Apo-parvalbumin as an Intrinsically Disordered Protein

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Recently defined family of intrinsically disordered proteins (IDP) includes proteins lacking rigid tertiary structure meanwhile fulfilling essential biological functions. Here we show that apo-state of pike parvalbumin (α- and β-isoforms, pI 5.0 and 4.2, respectively) belongs to the family of IDP, which is in accord with theoretical predictions. Parvalbumin (PA) is a 12-kDa calcium-binding protein involved into regulation of relaxation of fast muscles. Differential scanning calorimetry measurements of metal-depleted form of PA revealed the absence of any thermally induced transitions with measurable denaturation enthalpy along with elevated specific heat capacity, implying the lack of rigid tertiary structure and exposure of hydrophobic protein groups to the solvent. Calcium removal from the PAs causes more than 10-fold increase in fluorescence intensity of hydrophobic probe bis-ANS and is accompanied by a decrease in α-helical content and a marked increase in mobility of aromatic residues environment, as judged by circular dichroism spectroscopy (CD). Guanidinium chloride-induced unfolding of the apo-parvalbumins monitored by CD showed the lack of fixed tertiary structure. Theoretical estimation of energetics of the charge–charge interactions in the PAs indicated their pronounced destabilization upon calcium removal, which is in line with sequence-based predictions of disordered protein chain regions. Far-UV CD studies of apo-α-PA revealed hallmarks of cold denaturation of the protein at temperatures below 20°C. Moreover, a cooperative thermal denaturation transition with mid-temperature at 10–15°C is revealed by near-UV CD for both PAs. The absence of detectable enthalpy change in this temperature region suggests continuous nature of the transition. Overall, the theoretical and experimental data obtained show that PA in apo-state is essentially disordered nevertheless demonstrates complex denaturation behavior. The native rigid tertiary structure of PA is attained upon association of one (α-PA) or two (β-PA) calcium ions per protein molecule, as follows from calorimetric and calcium titration data. Proteins 2008. © 2008 Wiley-Liss, Inc.

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Proteins: Structure, Function, and Bioinformatics, v. 72, issue 3, p. 822-836