大肠埃希氏杆菌的外膜具有很高的通透性，允许小分子物质通过膜上的孔结构自由的双向扩散，因此大肠杆菌膜间质蛋白就直接暴露在外界环境条件的波动之下，不断受到诱导蛋白变性、聚集的因素的挑战。根据上面的分析，和现在普遍认可的蛋白在体内折叠、重折叠需要分子伴侣辅助的观点，膜间质蛋白应该受到大量膜间质分子伴侣蛋白的保护以维持膜间质的正常功能。然而，至今在膜间质中没有发现典型分子伴侣的同源蛋白。虽然膜间质中的蛋白二硫键异构酶和脯氨酸顺反异构酶最近被报导具有分子伴侣活性，但是这种活性的检测使用的不是天然的膜间质蛋白，而且没有捕获到复合物的形成，因此看起来仍不能够满足前面分析的对膜间质蛋白保护的需求。考虑到是否膜间质蛋白在其存在环境下需要保护的假设有失偏颇，我们从膜间质蛋白本身性质的检查入手进行探索。研究结果表明膜间质蛋白作为一个整体显示出对高温、低pH、有机溶剂和重金属几种不同性质的变性条件所诱导聚集的抗性。为了揭开这种现象背后的本质，我们提纯并鉴定了膜间质中四个主要的蛋白来进行进一步的检测。使用远紫外圆二色光谱和横向脲梯度电泳对蛋白的结构稳定性进行的考察发现，膜间质蛋白在比较温和的变性条件下即发生天然结构丧失；使用Bis-ANS疏水荧光探针探测该过程中蛋白表面的变化，发现膜间质蛋白随天然结构丧失而同步的暴露疏水表面；但是，在蛋白处于上述天然结构丧失、疏水表面暴露的状态时，其不与典型的分子伴侣蛋白形成复合物。根据这些发现，我们提出假说：膜间质蛋白在恶劣环境条件的选择压力下进化出对各种变性条件所诱导聚集的抗性，而不需要分子伴侣蛋白的有效保护。同时，由于本实验结果显示膜间质蛋白与常用的分子伴侣测活底物蛋白在聚集行为上存在极显著的区别，我们建议对最近报导的膜间质蛋白二硫键异构酶和脯氨酸顺反异构酶分子伴侣活性以膜间质蛋白为底物再进行验证，对其在膜间质是否发挥分子伴侣功能重新慎重考虑。
Periplasmic proteins of Gram-negative bacteria like Escherichia coli are subjected to immediate affect of environmental fluctuation that may unfold proteins, due to the permeability of the outer membrane to small molecules. They are thus supposedly protected by certain molecular chaperones. Nevertheless, no homologues of typical molecular chaperones have so far been found in periplasm, and the recently reported chaperone activities of periplasmic protein disulfide isomerase (PDI) and peptidyl prolyl isomerase (PPI) seem to be too weak to satisfy such assumed needs. In an attempt to reveal whether periplasmic proteins exhibit certain unusual properties, we discovered that such proteins as a whole are highly resistant to aggregation under a wide variety of denaturing conditions like high temperature, low pH, organic solvent and heavy metal. Furthermore, in an effort to unveil the nature behind this phenomenon we purified and examined four prominent periplasmic proteins. It is shown that these proteins unfold at rather mild denaturing conditions monitored by far-UV CD and trasverse urea-gradient PAGE; they synchronous expose hydrophobic surfaces during such unfolding process detected by bis-ANS hydrophobic fluorescence probe; but they do hardly form complexes with a typical molecular chaperone at those unfolded, hydrophobic surfaces exposed state. Based on these observations, we propose that the periplasmic proteins have been evolved to resist the formation of aggregates when subjected to various denaturing conditions and molecular chaperones may thus not be needed in periplasm. We also strongly suggest that the recently reported chaperone acitivity of periplasmic PDIase and PPIase assayed by traditional substrate should be reexamined using periplasmic proteins as substrate due to the significant difference between those two classes of proteins, the possible chaperone fuctions of PDIase and PPIase in vivo should be reappraised.
