Tworzydło Magdalena, Polit Agnieszka, Mikołajczak Jan, Wasylewski Zygmunt
Department of Physical Biochemistry, Faculty of Biotechnology, Jagiellonian University, Kraków, Poland.
FEBS J. 2005 Mar;272(5):1103-16. doi: 10.1111/j.1742-4658.2005.04540.x.
Cyclic AMP receptor protein (CRP) regulates the expression of more then 100 genes in Escherichia coli. It is known that the allosteric activation of CRP by cAMP involves a long-distance signal transmission from the N-terminal cAMP-binding domain to the C-terminal domain of CRP responsible for the interactions with specific sequences of DNA. In this report we have used a CRP mutant containing a single Trp13 located in the N-terminal domain of the protein. We applied the iodide and acrylamide fluorescence quenching method in order to study how different DNA sequences and cAMP binding induce the conformational changes in the CRP molecule. The results presented provide evidence for the occurrence of a long-distance conformational signal transduction within the protein from the C-terminal DNA-binding domain to the N-terminal domain of CRP. This conformational signal transmission depends on the promoter sequence. We also used the stopped-flow and Forster resonance energy transfer between labeled Cys178 of CRP and fluorescently labeled DNA sequences to study the kinetics of DNA-CRP interactions. The results thus obtained lead to the conclusion that CRP can exist in several conformational states and that their distribution is affected by binding of both the cAMP and of specific DNA sequences.
环腺苷酸受体蛋白（CRP）调控大肠杆菌中100多个基因的表达。已知cAMP对CRP的变构激活涉及从N端cAMP结合结构域到负责与DNA特定序列相互作用的CRP C端结构域的长距离信号传递。在本报告中，我们使用了一种CRP突变体，该突变体在蛋白质的N端结构域含有一个单一的色氨酸13（Trp13）。我们应用碘化物和丙烯酰胺荧光猝灭方法来研究不同的DNA序列和cAMP结合如何诱导CRP分子的构象变化。所呈现的结果为蛋白质内从CRP的C端DNA结合结构域到N端结构域发生长距离构象信号转导提供了证据。这种构象信号传递取决于启动子序列。我们还使用了CRP的标记半胱氨酸178（Cys178）与荧光标记的DNA序列之间的停流和福斯特共振能量转移来研究DNA-CRP相互作用的动力学。由此获得的结果得出结论，CRP可以以几种构象状态存在，并且它们的分布受cAMP和特定DNA序列结合的影响。
