Use of Fluorescence Resonance Energy Transfer in Studying Sac7d-induced DNA kink structure

• Main Author: 張芳慈
• Other Authors: 李其融
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/55971538258279774757

碩士 === 明新科技大學 === 化學工程與材料科技系 === 98 === Sac7d is a small, abundant, sequence-general DNA binding protein from the hyperthermophilic archaeon Sulfolobus acidocaldarius. A series of X-ray crystal structures of complexes show that Sac7d binds in the minor groove of DNA and causes a sharp kink in DNA (~60°) via the intercalation with both side chains of the Val26 and Met29. Here we investigated the Sac7d-induced DNA kink structure in solution by both bulk and single-molecule FRET measurements. To determine the end-to-end distance of the DNA which containing the Sac7d preferred binding sequence at its central part by measuring the change in EFRET between two Cyanine dye molecules covalently attached to the 5'-ends of the target DNA duplex. When Sac7d binds to the DNA, it bends the DNA. The bigger kink angle results in the shorter end-to-end distance and it cause the higher EFRET efficiency. Our results show that a strong decrease in the dye-to-dye distances was observed in the complex compared with the unbound DNA. The DNA kink angle induced by Sac7d has been estimated to be ~ 60° of 16 bp DNA by bulk FRET analysis. Binding of Sac7d M29F results in the larger bending of the same DNA molecule to be ~70°. These values compare well with the bending angles observed in the X-ray crystal structures. The peak FRET values of the major single-molecule populations are somewhat smaller than the corresponding bulk FRET efficiencies. It is shown that the Sac7d induced DNA bending angles extracted from EFRET efficiencies are smaller in single-molecule FRET study, but close to the expected values from the crystal structures. We also observed the dynamics of conformational changes of DNA duplex due to Sac7d association or dissociation of DNA in single-molecule study.