APPLICATION OF FLUORESCENCE ENERGY-TRANSFER AND POLARIZATION TO MONITOR ESCHERICHIA-COLI CAMP RECEPTOR PROTEIN AND LAC PROMOTER INTERACTION
APPLICATION OF FLUORESCENCE ENERGY-TRANSFER AND POLARIZATION TO MONITOR ESCHERICHIA-COLI CAMP RECEPTOR PROTEIN AND LAC PROMOTER INTERACTION
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DOI：
10.1073/pnas.87.5.1744
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发表时间：
1990-03-01
影响因子：
11.1
通讯作者：
LEE, JC
中科院分区：
文献类型：
--
作者：
HEYDUK, T;LEE, JC
建立了一种研究溶液中DNA-蛋白质相互作用的荧光方法。化学合成了含有大肠杆菌cAMP受体蛋白（CRP）主要结合位点的lac启动子的32个碱基对（bp）DNA片段，并在5“末端用荧光探针特异性标记。cAMP受体蛋白与该片段的结合可以方便地通过测量标记的DNA的荧光偏振的变化或通过测量从蛋白质色氨酸残基到DNA标记的荧光能量转移来进行。蛋白质-DNA复合物的形成作为cAMP浓度的函数进行监测。可以解析各种平衡常数以表征cAMP与CRP的结合以及随后CRP-cAMP和CRP-（cAMP）2与DNA的结合。这些结合研究表明，两种连接形式的CRP具有显着不同的特异性位点DNA的亲和力。这些结果表明，在原则上，荧光技术可以产生基本上任何溶液条件下的有效的平衡常数。这项技术也有可能提供有关蛋白质-DNA复合物结构的信息。
A fluorescence method was developed to study DNA-protein interactions in solution. A 32-base-pair (bp) DNA fragment of the lac promoter containing the primary binding site for Eschericia coli cAMP receptor protein (CRP) was chemically synthesized and labeled specifically at the 5'' end with fluorescent probe. Binding of cAMP receptor protein to this fragment can be conveniently followed by measuring changes in polarization of fluorescence of the labeled DNA or by measuring fluorescence energy transfer from protein tryptophan residues to the DNA label. Formation of protein-DNA complex was monitored as a function of cAMP concentration. Various equilibrium constants can be resolved to characterize the binding of cAMP to CRP and the subsequent binding of CRP-cAMP and CRP-(cAMP)2 to DNA. These binding studies showed that the two ligated forms of CRP have significantly different affinities for specific-site DNA. These results show that, in principle, the fluorescence technique can yield thermodynamically valid equilibrium constants under essentially any solution conditions. This technique also has the potential of providing information regarding the structure of protein-DNA complexes.