Sinibaldi Federica, Piro Maria C, Coletta Massimo, Santucci Roberto
Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Università di Roma 'Tor Vergata', Italy.
FEBS J. 2006 Dec;273(23):5347-57. doi: 10.1111/j.1742-4658.2006.05527.x. Epub 2006 Oct 24.
Structural information on partially folded forms is important for a deeper understanding of the folding mechanism(s) and the factors affecting protein stabilization. The non-native compact state of equine cytochrome c stabilized by salts in an acidic environment (pH 2.0-2.2), called the A-state, is considered a suitable model for the molten globule of cytochrome c, as it possesses a native-like alpha-helix conformation but a fluctuating tertiary structure. In this article, we extend our knowledge on anion-induced protein stabilization by determining the effect of anions carrying a double negative charge; unlike monovalent anions (which are thought to exert an 'ionic atmosphere' effect on the macromolecule), divalent anions are thought to bind to the protein at specific surface sites. Our data indicate that divalent anions, in comparison to monovalent ions, have a greater tendency to stabilize the native-like M-Fe(III)-H coordinated state of the protein. The possibility that divalent anions may bind to the protein at the same sites previously identified for polyvalent anions was evaluated. To investigate this issue, the behavior of the K88E, K88E/T89K and K13N mutants was investigated. The data obtained indicate that the mutated residues, which contribute to form the binding sites of polyanions, are important for stabilization of the native conformation; the mutants investigated, in fact, all show an increased amount of the misligated H-Fe(III)-H state and, with respect to wild-type cytochrome c, appear to be less sensitive to the presence of the anion. These residues also modulate the conformation of unfolded cytochrome c, influencing its spin state and the coordination to the prosthetic group.
部分折叠形式的结构信息对于更深入地理解折叠机制以及影响蛋白质稳定性的因素至关重要。在酸性环境（pH 2.0 - 2.2）中由盐稳定的马细胞色素c的非天然紧密状态，称为A状态，被认为是细胞色素c熔融球状体的合适模型，因为它具有类似天然的α-螺旋构象但三级结构波动。在本文中，我们通过确定携带双负电荷的阴离子的作用来扩展我们对阴离子诱导蛋白质稳定化的认识；与单价阴离子（被认为对大分子施加“离子氛围”效应）不同，二价阴离子被认为在特定表面位点与蛋白质结合。我们的数据表明，与单价离子相比，二价阴离子更倾向于稳定蛋白质的类似天然的M-Fe(III)-H配位状态。评估了二价阴离子可能在先前确定的多价阴离子相同位点与蛋白质结合的可能性。为了研究这个问题，研究了K88E、K88E/T89K和K13N突变体的行为。获得的数据表明，有助于形成聚阴离子结合位点的突变残基对于天然构象的稳定很重要；事实上，所研究的突变体都显示出错配的H-Fe(III)-H状态的量增加，并且相对于野生型细胞色素c，似乎对阴离子的存在不太敏感。这些残基还调节未折叠细胞色素c的构象，影响其自旋状态和与辅基的配位。
