【作者】 付新苗；

【导师】 昌增益；

【作者基本信息】 清华大学 ， 生物学， 2004， 博士

【摘要】 分子伴侣蛋白在生物体内的主要功能是帮助新生肽链的折叠以及防止变性蛋白的聚集等。小分子热休克蛋白是分子伴侣蛋白中的一个亚家族,结构特征是含有保守的α-crystallin结构域。本文的工作研究来自人类重要的病原微生物结核分支杆菌(Mycobacterium tuberculosis)的小分子热休克蛋白Hsp16.3调控分子伴侣活性的机制,主要包括三方面的内容。第一方面研究寡聚体解聚和分子伴侣活性的关系,主要结果为:⑴用化学交联法证明了Hsp16.3九聚体的解聚是发挥活性的必要条件;⑵发现Hsp16.3在维持九聚体大小不变的情况下可以通过调整寡聚体的动力学性质(解聚速度、亚基交换速度)来调节分子伴侣活性;⑶发现Hsp16.3能在生理温度范围内(25°C-37.5°C)调节分子伴侣活性,暗示该蛋白在体内具有结合变性蛋白的能力;⑷建立了一个描述寡聚体解聚和发挥分子伴侣活性的动力学模型,可以解释很多小分子热休克蛋白的实验现象。第二方面研究Hsp16.3蛋白的一级序列和寡聚体结构、分子伴侣活性的关系,主要结果为:⑴证明N端区域(35个氨基酸)包含底物结合位点;⑵N端区域作为一个整体对形成九聚体是必需的,首次观测到至少有三个亚基的N端区域对九聚体的形成不是必需的;⑶首次发现N端区域可以稳定作为大寡聚体的组装单位——三聚体;⑷发现C端尾巴保守的IXI motif对寡聚化是必需的;⑸鉴定了保守的LPGV motif的作用,证明59位的Gly位于亚基作用面上;⑹构建了有更高活性的小寡聚体,充分证明了小寡聚体是Hsp16.3的活性形式,而九聚体对活性不是必需的。第三方面研究Cys在分子伴侣蛋白家族中的进化,主要结果为:⑴发现Cys和Trp在该蛋白家族的含量显著低于其他蛋白家族;⑵首次观察到Cys所形成的二硫键对分子伴侣蛋白的损害作用,从而部分了解释了Cys在分子伴侣蛋白中含量少的进化现象,并揭示了构象柔性对分子伴侣蛋白的重要性。更多还原

【Abstract】 Molecular chaperones are predominantly involved in assisting newly synthesized protein folding and in preventing denaturing proteins from misfolding and aggregation. Small heat shock proteins (sHsps), as one sub-class of molecular chaperones, are known for the existence of a highly conserved 80-100 amino acids α-crystallin domain. In present dissertation, I am attempting to reveal the mechanism of Hsp16.3, one sHsps from the human pathogen Mycobacterium tuberculosis, in modulating its chaperone-like activities to suppress the aggregation of denaturing proteins, as shown by the following three sections.The oligomeric dissociation was previously revealed to be correlated with chaperone-like activities of sHsps. Here our further studies by chemical cross-linking showed that the oligomeric dissociation of Hsp16.3 is a prerequisite for its chaperone-like activities. Furthermore, we demonstrated that Hsp16.3 is able to modulate its chaperone-like activities by adjusting the rate of oligomeric dissociation while maintaining its static oligomeric size unchangeable. As a result, a kinetic model to describe the dynamic oligomeric dissociation and chaperone activities is proposed to explain some contradictory observations. In addition, Hsp16.3 was found to modulate its chaperone-like activities in a range of physiological temperatures (25°C-37.5°C), thus indicating that the protein is capable of binding non-native proteins in vivo. In an attempt to reveal the relation of oligomeric structure of Hsp16.3 with its primary structure, we found that the N-terminal region is a substrate-binding site and essential for chaperone-like activities. Remarkably, these regions in Hsp16.3 nine subunits, being essential as a whole for the nonameric assembly and important for stabilization of trimers, of which in at least three subunits are unessential for such an assembly. The C-terminal extension, especially the conserved IXI/V motif in this region, is <WP=5>also critical for the nonameric assembly of Hsp16.3, however, the dissociated oligomers with this motif truncated exhibits greatly enhanced chaperone activities. Gly59 in the conserved motif LPGV was found to be located in the subunit interface of Hsp16.3, of which the replacement by Trp results in the disappearance of nonamer and enhanced chaperone-like activities. In light of these observations, we propose that the nonamric structure of Hsp16.3 is not a prerequisite for its chaperone-like activities and the dissociated oligomers are active. The structural feature of molecular chaperone family was summarized as highly conformational flexibility and spontaneously folding/assembly, however, the mechanism is far from clear. The frequency of Cys and Trp in molecular chaperones is significantly less than that in other protein families. Further more, we for the first time presented evidence to show that disulfide bonds will convert Hsp16.3 from a chaperone to a non-chaperone, thus partially explaining the above intriguing evolution observation and implying that the conformational flexibility is important for molecular chaperones.更多还原