肺炎链球菌中HtrA和PpmA的结构研究

发布时间：2018-05-05 13:00

本文选题：温度依赖 + 丝氨酸蛋白酶　；参考：《中国科学技术大学》2010年硕士论文

【摘要】：1.肺炎链球菌中温度依赖的蛋白HtrA的结构研究 HtrA (high-temperature requirement A)是一种温度依赖的热休克蛋白,它通过感应外界温度的变化来调节两种功能的转化,分别是:正常温度时行使分子伴侣功能,以及温度升高时的丝氨酸蛋白酶活性。这种功能上的精确转化使得HtrA在维持蛋白质的稳定性以及降解热变形的蛋白以维持细胞的稳定性过程中起关键作用。它被认为是细菌周质间隙中蛋白质质量调控体系中的关键蛋白。研究证实HtrA对于帮助细菌和细胞抵抗高温损伤都有重要作用。HtrA家族蛋白广泛分布在从古细菌到人类的许多的组织中。近期的生物化学和结构学研究证实,不管是细菌抵抗一系列的环境压力还是蛋白质质量调控,都是通过HtrA家族蛋白的结构和功能的精确变化来完成的。然而,其精确的蛋白质质量调控方式还有待研究。为了更好的解析HtrA家族蛋白的作用作用方式,需要进一步对其进行生物化学和结构学研究,帮助我们对其分子功能以及其特殊的分子调控机制有更好的理解。我们已经得到了肺炎链球菌的基因组DNA,经过PCR扩增出一个htrA全长基因和丝氨酸蛋白酶功能域片段,随后分别插入p28质粒进行表达载体的构建。其中全长蛋白经过表达优化与纯化后,可以得到大量的可溶状态。蛋白质初筛后得到晶体,目前我们尝试优化了各种条件,但是没有优化到合适于X射线衍射的蛋白晶体。 2.肺炎链球菌中假定的蛋白酶成熟蛋白PpmA的结构研究在过去的20年中,肽基脯氨酰顺反异构酶积累了大量的研究数据,分别在生理,细胞生化,分子动力学以及蛋白质结构研究中取得了相当多的进展。尽管肽基脯氨酰顺反异构酶催化的顺反异构作用本身并不复杂,但是但目前为止对于详细的蛋白催化机理仍没有得到完全的解释。我们研究的PpmA (Potential proteinase maturation protein A)属于被广泛研究的肽基脯氨酰顺反异构酶的小细胞蛋白家族。我们已经得到了肺炎链球菌的基因组DNA,进行PCR扩增出一个ppmA全长基因和一个功能域片段,随后分别插入p28质粒进行表达载体的构建。其中PpmA全长蛋白经过表达优化与纯化后,可以得到大量的可溶状态,但是晶体筛选后没有得到蛋白晶体。
[Abstract]:1. Study on the structure of Temperature-dependent protein HtrA in Streptococcus pneumoniae HtrA high-temperature requirement A is a temperature-dependent heat shock protein, which regulates the transformation of two functions by inducting the change of external temperature, namely, the function of chaperone at normal temperature and the activity of serine protease at elevated temperature. This precise functional transformation makes HtrA play a key role in maintaining the stability of proteins and the degradation of thermodeformed proteins to maintain the stability of cells. It is considered to be a key protein in the protein quality control system in the periplasmic space of bacteria. It has been confirmed that HtrA plays an important role in helping bacteria and cells resist high temperature damage. HtrA family proteins are widely distributed in many tissues from ancient bacteria to humans. Recent biochemistry and structural studies have confirmed that both bacterial resistance to a series of environmental pressures and protein quality regulation are accomplished by precise changes in the structure and function of HtrA family proteins. However, the precise regulation of protein quality remains to be studied. In order to elucidate the action of HtrA family proteins, it is necessary to further study their biochemistry and structure to help us understand their molecular functions and their special molecular regulation mechanisms. We have obtained the genomic DNA of Streptococcus pneumoniae and amplified a full-length htrA gene and a functional domain of serine protease by PCR. Then we inserted p28 plasmid to construct the expression vector. After expression optimization and purification, a large number of soluble state can be obtained. At present, we have tried to optimize various conditions, but we have not optimized the protein crystal suitable for X-ray diffraction. 2. Study on the structure of putative protease mature protein PpmA in Streptococcus pneumoniae In the past 20 years, a large amount of research data have been accumulated on the peptide proline cis-trans isomerase, and considerable progress has been made in the studies of physiology, cell biochemistry, molecular dynamics and protein structure, respectively. Although the cis-trans isomerization catalyzed by peptidyl prolyl cis-trans isomerase is not complicated, the detailed mechanism of protein catalysis has not been fully explained. Our PpmA potential proteinase maturation protein A belongs to the small cell protein family of peptidyl prolyl cis-trans isomerase. We have obtained the genomic DNA of Streptococcus pneumoniae, amplified a full-length ppmA gene and a functional domain fragment by PCR, then inserted p28 plasmid to construct the expression vector. After expression optimization and purification of PpmA full-length protein, a large number of soluble state could be obtained, but no protein crystal was obtained after crystal screening.
【学位授予单位】：中国科学技术大学
【学位级别】：硕士
【学位授予年份】：2010
【分类号】：R378

【共引文献】