Folding Mechanism of beta-Lactogobulin

蛋白质折叠是将蛋白质初级氨基酸序列中的遗传信息传递到其独特的三维结构中的一种机制，阐明蛋白质折叠机制对于理解蛋白质的结构和功能至关重要。β -乳球蛋白是阐明蛋白质从α -螺旋到β -片（α - β）转变机制的一个有趣模型，这是理解许多蛋白质分子折叠和生物学功能的关键问题。为了阐明β -乳球蛋白折叠的机制，我们开展了国际科研计划，得到了以下结果：牛β -乳球蛋白A已在嗜甲基酵母毕赤酵母中表达。在补料分批发酵罐中，经过72hr的甲醇诱导，分泌蛋白达到1g /L的水平。重组蛋白的物理特性与天然牛蛋白没有明显区别。重组的牛β -乳球蛋白A在天然状态和2,2,2-三氟乙醇（TEE）诱导的高螺旋状态下，用^1H、^<13>3C和^<15 >5n多维核磁共振谱进行了表征。天然状态下的二级结构与晶体结构基本一致。另一方面，TEE状态下的β -乳球蛋白由许多α -螺旋片段组成。我们基于异核磁共振技术确定了天然状态下的溶液结构。单体-乳球蛋白在pH为2.0的水溶液中的整体结构与pH为6.5.4时的x射线结构非常相似。我们通过核磁共振测量和H/D交换反应分析了β -乳球蛋白的折叠动力学。结果表明，在重折叠过程中，原生类核心β -sheet和一些非原生螺旋由于疏水坍塌而迅速形成，随后，剩余的β -sheet通过与先前存在的核心β -sheet相互作用而被“诱导”。少

Elucidation of the mechanisms of protein folding, by which the genetic information contained in the primary amino acid sequence of a protein is transmitted to its unique three-dimensional structure, is essential for understanding the structure and function of proteins. beta-Lactoglobulin is an intriguing model for clarifying the mechanism of the alpha-helix to beta-sheet (alpha-beta) transition of proteins, a key issue for understanding the folding and biological function of a number of protein molecules. We carried out the International Scienlific Research Program in order to clarify the mechanism of beta-lactoglobulin folding and obtained the following results.1. Bovine beta-lactoglobulin A has been expressed in the methylotropic yeast Pichia pastoris. In a fed-batch fermenter, after 72hr of methanol induction, the secreted protein reached levels of 1g./L.The physical characteristics of recombinant protein were indistinguishable from those of the native bovine protein.2. The recombin … More ant bovine beta-lactoglobulin A in the native state and in the highly helical state induced by 2,2,2-trifluoroethanol (TEE) were characterized by ^1H, ^<13>3C, and ^<15>5N multidimensional NMR spectroscopy. Overall secondary structures in the native state were similar to those of the crystal structure. On the other hand, beta-lactoglobulin in the TEE state was composed of many alpha-helical segments.3. We determined the solution structure in the native state based on heteronuclear NMR techniques. The overall structure of monomeric beta-lactoglobulin at pH 2.0 in aqueous solution is very similar to the X-ray structure obtained at pH 6.5.4. We analyzed the folding kinetics of beta-lactoglobulin by NMR measurements coupled with the H/D) exchange reaction. The results suggest that, during refolding, the native-like core beta-sheet and several non-native helices are formed rapidly due to hydrophobic collapse, and subsequently, the remaining beta-sheet is "induced" through interaction with the preexisting core beta-sheet. Less