FLUORESCENCE ENERGY TRANSFER STUDIES OF PROTEIN INDUCED DNA BENDING

我们已经使用时间分辨荧光能量转移， 研究溶液中蛋白质诱导DNA弯曲。 我们正在测量 在两种不同的蛋白质与DNA和DNA与DNA的距离中， 蛋白质-DNA复合物显示出显著的蛋白质诱导的DNA 结晶状态下的弯曲：CAP-DNA复合物和TBP-DNA 复杂. 我们正在确定DNA弯曲的角度和轨迹， 溶液，评估蛋白质诱导的DNA的盐依赖性 弯曲，评估蛋白突变体对蛋白诱导的 DNA弯曲和评估DNA突变体对 蛋白质诱导的DNA弯曲 我们使用AEDANS？？ex=340nm; ? em= 480 nm）作为供体荧光染料和荧光素（？ex=490 nm; ? em=520 nm）作为受体荧光染料。 我们已将AEDANS 通过定点突变在蛋白质的特定位点引入 独特的表面半胱氨酸， 修饰，我们已经在特定位点掺入荧光素， DNA的全合成。 初步结果已经报告。

We have used time-resolved fluorescence energy transfer to investigate protein-induced DNA bending in solution. We are measuring multiple protein-to-DNA and DNA-to-DNA distances in each of two protein-DNA complexes that exhibit dramatic protein-induced DNA bending in the crystalline state: the CAP-DNA complex and the TBP-DNA complex. We are determining DNA bend angles and trajectories in solution, assessing the salt-dependence of the protein-induced DNA bending, assessing the effects of protein mutants on protein-induced DNA bending and assessing the effects of DNA mutants on protein-induced DNA bending. We are using AEDANS ??ex=340nm; ?em=480nm) as the donor fluorochrome and fluorescein (?ex=490 nm; ?em=520 nm) as the acceptor fluorochrome. We have incorporated AEDANS at specific sites in protein by site-directed mutagenesis to introduce unique surface cysteines followed by cysteine-specific chemical modification, and we have incorporated fluorescein at specific sites in DNA by total synthesis. Preliminary results have been reported.