fluorescence). The thermal denaturation curves for the parvalbumins show two-stepped character. The main change of the protein conformation occurs at the higher temperature step. Comparison of the fluorescence data with the microcalorimetry results shows that the maxima of the asymmetric heat sorption peaks for pike parvalbumins correlate with the mid-points of the higher temperature steps of the fluorimetric curves.