In Search for Globally Disordered Apo-parvalbumins: Case of Parvalbumin Β-1 from Coho Salmon

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IDPintrinsically disordered protein, intrinsically disordered protein, PAparvalbumin/oncomodulin, parvalbumin/oncomodulin, intact PAparvalbumin isolated from muscles, parvalbumin isolated from muscles, rWT PArecombinant wild-type parvalbumin, recombinant wild-type parvalbumin, α-PAα isoform of parvalbumin, α isoform of parvalbumin, β-PAβ isoform of parvalbumin, β isoform of parvalbumin, csPAcoho salmon PA, coho salmon PA, apo-csPAmetal-free form of coho salmon PA, prepared as described in Materials and methods chapter, metal-free form of coho salmon PA, prepared as described in Materials and methods chapter, IPTGisopropyl β-d-1-thiogalactopyranoside, isopropyl β-d-1-thiogalactopyranoside, PMSFphenylmethanesulfonyl fluoride, phenylmethanesulfonyl fluoride, 2-ME2-mercaptoethanol, 2-mercaptoethanol, DTTDL-dithiothreitol, DL-dithiothreitol, DTNB5, 5′-dithiobis(2-nitrobenzoic acid), 5, 5′-dithiobis(2-nitrobenzoic acid), EDTAethylenediaminetetraacetic acid, ethylenediaminetetraacetic acid, TCAtrichloroacetic acid, trichloroacetic acid, Tristris(hydroxymethyl)aminomethane, tris(hydroxymethyl)aminomethane, HEPESN-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid), N-(2-hydroxyethyl)piperazine-N′-(2-ethanesulfonic acid), MOPS3-(N-morpholino)propanesulfonic acid, 3-(N-morpholino)propanesulfonic acid, bis-ANS4, 4′-dianilino-1, 1′-binaphthyl-5, 5′-disulfonic acid, 4, 4′-dianilino-1, 1′-binaphthyl-5, 5′-disulfonic acid, GdmClguanidine hydrochloride, guanidine hydrochloride, SDS-PAGEsodium dodecyl sulfate polyacrylamide gel electrophoresis, sodium dodecyl sulfate polyacrylamide gel electrophoresis, CDcircular dichroism spectroscopy, circular dichroism spectroscopy, DSCdifferential scanning calorimetry, differential scanning calorimetry, DLSdynamic light scattering, dynamic light scattering, SIsequence identity, sequence identity, CH-plotscharge–hydropathy plots, charge–hydropathy plots, CDFCumulative Distribution Function, Cumulative Distribution Function, Protein stability, Intrinsically disordered proteins, Molten globule state, Calcium-binding proteins, EF-hand, Parvalbumin, Oncomodulin

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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.

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Cell Calcium, v. 67, p. 53-64