Permyakov Sergei E, Bakunts Anush G, Denesyuk Alexander I, Knyazeva Ekaterina L, Uversky Vladimir N, Permyakov Eugene A
Institute for Biological Instrumentation of the Russian Academy of Sciences, Pushchino, Moscow Region 142290, Russia.
Proteins. 2008 Aug 15;72(3):822-36. doi: 10.1002/prot.21974.
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 (alpha- and beta-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 alpha-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-alpha-PA revealed hallmarks of cold denaturation of the protein at temperatures below 20 degrees C. Moreover, a cooperative thermal denaturation transition with mid-temperature at 10-15 degrees 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 (alpha-PA) or two (beta-PA) calcium ions per protein molecule, as follows from calorimetric and calcium titration data.
最近定义的内在无序蛋白（IDP）家族包括缺乏刚性三级结构但履行基本生物学功能的蛋白质。在这里我们表明，梭子鱼小清蛋白的脱辅基状态（α-和β-亚型，pI分别为5.0和4.2）属于IDP家族，这与理论预测一致。小清蛋白（PA）是一种12 kDa的钙结合蛋白，参与快速肌肉松弛的调节。对PA的金属耗尽形式进行差示扫描量热法测量，结果显示没有任何具有可测量变性焓的热诱导转变，同时比热容升高，这意味着缺乏刚性三级结构且疏水蛋白基团暴露于溶剂中。从PA中去除钙会导致疏水探针双-ANS的荧光强度增加10倍以上，并伴随着α-螺旋含量的降低以及芳香族残基环境流动性的显著增加，这通过圆二色光谱（CD）判断得出。通过CD监测的氯化胍诱导的脱辅基小清蛋白的去折叠表明缺乏固定的三级结构。对PA中电荷-电荷相互作用能量学的理论估计表明，去除钙后它们会明显不稳定，这与基于序列的无序蛋白链区域预测一致。对脱辅基-α-PA的远紫外CD研究揭示了该蛋白在低于20摄氏度的温度下冷变性的特征。此外，近紫外CD显示两种PA在10 - 15摄氏度的中温下都有协同热变性转变。在这个温度区域没有可检测到的焓变表明转变具有连续性。总体而言，所获得的理论和实验数据表明，脱辅基状态的PA本质上是无序的，但仍表现出复杂的变性行为。根据量热法和钙滴定数据，每个蛋白质分子结合一个（α-PA）或两个（β-PA）钙离子后，PA会获得天然的刚性三级结构。
