蛋白质的氧化重折叠

经过近几十年来广泛而深入的研究，蛋白质氧化重折叠的机制已得到相当详细的阐明。1在已研究过的蛋白质中，大多数蛋白质都是沿着多途径而非单一、特定的途径进行氧化重折叠，这与折叠能量景观学说是一致的。2正是氨基酸残基间的天然相互作用而不是非天然的相互作用控制蛋白质的折叠过程。这一结论与含非天然二硫键的折叠中间体在牛胰蛋白酶抑制剂（BPTI）折叠中所起的重要作用并非相互排斥，因为后者仅仅是进行链内二硫键重排的化学反应所必需，与控制肽链折叠无直接关系。3根据对BPTI的研究，二硫键曾被认为仅仅具有稳定蛋白质天然结构的作用，既不决定折叠途径也不决定其三维构象。这一观点不适用于其它蛋白质。对凝乳酶原的研究表明，天然二硫键的形成是恢复天然构象的前提。天然二硫键的形成与肽键的正确折叠相辅相成，更具有普遍意义。4在氧化重折叠的早期，二硫键的形成基本上是一个随机过程，随着肽链的折叠二硫键的形成越来越受折叠中间体构象的限制。提高重组蛋白质的复性产率是生物技术领域中的一个巨大的挑战。除了分子聚集外，在折叠过程中所形成的二硫键错配分子是导致低复性率的另一个主要原因。氧化重折叠机制的阐明为解决此问题提供了有益的启示。如上所述，在折叠的后期，二硫键的形成决定于折叠中间体的构象，类天然、有柔性的结构有利于天然二硫键形成和正确折叠，具有这类结构的分子为有效的折叠中间体，最终都能转变为天然产物；而无效折叠中间体往往具有稳定的结构，使巯基、二硫键内埋妨碍二硫键重排，并因能垒的障碍不利于进一步折叠。因此，降低无效折叠中间体的稳定性使之转变为有效折叠中间体是提高含二硫键蛋白质复性率的一条基本原则，实验证明，碱性pH、低温、降低蛋白质稳定性的试剂、蛋白质二硫键异构酶、改变蛋白质一级结构是实现这一原则的有效手段。此外，这里还就氧化重折叠的基础和应用研究的前景进行了讨论。

Oxidative Refolding of Proteins

The mechanism of oxidative refolding of proteins was elucidated in more detail from the intensive and extensive studies in the past decades. 1 Most of the proteins examined so far proceed oxidative refolding via multiple pathways rather than a single and specific pathway. This is consistent with the folding energy landscape theory. 2 It is the native interactions rather than the non native interactions that direct the folding process. This is not necessarily incompatible with the importance of the non native disulfide intermediates in the bovine pancreatic trypsin inhibitor (BPTI) pathway, which are just a chemical necessity in the intramolecular arrangement to facilitate native disulfide formation. 3 Based on the BPTI refolding it was suggested that disulfide bonds have a stabilizing effect on the native state without determining either the folding pathway or the final three dimensional structure of the protein. This point of view is not applicable to other proteins. Studies on the refolding of prochymosin unequivocally demonstrated that the formation of native disulfides is the prerequisite to the recovery of the native conformation. It is more likely that the interdependence between the native disulfide formation and the formation of native structure is a general rule. 4 At the early stage of oxidative refolding disulfide formation is essentially a random process, with the progress of refolding further disulfide formation is increasingly dependent on the conformations of the intermediates. Enhancing the renaturation yield of recombinant proteins is a major challenge in biotechnology. In addition to aggregation, the formation of species with mispaired disulfide bonds is a leading cause of decreased yield. Progress in understanding the mechanism of oxidative refolding has provided insight into how to solve this problem. As described above, at the later stage of refolding disulfide formation depends on the conformations of intermediates. The intermediates with native like and flexible structure favourable for native disulfide formation and correct refolding are productive intermediates, while the unproductive intermediates tend to adopt stable conformations, which render the thiol groups and disulfide bond(s) inaccessible and further folding unfavourable energetically. Therefore, the principle to enhance the renaturation yield of disulfide containing proteins is to cause the productive intermediates to predominate by destabilizing the unproductive intermediates. To approach this, alkaline pH, low temperature, labilizing agents, protein disulfide isomerase and its analogues and alteration of primary structure have been proved useful to adjusting the structure of the unproductive intermediates so as to facilitate thiol/disulfide interchange and in turn the native disulfide formation. The prospects for the oxidative refolding of proteins both in basic and applied researches are discussed in this review article.