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

本研究对细胞周围病变细菌放线菌（actinomycetemcomitans）中的铁结合蛋白（Dps （DNA保护）蛋白和铁蛋白）进行了x射线结构分析，这些铁结合蛋白在保护细胞大分子免受活性氧损伤中起重要作用。ftn1可以成功结晶，但ftn2只能变成无定形。在含28%聚乙二醇400的0.1 M HEPES-Na缓冲液pH 7.5和0.2 M二水合氯化钙溶液中，采用蒸汽扩散悬滴法培养放线菌a . dps样蛋白晶体。在蛋白质浓度为~10 mg/ml的条件下，培养出小而完美的六边形棒状细胞。这些棒在内部衍射强度为1.9 A，位于六边形空间群P63中，胞元尺寸为A = b = 128.5 A, c = 91。lA和γ=120°。数据集的完成率为93.9%，共收集到1.9 A，多重度为2.9,R_<merge>为0.071。以李斯特菌（Listeria innocua）的十二聚体铁蛋白（tiritin like protein）为搜索模型，采用分子置换法求解放线菌A.actinomycetemcomitans的dps样蛋白结构。不对称单元包含四个独特的亚单元，它们围绕晶体学的三重轴排列。我们还分析了人类β -防御素-2 （hBD-2）作为新的抗菌物质的候选物对口腔细菌的抗菌活性。hBD-2对放线菌、牙龈卟啉单胞菌、变形链球菌的抑菌活性与二甲胺四环素在等摩尔浓度下的抑菌活性大致相当。我们进一步研究了蔓越莓的成分和尿液代谢物的影响，蔓越莓富含多酚，对大肠杆菌的生物膜形成有多种有益人体健康的作用。我们发现阿魏酸、同型香豆酸、4-香豆酸、异阿魏酸和香草酸对大肠杆菌生物膜的形成具有抑制作用。少

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