Abstract
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.