8-anilino-1-naphthalene sulfonic acid (ANS) induces folding of acid unfolded cytochrome c to molten globule state as a result of electrostatic interactions
8-anilino-1-naphthalene sulfonic acid (ANS) induces folding of acid unfolded cytochrome c to molten globule state as a result of electrostatic interactions
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DOI：
10.1021/bi9907835
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发表时间：
1999-10-12
期刊：
影响因子：
2.9
通讯作者：
Bhakuni, V
中科院分区：
文献类型：
--
作者：
Ali, V;Prakash, K;Bhakuni, V
8-苯胺-1-萘磺酸（ANS）与蛋白质的疏水相互作用是表征/检测蛋白质部分折叠态的广泛方法之一。本文采用光学方法和电喷雾电离质谱（ESI MS）相结合的方法，系统研究了带电疏水荧光染料ANS对pH为2.0时酸折叠马心脏细胞色素c结构性质的影响。发现ANS在酸折叠蛋白中诱导了与天然蛋白相似的二级结构和色氨酸残基荧光猝灭。然而，三级结构被发现被破坏，从而表明ANS在酸折叠蛋白中稳定熔融球状状态。为了了解ANS诱导酸折叠细胞色素c折叠的机制，我们使用中性疏水染料尼罗红和ANS进行了ESI质谱、soret吸收和色氨酸荧光对比研究。这些研究表明，在低pH下，带负电荷的ANS分子与酸折叠细胞色素c中带正电荷的氨基酸残基之间的静电相互作用可能是ANS诱导酸折叠蛋白折叠成部分折叠致密态或熔融球态的原因。这是ANS诱导未折叠蛋白质折叠的第一个实验证明，并对在低pH条件下观察到的部分折叠蛋白质中间体的表征/测定的有效性提出了质疑。
Hydrophobic interaction of 8-anilino-1-naphthalene sulfonic acid (ANS) with proteins is one of the widely used methods for characterizing/detecting partially folded states of proteins. We have carried out a systematic investigation on the effect of ANS, a charged hydrophobic fluorescent dye, on structural properties of acid-unfolded horse heart cytochrome c at pH 2.0 by a combination of optical methods and electrospray ionization mass spectroscopy (ESI MS). ANS was found to induce, a secondary structure similar to native protein and quenching of fluorescence of tryptophan residue, in the acid-unfolded protein. However, the tertiary structure was found to be disrupted thus indicating that ANS stabilizes a molten globule state in acid-unfolded protein. To understand the mechanism of ANS-induced folding of acid-unfolded cytochrome c, comparative ESI MS, soret absorption, and tryptophan fluorescence studies using nile red, a neutral hydrophobic dye, and ANS were carried out. These studies suggested that, at low pH, electrostatic interactions between negatively charged ANS molecules and positively charged amino acid residues present in acid-unfolded cytochrome c are probably responsible for ANS-induced folding of acid-unfolded protein to partially folded compact state or molten globule state. This is the first experimental demonstration of ANS induced folding of unfolded protein and puts to question the usefulness of ANS for characterization/determination of partially folded intermediates of proteins observed under low pH conditions.