生命科学学院知识库

蛋白质是生命活动的主要承担者，其功能活性完全依赖于特定的空间结构。错误的蛋白质折叠通常导致疾病发生。然而，蛋白质的空间结构是如何由其一级氨基酸序列折叠而成的，是一个尚未解决的基础生物学问题。对于含二硫键的蛋白质来说，二硫键的存在增加了其结构的复杂性。这部分蛋白质的折叠过程中，由于构象折叠和二硫键形成之间的互相干扰，理论上使得其折叠过程变得更加困难。为了探讨蛋白质折叠过程中构象折叠与二硫键形成之间的协调机制，我们以C-反应蛋白（C-reactive protein, CRP）的胞内折叠入手展开了一系列研究。CRP是人体重要的急性期血浆蛋白，是由5个相同的亚基非共价聚合而成的盘状五聚体，每个亚基内含有一对二硫键。在机体受到感染和炎症损伤时，CRP的表达量会短时间提升上千倍。这暗示其在胞内具有高效的折叠组装机制。另外，CRP的天然结构能以E.coli体系进行重组表达和分泌。
本文工作中，我们通过一系列CRP突变体在COS-7与E.coli细胞中的表达，捕捉了CRP天然结构形成过程中可能的结构中间体，并通过对其折叠过程中二级结构元件定位顺序的研究，最终描绘了CRP的胞内折叠组装机制。研究结果表明，胞内五聚体CRP天然结构的形成分两大步骤进行。首先，CRP肽链折叠成近似天然构象的亚基；然后，已折叠的亚基再用于五聚体的组装。CRP亚基的折叠也分为两个连续的阶段。在CRP亚基折叠的早期过程，strands C-I自发折叠并形成了一个疏水骨架结构，此时C端a.a.168-176 helix也完成了自发定位，二者共同驱动strands C&H相互靠近并形成了Cys36-Cys97亚基内二硫键。相比之下，折叠的第二阶段是非自发过程，涉及到大范围的结构重构。此阶段中，Cys36-Cys97稳定了前期形成的骨架结构，并允许strands J&K通过钙离子结合的介导而顺利地整合至疏水骨架中，并最终完成了CRP亚基折叠的全过程。
因此，我们的结果表明，CRP亚基折叠的早期阶段，构象的自发折叠驱动了二硫键的形成；而在非自发的后期折叠阶段，已经形成的二硫键反过来驱动了构象的折叠与整合。这一结论较为清晰地描绘了蛋白质氧化折叠中构象折叠与二硫键形成之间的相互协调关系。在一定程度上补充和完善了该研究领域先前的相关结论。除此之外，我们的结论还证明，胞内CRP天然结构的形成是经历特定中间产物的单一途径，而非经历多种途径的随机选择过程。这也很好地解释了为什么CRP能在胞内如此快速地折叠组装。
 

As the main executor of life activities, protein functions entirely dependent on specific spatial structure. Mistakes in protein folding always induce diseases. Nevertheless, it remains unclear how protein’s primary sequence determines its spatial structure. For proteins containing disulfide bond, the structure complexity is further increased. During the protein oxidative folding, the conformational folding and disulfide bonding may interfere with each other, theoretically making the folding process more difficult. To explore the co-ordination mechanisms between conformational folding and disulfide bonding in protein oxidative folding, we use C-reactive protein (CRP) as a case to study its folding mechanism in live cells. As an important acute phase protein in human body, CRP is a pentamer polymerized by five identical subunits in a no-covalent way. Each CRP subunit was equipped with a pair of intrasubunit disulfide bond. When the infections or inflammations happen, CRP expression will increase hundreds of times in a short period of time, indicating an efficient folding and assembly mechanism in vivo. Moreover, native CRP can be produced and secreted by E.coli with recombinant expression.
In the present work, we expressed a series of CRP mutants with COS-7 and E.coli cells. Possible folding intermediates were captured and the folding orders of each secondary elements were carefully analysed, which allowed us to describe the exact folding and assembly mechanism in live cells. Our results reveal that CRP generates its pentameric structure with two sequencial steps. Firstly, CRP peptide folds into a near-native subunit. Then the assembly of pentamer occurs using the folded subunits. For the folding process of CRP subunits, it also contains two sequencial stages. At the early stage, strands C-I fold spontaneously into a hydrophobic core, companied with the correct position of a C-terminal helix. These together draw close the strands C&H and directly induce the formation of Cys36-Cys97 disulfide bond. By contrast, the second stage of CRP subunit folding involves extensive structure remolding and is not spontaneous. In this stage, the formed Cys36-Cys97 disulfide bond stabilizes the folded core and allows the strands J&K to integrate into the core in a calcium binding-guide way. Finally, the whole process of CRP subunit folding is completed.
As a whole, we show that spontaneous conformational folding drives the formation of disulfide bond in the early stage of CRP subunit folding. In the subsequent folding process which is no-spontaneous, instead, the formed disulfide bond drives the conformational folding and assembly. Conclusions here thus clearly depicts a coordination relationship between conformation folding and disulfide bonding in the protein oxidative folding, which supplements and perfects the previous conclusions in this field to some extent. In addition, our results also argue that CRP generates its native structure following a specific pathway involving obligatory intermediates rather than undergoing numerous folding routes with random selections.