利用結構生物學技術進行古生菌之核醣核酸結合蛋白質功能與結構之研究;Structural and Functional studies of DNA binding proteins from hyperthermophilic Sulfolobus solfatarius by using X-ray crystallography, FRET and EM

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Title:	利用結構生物學技術進行古生菌之核醣核酸結合蛋白質功能與結構之研究;Structural and Functional studies of DNA binding proteins from hyperthermophilic Sulfolobus solfatarius by using X-ray crystallography, FRET and EM
Authors:	陳青諭
Contributors:	國立中央大學生命科學系
Keywords:	化學;生物技術(理)
Date:	2013-12-01
Issue Date:	2014-03-17 11:34:41 (UTC+8)
Publisher:	行政院國家科學委員會
Abstract:	研究期間：10208~10307;Sulfolobus solfataricus is a thermoacidophilic archaeon which grows optimally at approximately 75~85 °C and pH 2~4. It contains a number of abundant, small and basic DNA-binding proteins ranging in molecular weight from 7 to 10 KDa (e.g. Sso10b1/Sso10b2, Sso7d and Sso7c).Sso7d/Sac7d have been well-studied extensive. Sso10b proteins bind non-specifically to duplex DNA, but the exact DNA-binding properties of these proteins are not yet clear. DNA binding by Sso7c and solution structure of Sso7c has been studies by NMR. Here we plan to obtain the crystal of the DNA binding protein alone or in complex with the dsDNA by vapor diffusion method and determiner the crystal structure by X-ray diffraction. The crystals of Sso10b2-DNA complexes and protein Sso7c have been obtained and diffract X-ray to high resolution. Further heavy atom derivatives preparation for phase determination is in progress now. The crystals of Sso7d in complex with several DNA fragments are also being prepared .Previous EM studies of Sso10b-DNA complexes have revealed the formation of nucleoprotein filaments. All Sso7d, Sso7c, Sso10b can bind to dsDNA in vitro in a sequence-general manner. In this study, I will also study the protein-DNA interactions in the presence of these three different DNA binding proteins by using conventional TME and croy-EM. Our results will help understand the molecular basis of how the chromosomal protein molecules interact with DNA. The crystal structures of several Sac7d/Sso7d-DNA complexes have revealed an unexpected DNA binding mode in which a sharp kink in DNA was observed. In this study, we further investigated the Sac7d-induced DNA kink structure in solution by both bulk and single-molecule FRET measurements. Our preliminary results show that a strong decrease in the dye-to-dye distances was observed in the complex compared with the unbound DNA. We also observed the dynamics of conformational changes of DNA duplex due to Sac7d association or dissociation of DNA in single-molecule study. The photochemical study showed evidence for the photoinduced specific electron transfer process from Trp24 to bromo-uracil and T-T dimmer containing DNA in the Sso7d-DNA complex. It also has been demonstrated that the charge transfer process in DNA could be observed at the single-molecule level by using total internal reflection fluorescence microscopy. Here, we propose to combination these two research approaches to direct visualization of photo-induced electron transfer from Sso7d to DNA at single-molecule level.
Relation:	財團法人國家實驗研究院科技政策研究與資訊中心
Appears in Collections:	[Department of Life Science] Research Project

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