Departament de Química, Universitat de les Illes Balears Palma de Mallorca, Spain.
Molecular Structure Division, National Institute for Medical Research London, UK.
Front Mol Biosci. 2014 Sep 30;1:13. doi: 10.3389/fmolb.2014.00013. eCollection 2014.
Yfh1, the yeast ortholog of frataxin, is a protein of limited thermodynamic stability which undergoes cold denaturation at temperatures above the water freezing point. We have previously demonstrated that its stability is strongly dependent on ionic strength and that monovalent or divalent cations are able to considerably stabilize the fold. Here, we present a study of the folded state and of the structural determinants that lead to the strong salt dependence. We demonstrate by nuclear magnetic resonance that, at room temperature, Yfh1 exists as an equilibrium mixture of a folded species and a folding intermediate in slow exchange equilibrium. The equilibrium completely shifts in favor of the folded species by the addition of even small concentrations of salt. We demonstrate that Yfh1 is destabilized by a localized energetic frustration arising from an "electrostatic hinge" made of negatively charged residues mapped in the β-sheet. Salt interactions at this site have a "frustration-relieving" effect. We discuss the consequences of our findings for the function of Yfh1 and for our understanding of protein folding stability.
Yfh1 是酵母的 frataxin 同源物，是一种热力学稳定性有限的蛋白质，在高于水冰点的温度下会发生冷变性。我们之前已经证明，其稳定性强烈依赖于离子强度，单价或二价阳离子能够显著稳定其构象。在这里，我们研究了折叠状态和导致强烈盐依赖性的结构决定因素。我们通过核磁共振证明，在室温下，Yfh1 以折叠物种和折叠中间体的平衡混合物形式存在，在缓慢的交换平衡中。通过添加甚至少量的盐，平衡完全向折叠物种倾斜。我们证明 Yfh1 由于β-折叠中带负电荷的残基形成的“静电铰链”而受到局部能量障碍的破坏。该位点的盐相互作用具有“缓解障碍”的作用。我们讨论了我们的发现对 Yfh1 功能和我们对蛋白质折叠稳定性理解的影响。
