Time-resolved fluorescence resonance energy transfer studies of DNA bending in double-stranded oligonucleotides and in DNA-protein complexes.
Time-resolved fluorescence resonance energy transfer studies of DNA bending in double-stranded oligonucleotides and in DNA-protein complexes.
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双链寡核苷酸和 DNA-蛋白质复合物中 DNA 弯曲的时间分辨荧光共振能量转移研究。
DOI：
10.1002/bip.10138
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发表时间：
2001
期刊：
影响因子：
--
通讯作者：
Williams,S
中科院分区：
文献类型：
--
作者：
Parkhurst,LJ;Parkhurst,KM;Powell,R;Wu,J;Williams,S
时间分辨Förster共振能量转移(TrFRET)被用来获得染料间距分布。这些分布给出了最可能的距离以及表征分布宽度的参数。后一个参数不仅包含与大分子相连的染料的柔韧性信息，而且还包含与大分子柔韧性有关的信息。最可能的染料间距离以及对DNA静态弯曲和DNA柔韧性的洞察。时间分辨的荧光各向异性和静态各向异性的测量可以结合起来，以提供系留染料似乎抖动的锥角的测量。当这种运动比表征转移的平均寿命快一个数量级时，可以计算出各种相互染料取向的偶极取向参数2的平均值。结果得到的2分布比极限值0和4窄得多，从而可以更精确地确定距离和距离变化。静态和时间分辨的荧光数据可以结合起来限制DNA-蛋白质动力学的分析，以提供结合和沿反应坐标的构象变化的热力学参数。该参数可用于在trFRET数据的全局拟合中模拟具有不同DNA弯曲角的多个DNA-蛋白质复合体。这种全局拟合方法表明，当被TATA结合蛋白(TBP)结合时，单碱基DNA变体中的弯曲范围可以用两种限制形式来理解。时间分辨FRET与稳态FRET相结合，不仅可以显示渗透分子如何影响DNA与蛋白质的结合，而且还可以显示DNA弯曲如何依赖于DNA-蛋白质复合体中的渗透分子浓度。
Time-resolved Förster resonance energy transfer (trFRET) has been used to obtain interdye distance distributions. These distributions give the most probable distance as well as a parameter,, that characterize the width of the distribution. This latter parameter contains information not only on the flexibility of the dyes tethered to macromolecules, but on the flexibility of the macromolecules. Both the most probable interdye distance as well as provide insight into DNA static bending and DNA flexibility. Time-resolved fluorescence anisotropy and static anisotropy measurements can be combined to provide a measure of the cone angle within which the tethered dyes appear to wobble. When this motion is an order of magnitude faster than the average lifetime that characterizes transfer, an average value of the dipolar orientational parameter 2 can be calculated for various mutual dye orientations. The resulting 2 distribution is very much narrower than the limiting values of 0 and 4, allowing more precise distances and distance changes to be determined. Static and time-resolved fluorescence data can be combined to constrain the analyses of DNA–protein kinetics to provide thermodynamic parameters for binding and for conformational changes along a reaction coordinate. The parameter can be used to model multiple DNA–protein complexes with varying DNA bend angles in a global fitting of trFRET data. Such a global fitting approach has shown how the range of bends in single base DNA variants, when bound by the TATA binding protein (TBP), can be understood in terms of two limiting forms. Time-resolved FRET, combined with steady-state FRET, can be used to show not only how osmolytes affect the binding of DNA to proteins, but also how DNA bending depends on osmolyte concentration in the DNA–protein complexes.© 2002 Wiley Periodicals, Inc. Biopoly (Nucleic