- 學位論文
克雷伯氏肺炎桿菌之黃素氧還蛋白結構與功能及其藥物設計研究
Structures and Functional Studies of Flavodoxin from Klebsiella pneumoniae — a potential therapeutic target
摘要
克雷伯氏肺炎桿菌係一革蘭氏陰性細菌，它造成院區感染及感染免疫系統受損的病人，在肝膿瘍病人造成的死亡率為10%，對併發性的髓膜炎病人則有30-40%死亡率，近廿年來主要由克雷伯氏肺炎菌造成之肝膿瘍及併發症已為台灣最普遍的社區感染菌。它已取代鏈球肺炎菌，成為台灣成人社區感染髓膜炎首號病菌。研究方向主要針對具病理相關蛋白質，並輔以結構基因體學特質。選定探討之蛋白質主要由具毒性相關基因組群及抗藥性組群中挑選，此外也將探討序列守衡之虛擬蛋白質(CHP)結構，決定此類CHP蛋白結構將有助於瞭解其功能並可能找到新摺疊體。文獻指出flavodoxin在人體中是不存在的，是做為藥物設計很好的蛋白，本篇以flavodoxin (KP0001)為主要研究蛋白。在本研究當中,我們已經成功地利用大腸桿菌(E. coli)將KP0001 (Flavodoxin)蛋白表現出來,並經由各種純化步驟得到大量的高純度蛋白.在蛋白質結構的初步鑑定上,經由圓二色光譜儀(circular dichroism, CD)和化學位移預測法(chemical shift index, CSI)我們可以得知, KP0001 (Flavodoxin)蛋白主要是由5個β摺板(β-sheet)以及4個α螺旋所組成。在穩定性部分，則是利用溫度變性實驗(thermal denaturation)以及尿素變性實驗(Urea-induced denaturation)加以探討。至於與FMN 交互作用部分,則是透過恆溫滴定熱卡計(isothermal titration calorimetry, ITC)得到結合常數。在且利用HSQC探討KP0001(Flavodoxin)與FAD、FMN、flavobin以及二價鐵離子之間的交互作用。此外,我們亦根據在核磁共振(NMR)實驗上所得之數據,經由電腦計算的方式(ARIA-CNS)將KP0001(Flavodoxin)蛋白在水溶液中的三度空間結構(three-dimensional structure)解出,未來預期能夠全盤了解此一重要蛋白質之詳細作用機轉。
並列摘要
Klebsiella pneumoniae is an enteric gram-negative bacillus causing hospital-acquired infections and infections in debilitated, or immunocompromised, patients. Mortality rates as high as 10% for primary liver abscess, and among them, 30– 40% for those with metastatic meningitis have been reported. In the past two decades, primary K. pneumoniae liver abscess and its septic metastatic complications have emerged as one of the most common community-acquired bacterial diseases in Taiwan, and K. pneumoniae has replaced Streptococcus pneumoniae as the leading pathogen of adult community-acquired bacterial meningitis in Taiwan. This thesis focuses on determining the structures and function of flavodoxin. From previous studies, it has been confirmed that flavodoxin does not exit in human; therefore, this protein can be well used in pharmaceutical purpose. We have expressed the KP0001 (Flavodoxin) protein in Escherichia coli in high yields and recovered the KP0001 (Flavodoxin) protein by purification, using heparin-sepharose affinity resin. Far UV circular dichroism data and 1Hα, 13Cα, 13Cβ and 13CO chemical shift indices suggested that the KP0001 (Flavodoxin) protein is an α/β protein consisting of 5β-strands and 4α-helix. In order to test the stability of the protein, we chose to use thermal denaturation and Urea-induced denaturation to examine the property of protein. On the other hand, we also used isothermal titration calorimetry, ITC, to determine the binding affinity constant between the interaction of KP0001 with FMN. Furthermore, HSQC is used to search upon the interaction between KP0001 (flacodoxin), FAD, FMN, flavobin and Fe2+.Using a variety of triple resonance NMR experiments, assignments of the 1H, 15N and 13C resonances in the KP0001 (Flavodoxin) protein have been completely accomplished. The three-dimensional structure of the KP0001 (Flavodoxin) protein was calculated using distance-geometry followed by simulated annealing techniques with ARIA. In the future, it is expected that detailed activity of such an important protein can be fully understood and set as a foundation for more future drug development.