胃螺旋幽門桿菌蛋白質HP0404結構與功能的探討

Solution-structural and functional studies of protein HP0404 from helicobacter pylori

林鼎翔(Ting-Shiang Lin)

指導教授 ： 程家維

國立清華大學/生命科學院/生物科技研究所/碩士(2006年)

摘要

胃螺旋幽門桿菌 (Helicobacter pylori)是一種格蘭氏陰性菌、成長慢、具有鞭毛及螺旋外狀的細菌，他會引起嚴重的胃潰瘍、十二指腸相關的疾病和胃癌。根據蛋白質序列比對的結果，由於一段特殊的片段片段 (motif)：His-X-His-X-His-X-X (其中X是疏水性的胺基酸)我們發現HP0404蛋白質是一種屬於HIT (histidine triad)蛋白質家族裡的蛋白質激酶的抑制劑 (protein kinase C inhibitor)。HIT 蛋白質家族主要分為三個分支：Fhit (fragile histidine triad)分支，這一類主要是在動物和真菌中被發現。HINT (histidine triad nucleotide-binding protein)分支，這一類的蛋白質幾乎出現在所有的細胞中，另外最後一個是Galt (Galactose-1-phosphate uridylytransferase)分支。在這篇論文中，我們選擇HP0404作為我們的研究目標，利用NMR (nuclear magnetic resonance)來研究其結構， 利用C13 和N15標定的HP0404蛋白質樣品做NMR的實驗，並且也辨認出來HP0404的氫、碳、氮的化學位移的數值，光譜的分析的部分利用將同核二維的核磁共振光譜搭配了C13和 N15的三維核磁共振光譜(實驗都在298K PH=6.5的環境下進行)，在這裡，我們呈現HP0404蛋白質的序列分析的結果，並且將分析的結果標示在HSQC (heteronuclear single quantum correlation)光譜圖上。並且也做了腺甘三磷酸(adenosine 5′-triphospate)滴定的實驗，發現I17、N20、S25、S44、K68、T81、N86 和H97這些胺基酸在蛋白質和ATP作用中也許扮演著重要的角色。希望得到的結果可以對之後HP0404蛋白質結構和功能研究有更進一步的幫助。

關鍵字

核磁共振光譜儀 ； 螺旋幽門桿菌

並列摘要

H. Pylori (Helicobacter pylori) is a Gram-negative, microaerophilic, and slow-growing bacterium, which can cause serious gastric ulcer, duodenum-associated disease and gastric cancer. According to the sequence alignment, HP0404 which is produced by H. pylori is the PKC inhibitor of HIT (histidine triad) protein family, because of the special sequence motif, His-X-His-X-His-X-X (X is a hydrophobic amino acid). HIT proteins fall into three branches, the Fhit (fragile histidine triad) branch that is found only in animals and fungi and histidine triad nucleotide-binding protein (Hint) branch that has represented in all cellular life and the Galt(Galactose-1-phosphate uridylytransferase) brench. Here we chose HP0404 as interest target to investigate the structure by using NMR spectroscopy (nuclear magnetic resonance). The C13- and N15-label protein sample of HP0404 have been produced for the NMR experiment. Proton, nitrogen, and carbon chemical shift assignment have been made for HP0404. assignment were made from a combination of homonuclear two-dimensional and N15- and C13- edited three –dimensional spectra at PH6.5 and 298K. Here we present the sequential assignment of HP0404 and make the residue annotation on the HSQC (heteronuclear single quantum correlation) spectra. We also did the ATP titration experiment for functional study and found residue I17, N20, S25, S44, K68, T81, N86 and H97 maybe important for interacting with ATP. This annotation maybe could offer slight help for further investigation between structure and function of HP0404.

並列關鍵字

NMR ； Helicobacter pylori

參考文獻

Reference List

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