结核分枝杆菌ABC转运蛋白COG1732底物结合结构域的晶体学研究

发布时间：2018-03-28 06:01

本文选题：ABC转运蛋白　切入点：结核分枝杆菌　出处：《兰州大学》2017年硕士论文

【摘要】：ABC转运蛋白是一类古老的、广泛存在的,出现在古细菌、真细菌及真核生物中的跨膜转运蛋白。这类蛋白的主要功能是通过水解ATP驱动物质的跨膜转运,它们一般由三个结构域组成,包括跨膜结构域(TMDs),核酸结合结构域(NBDs)和底物结合结构域(SBDs)。结核分枝杆菌(Mycobacterium tuberculosis)是导致结核病的高致病性病原,它的细胞膜具有革兰氏阳性菌和革兰氏阴性菌的共同特点,可在全身各个器官增殖,在肺中最为多见,而ABC转运蛋白所介导的物质转运可能是其适应不同宿主细胞内环境的重要机制。本研究主要针对结核分支杆菌中的一个ABC转运蛋白的底物结合蛋白质COG1732的生化功能及三维结构展开。序列分析表明该蛋白与相容性溶质结合蛋白有27.8%的序列相同度,暗示了该蛋白可能与结核分枝杆菌对高渗透环境的适应有关,但是通过荧光淬灭实验我们发现该蛋白并不能结合甜菜碱、胆碱等相容性溶质。进一步通过化合物筛选我们发现一系列与COG1732结合的酚类化合物2`,6`-Dihydroxyacetophenone(2`,6`-HAP)、Monoacetylphloroglucinol(MAPG)、2`-Hydroxyacetophenone(2`-HAP)、4`-Hydroxyacetophenone(4`-HAP)、2`,4`-Dihydroxyacetophloroglucinol(DAPG)、Phloretin、2`,4`-Dihydroxyacetophenone(2`,4`-HAP)这些化合物与COG1732结合的解离常数在20 uM-200 uM之间。同时我们也尝试了对COG1732的结晶,通过对重组蛋白序列的优化,获得了高质量的蛋白晶体,这为揭示COG1732的生化功能以及其结合配体分子的结构生物学基础奠定了坚实的基础。
[Abstract]:ABC transporters are a class of ancient, ubiquitous transmembrane transporters found in archaic bacteria, eukaryotes and eukaryotes. The main function of these transporters is the transmembrane transport of substances driven by hydrolysis of ATP. They are generally composed of three domains, including transmembrane domain (TMD), nucleic acid binding domain (NBDs) and substrate binding domain (SBDsN). Mycobacterium tuberculosism (Mycobacterium tuberculosissis) is a highly pathogenic pathogen leading to tuberculosis. Its cell membrane has the common characteristics of Gram-positive bacteria and Gram-negative bacteria, and can proliferate in all organs of the body, most commonly seen in the lung. ABC transporter mediated substance transport may be an important mechanism for its adaptation to the intracellular environment of different hosts. This study focused on the biochemical work of COG1732, the substrate binding protein of a ABC transporter in Mycobacterium tuberculosis. Sequence analysis showed that the protein had the same sequence with compatible solute binding protein in the order of 27.8%. It suggests that the protein may be related to the adaptation of Mycobacterium tuberculosis to high osmotic environment, but the fluorescence quenching test shows that the protein does not bind betaine. Choline and other compatible solutes. Further screening, we found that a series of phenolic compounds 2 '-dihydroxyacetophenone 2' -Dihydroxyacetoneone 2 '-Dihydroxyacetone acetophenone 2`-Hydroxyacetophenone2' -HAPOX 4 '-Hydroxyacetophenone4' -HAP1 '-Dihydroxyacetophenone4' -Dihydroxyacetophenone4 '-Dihydroxyacetophenone 2' -Dihydroxyacetophenone 2 '-dihydroxyacetophenone 2' -Dihydroxyacetophenone2'4 '-Dihydroxyacetophenone 2' -dihydroxyacetophenone2'4 '-Dihydroxyacetophenone 2' 4 '-Dihydroxyacetophenone 2' 4 '-dihydroxyacetophenone 2' -dihydroxyacetophenone 2 '-dihydroxyacetophenone 2' -dihydroxyacetophenone 2 '-dihydroxyacetophenone 2' -dihydroxyacetophenone 2 '-dihydroxyacetophenone 2' -dihydroxyacetophenone 2 '-dihydroxyacetophenone 2' -dihydroxyacetophenone 2 '-dihydroxyacet@@. High quality protein crystals were obtained by optimizing the sequence of recombinant proteins, which laid a solid foundation for revealing the biochemical functions of COG1732 and the structural and biological basis of its binding ligands.
【学位授予单位】：兰州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R378.911

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