Hutcheon George W, Vasisht Nishi, Bolhuis Albert
Department of Biological Sciences, University of Warwick, CV4 7AL, Coventry, UK.
Extremophiles. 2005 Dec;9(6):487-95. doi: 10.1007/s00792-005-0471-2. Epub 2005 Aug 2.
Intracellular and extracellular proteins from halophilic archaea face very saline conditions and must be able to maintain stability and functionality at nearly saturated salt concentrations. Haloarchaeal proteins contain specific adaptations to prevent aggregation and loss of activity in such conditions, but these adaptations usually result in a lack of stability in the absence of salt. Here, we present the characterisation of a secreted alpha-amylase (AmyH) from the halophilic archaeon Haloarcula hispanica. AmyH was shown to be very halophilic but, unusually for a halophilic protein, it retained activity in the absence of salt. Intrinsic fluorescence measurements and activity assays showed that AmyH was very stable in high-salt buffer and even maintained stability upon the addition of urea. Urea-induced denaturation was only achieved in the absence of NaCl, demonstrating clearly that the stability of the protein was salt-dependent. Sequencing of the amyH gene showed an amino acid composition typical of halophilic proteins and, moreover, the presence of a signal peptide containing diagnostic features characteristic of export via the Twin-arginine translocase (Tat). Analysis of the export of AmyH showed that it was translocated post-translationally, most likely in a folded and active conformation, confirming that AmyH is a substrate of the Tat pathway.
嗜盐古菌的细胞内和细胞外蛋白质面临着高盐环境，必须能够在接近饱和盐浓度的条件下维持稳定性和功能。嗜盐古菌蛋白质具有特定的适应性，以防止在这种条件下发生聚集和活性丧失，但这些适应性通常会导致在无盐情况下缺乏稳定性。在这里，我们展示了嗜盐古菌西班牙嗜盐碱杆菌分泌的一种α-淀粉酶（AmyH）的特性。结果表明，AmyH具有很强的嗜盐性，但与嗜盐蛋白质不同的是，它在无盐情况下仍保持活性。内在荧光测量和活性测定表明，AmyH在高盐缓冲液中非常稳定，甚至在添加尿素后仍能保持稳定性。只有在没有氯化钠的情况下才能实现尿素诱导的变性，这清楚地表明该蛋白质的稳定性依赖于盐。amyH基因测序显示其氨基酸组成具有嗜盐蛋白质的典型特征，此外，还存在一个信号肽，其中包含通过双精氨酸转运体（Tat）输出的诊断特征。对AmyH输出的分析表明，它是在翻译后转运的，很可能是以折叠和活性构象转运的，这证实了AmyH是Tat途径的底物。
