Bandyopadhyay Amal K, Krishnamoorthy G, Padhy Lakshmi C, Sonawat Haripalsingh M
Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai 400005, India.
Extremophiles. 2007 Jul;11(4):615-25. doi: 10.1007/s00792-007-0075-0. Epub 2007 Apr 4.
The [2Fe-2S] ferredoxin from the extreme haloarchaeon Halobacterium salinarum is stable in high (>1.5 M) salt concentration. At low salt concentration the protein exhibits partial unfolding. The kinetics of unfolding was studied in low salt and in presence of urea in order to investigate the role of salt ions on the stability of the protein. The urea dependent unfolding, monitored by fluorescence of the tryptophan residues and circular dichroism, suggests that the native protein is stable at neutral pH, is destabilized in both acidic and alkaline environment, and involves the formation of kinetic intermediate(s). In contrast, the unfolding kinetics in low salt exhibits enhanced rate of unfolding with increase in pH value and is a two state process without the formation of intermediate. The unfolding at neutral pH is salt concentration dependent and occurs in two stages. The first stage, involves an initial fast phase (indicative of the formation of a hydrophobic collapsed state) followed by a relatively slow phase, and is dependent on the type of cation and anion. The second stage is considerably slower, proceeds with an increase in fluorescence intensity and is largely independent of the nature of salt. Our results thus show that the native form of the haloarchaeal ferredoxin (in high salt concentration) unfolds in low salt concentration through an apparently hydrophobic collapsed form, which leads to a kinetic intermediate. This intermediate then unfolds further to the low salt form of the protein.
来自极端嗜盐古菌盐沼盐杆菌的[2Fe-2S]铁氧化还原蛋白在高盐浓度（>1.5 M）下稳定。在低盐浓度下，该蛋白会出现部分解折叠。为了研究盐离子对蛋白质稳定性的作用，我们研究了低盐条件下以及存在尿素时的解折叠动力学。通过色氨酸残基的荧光和圆二色性监测尿素依赖性解折叠，结果表明天然蛋白在中性pH下稳定，在酸性和碱性环境中均不稳定，并且涉及动力学中间体的形成。相比之下，低盐条件下的解折叠动力学显示，随着pH值升高，解折叠速率加快，且是一个无中间体形成的两态过程。中性pH下的解折叠取决于盐浓度，且分两个阶段进行。第一阶段包括一个初始快速阶段（表明形成了疏水塌缩状态），随后是一个相对缓慢的阶段，并且取决于阳离子和阴离子的类型。第二阶段要慢得多，随着荧光强度增加而进行，并且在很大程度上与盐的性质无关。因此，我们的结果表明，嗜盐古菌铁氧化还原蛋白的天然形式（在高盐浓度下）在低盐浓度下通过明显的疏水塌缩形式解折叠，这会导致形成一个动力学中间体。然后这个中间体进一步解折叠为蛋白质的低盐形式。
