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X-ray Crystal Structural Analysis of Bacterial Iron Binding Protein and Development for New Antibiotics

细菌铁结合蛋白的X射线晶体结构分析及新型抗生素的开发

基本信息

批准号：
15390566
负责人：
KOKEGUCHI Susumu
金额：
$ 8.06万
依托单位：
OKYAMA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2003
资助国家：
日本
起止时间：
2003 至 2006
项目状态：
已结题

项目摘要

In this study, we investigated the X-ray structural analyses on the iron-binding proteins(Dps (DNA protection during starvation) protein and ferritin protein) from peridontopathic bacteria, Actinobacillus actinomycetemcomitans, which play an important role in protecting cellular macromolecules from damage by reactive oxygen species.A.actinomycetemcomitans expressed two ferritin protein(Ftn1 and Ftn2). The Ftn 1 could be successfully crystallized, but Ftn 2 became only amorphous form. Crystals of the Dps-like protein of A. actinomycetemcomitans were grown by the hanging drop method of vapour diffusion from a solution containing 28% polyethylene glycol 400 in 0.1 M HEPES-Na buffer pH 7.5 with 0.2 M calcium chloride dihydrate. Small but perfect hexagonal rods were grown with a protein concentration of~10 mg/ml. These rods diffract strongly to 1.9 A in-house and were found to be in the hexagonal space group P63 with cell dimensions a = b = 128.5 A, c = 91.lA and γ=120°. A 93.9% complete da … More ta set was collected to 1.9 A with a multiplicity of 2.9 and an R_<merge> of 0.071. The structure of the Dps-like protein of A.actinomycetemcomitans was solved by molecular replacement using the dodecameric ferritin like protein of Listeria innocua as the search model. The asymmetric unit contains four unique subunits arranged around the crystallographic 3-fold axis.We also analyzed the antimicrobial activity against oral bacteria of human beta-defensin-2 (hBD-2) as a candidate of new antimicrobial substances. hBD-2 shouwed antimicrobial activity gainst Actinobacillus actinomycetemcomitans, Porphyromonas gingivalis, Streptococcus mutans, which was approximately equal to that of minocycline at equimolar concentrations. And we further examined the effect of ingredients and urinary metabolites of cranberry, which is rich in polyphenols, has been found to have various effects beneficial to human health, on biofilm formation by Escherichia coli. We found that ferulic acid, homovanillic acid, 4-coumaric acid, isoferulic acid and vanillic acid with inhibitory activity on Escherichia coli biofilm formation. Less

在这项研究中，我们研究了牙周病放线杆菌的铁结合蛋白(DPS蛋白和铁蛋白)的X射线结构分析，这些蛋白在保护细胞大分子免受活性氧物种的损伤方面发挥着重要作用。FTN-1结晶成功，FTN-2为非晶态。用气相扩散悬滴法从28%聚乙二醇400的0.1M HEPES-Na缓冲液(pH 7.5)和0.2M二水氯化钙溶液中生长伴生放线菌类DPS蛋白晶体。在蛋白质浓度为~10 mg/ml的条件下，生长出细小而完美的六角棒，这些棒的内部衍射率为1.9A，属于六方空间群P63，晶胞尺寸a=b=128.5 A，c=91.1A，γ=12 0°。A 93.9%完成da…更多的ta集合被收集到1.9A，复数为2.9，R_&lt；合并&>0.071。以诺氏李斯特菌的铁蛋白十二聚体为搜索模型，采用分子置换的方法对伴生放线菌的类DPS蛋白的结构进行了解析。我们还分析了人β-防御素-2(HBD-2)作为一种新型抗菌物质对口腔细菌的抗菌活性。HBD-2对伴生放线杆菌、牙龈卟啉单胞菌、变形链球菌的抗菌活性与等摩尔浓度的米诺环素相当。我们还进一步研究了富含多酚的酸莓的成分和尿液代谢产物对大肠杆菌生物被膜形成的影响。我们发现阿魏酸、高香草酸、4-香豆酸、异阿魏酸和香草酸对大肠杆菌生物被膜的形成有抑制作用。较少