Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/98125
標題: 柑橘黃單胞菌Xanthomonas axonopodis效應蛋白XopAI晶體結構與螢光光譜分析
Crystal Structure and Fluorescence Spectrometry Analysis on the Effector Protein XopAI from Xanthomonas axonopodis
作者: 李昀霈
Yun-Pei Li
關鍵字: 柑橘潰瘍;柑橘黃單胞菌;第三型分泌效應系統;XopAI;螢光光譜分析法;蛋白質晶體結構;Citrus canker;Xac;T3SS;XopAI;Fluorescence spectroscopy;Crystal Structure
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摘要: 
黃單孢菌(Xanthomonas axonopodis pv. Citri, Xac)是柑橘類植物潰瘍的主要致病原因,能利用第三型分泌系統(Type III secrestion system, T3SS)的複合蛋白質跨膜通道,將效應蛋白注入到宿主細胞中,影響宿主細胞的防禦機制,使細菌能有效感染宿主,因此,若能抑制效應蛋白作用或使效應蛋白攜帶藥物,將能降低柑橘潰瘍(Citrus canker)發生。Xac主要效應蛋白有四種,當中XopAI作用機制尚不明確。目前已知XopAI蛋白晶體堆疊方式,會將N端的Ser60、Arg62、Pro63、Leu64、Leu67與另一個XopAI分子的C端的凹槽處相互作用,其結果會影響到實際XopAI蛋白質分子與輔因子之間的結合,因此,在本實驗中我們為了瞭解它的作用機制,我們將它去除N端前70個胺基酸(XopAI-delN70)進行表現,並進行結晶體實驗解出其結構,與藉由螢光光譜分析法(Fluorescence spectroscopy)分析與輔因子之間關係。由實驗結果得知,當XopAI去除N端前70個胺基酸後,其蛋白質晶體結構空間群(Space group)為P21,具有四種蛋白質構型,其C端凹槽處為保留區,可能為它的活性區域,比對結果,其C端序列與HopU1、Vibrio splendidus的NAD+-Arg ADP-核醣基轉移酶(ADP- ribosyltransferase, ADP-RT)蛋白質為相似。在螢光光譜分析結果中,我們證實XopAI-delN70的Tryptophan於加入輔因子後螢光量會明顯下降,當中以NAD+影響程度最高,且再加入Arg還能提升兩者的作用,最後,利用GraphPad分析0.25 μM XopAI-delN70與NAD+的解離常數(Kd),計算結果Kd值介於955.8~1618 μM之間。

Xanthomonas axonopodis pv. citri (Xac) is the main cause of citrus canker infection with Citrus plants, it can use the Type III secrestion system (T3SS) of transmembrane protein (TP) to inject Bacterial effector protein into the inside of plant cells, affecting the expression levels of host plant defense-related genes of plant to facilitate bacteria infection plant. Therefore, if we can suppress the influence of bacterial effector protein or let bacterial effector protein carry drugs will reduce the occurrence of citrus canker. Xac possesses four unique effectors, of which the XopAI mechanism is unknown. The XopAI crystals structure are stacked in such that the XopAI uses the N-terminal residues (Ser60, Arg62, Pro63, Leu64, Leu67) to interact with the N-terminal groove of other XopAI, but this result does not match the role of real XopAI and cofactors. Therefore, in order to understand the XopAI mechanism, we removed it the first 70 amino acids of the N-terminus (XopAI-delN70), and performed protein expression. After that, the structure was obtained by using Protein crystallization experiment, and the relationship between it and cofactors was analyzed by fluorescence spectroscopy. From the experimental results that the space group of XopAI-delN70 is P21, which has four protein configurations, and the C-terminal groove is a retention region, which may be its active region, and its sequence is similar to Type III effector HopU1 and Vibrio splendidus with Novel NAD+-Arg ADP-ribosyltransferase (ADP-RT). The results of fluorescence spectroscopy confirmed that the fluorescence of the Tryptophan on the XopAI-delN70 surface was significantly decreased after adding the cofactor, among which NAD+ had the highest degree of influence, and the addition of Arg could enhance the effect. The GraphPad was used to analyze 0.25 μM XopAI-delN70 and NAD+. As a result of calculation, the Kd value is between 955.8~1618 μM.
URI: http://hdl.handle.net/11455/98125
Rights: 同意授權瀏覽/列印電子全文服務,2021-08-20起公開。
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