Sanfelice Domenico, Puglisi Rita, Martin Stephen R, Di Bari Lorenzo, Pastore Annalisa, Temussi Piero Andrea
MRC National Institute for Medical Research, The Ridgeway, London, United Kingdom.
MRC National Institute for Medical Research, The Ridgeway, London, United Kingdom; Dipartimento di Chimica e Chimica Industriale, Pisa, Italy; Scuola Normale Superiore, Pisa, Italy.
PLoS One. 2014 May 6;9(5):e95801. doi: 10.1371/journal.pone.0095801. eCollection 2014.
Frataxins are a family of metal binding proteins associated with the human Friedreich's ataxia disease. Here, we have addressed the effect of non-specifically binding salts on the stability of the yeast ortholog Yfh1. This protein is a sensitive model since its stability is strongly dependent on the environment, in particular on ionic strength. Yfh1 also offers the unique advantage that its cold denaturation can be observed above the freezing point of water, thus allowing the facile construction of the whole protein stability curve and hence the measurement of accurate thermodynamic parameters for unfolding. We systematically measured the effect of several cations and, as a control, of different anions. We show that, while strongly susceptible to ionic strength, as it would be in the cellular environment, Yfh1 stability is sensitive not only to divalent cations, which bind specifically, but also to monovalent cations. We pinpoint the structural bases of the stability and hypothesize that the destabilization induced by an unusual cluster of negatively charged residues favours the entrance of water molecules into the hydrophobic core, consistent with the generally accepted mechanism of cold denaturation.
铁硫蛋白是一类与人类弗里德赖希共济失调症相关的金属结合蛋白。在此，我们研究了非特异性结合盐对酵母同源蛋白Yfh1稳定性的影响。该蛋白是一个敏感模型，因为其稳定性强烈依赖于环境，特别是离子强度。Yfh1还具有独特优势，即在水的冰点以上就能观察到其冷变性，从而便于构建完整的蛋白质稳定性曲线，进而测量准确的去折叠热力学参数。我们系统地测量了几种阳离子以及作为对照的不同阴离子的影响。我们发现，尽管Yfh1在细胞环境中对离子强度非常敏感，但其稳定性不仅对特异性结合的二价阳离子敏感，对单价阳离子也敏感。我们确定了稳定性的结构基础，并推测由异常的带负电荷残基簇引起的去稳定化有利于水分子进入疏水核心，这与普遍接受的冷变性机制一致。
