Mapping cyclic nucleotide-induced conformational changes in cyclicAMP receptor protein by a protein footprinting technique using different chemical proteases.
Mapping cyclic nucleotide-induced conformational changes in cyclicAMP receptor protein by a protein footprinting technique using different chemical proteases.
复制标题
使用不同的化学蛋白酶通过蛋白质足迹技术绘制环核苷酸诱导的 cyclAMP 受体蛋白构象变化。
DOI：
10.1110/ps.8.3.518
复制
发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Heyduk,T
中科院分区：
文献类型：
--
作者：
Baichoo,N;Heyduk,T
CyclicAMP受体蛋白（CRP）调节大肠杆菌中许多基因的转录。cAMP和cGMP都能与CRP结合，但只有cAMP能诱导构象变化，从而显著增加该蛋白的特异性DNA结合活性。我们之前已经证明，我们的蛋白质足迹技术足够敏感，可以通过cAMP检测CRP的构象变化[Baichoo N， Heyduk T. 1997]。生物化学36:10830 - 10836]。在这项工作中，用铁络合二乙烯三胺五乙酸（[Fe-DTPA]2−）、铁络合乙二胺二乙酸（[Fe-EDDA]）、铁络合甲磺酸去铁胺（[Fe-HDFO]+）和铜络合邻菲罗啉（[(OP)2Cu]+）作为蛋白酶，对cAMP和cGMP结合诱导的CRP构象变化进行了绘制和定量分析。这些化学蛋白酶的大小、电荷和疏水性各不相同。cAMP与CRP的结合导致所有四种蛋白酶对裂解的敏感性发生变化。通过[Fe-EDDA]和[Fe-DTPA]2−切割CRP-cAMP，检测到dna结合的F α-螺旋、结构域间铰链和参与亚基间相互作用的C α-螺旋末端的超敏反应。[Fe-EDDA]和[Fe-DTPA]2−也检测到参与DNA识别的D和E α-螺旋的切割减少。[Fe-HDFO]+对CRP-cAMP的切割检测到β-链8、B α-螺旋以及部分F和C α-螺旋的超敏反应。[Fe-HDFO]+还检测到β-链4至5及其中间环β-链7（核苷酸结合袋的一部分）以及D和E α-螺旋的剪切保护作用。[(OP)2Cu]+对CRP-cAMP的切割检测到β-链9和11以及D和E α-螺旋的超敏反应。[(OP)2Cu]+在C α-螺旋、结构域间铰链和β-链2-7中也检测到保护作用。cGMP与CRP结合仅导致[(OP)2Cu]+对裂解敏感性的改变，这在β-链3-7、结构域间铰链和C α-螺旋中检测到轻微的保护作用。这些结果表明，cAMP的结合导致CRP在核苷酸结合域、结构域间铰链、DNA结合域和亚基间相互作用相关区域的结构变化。由cGMP结合引起的结构变化似乎非常微小，并且局限于核苷酸结合结构域、结构域间铰链和参与亚基相互作用的区域。在蛋白质足迹中使用不同的裂解剂似乎比单独使用一种蛋白酶能提供更详细的结构变化图。
CyclicAMP receptor protein (CRP) regulates transcription of numerous genes in Escherichia coli. Both cAMP and cGMP bind CRP, but only cAMP induces conformational changes that dramatically increase the specific DNA binding activity of the protein. We have shown previously that our protein footprinting technique is sensitive enough to detect conformational changes in CRP by cAMP [Baichoo N, Heyduk T. 1997. Biochemistry 36:10830–10836]. In this work, conformational changes in CRP induced by cAMP and cGMP binding were mapped and quantitatively analyzed by protein footprinting using iron complexed to diethylenetriaminepentaacetic acid ([Fe-DTPA]2−), iron complexed to ethylenediaminediacetic acid ([Fe-EDDA]), iron complexed to desferrioxamine mesylate ([Fe-HDFO]+), and copper complexed to o-phenanthroline ([(OP)2Cu]+) as proteases. These chemical proteases differ in size, charge, and hydrophobicity. Binding of cAMP to CRP resulted in changes in susceptibility to cleavage by all four proteases. Cleavage by [Fe-EDDA] and [Fe-DTPA]2− of CRP-cAMP detected hypersensitivities in the DNA-binding F α-helix, the interdomain hinge, and the ends of the C α-helix, which is involved in intersubunit interactions. [Fe-EDDA] and [Fe-DTPA]2− also detected reductions in cleavage in the D and E α-helices, which are involved in DNA recognition. Cleavage by [Fe-HDFO]+ of CRP-cAMP detected hypersensitivities in β-strand 8, the B α-helix, as well as in parts of the F and C α-helices. [Fe-HDFO]+ also detected protections from cleavage in β-strands 4 to 5 and their intervening loop, β-strand 7, which is part of the nucleotide binding pocket, as well as in the D and E α-helices. Cleavage by [(OP)2Cu]+ of CRP-cAMP detected hypersensitivities in β-strands 9 and 11 as well as in the D and E α-helices. [(OP)2Cu]+ also detected protections in the C α-helix , the interdomain hinge, and β-strands 2–7. Binding of cGMP to CRP resulted in changes in susceptibility to cleavage only by [(OP)2Cu]+, which detected minor protections in β-strands 3–7, the interdomain hinge, and the C α-helix. These results show that binding of cAMP causes structural changes in CRP in the nucleotide binding domain, the interdomain hinge, the DNA binding domain, and regions involved in intersubunit interaction. Structural changes induced by binding of cGMP appear to be very minor and confined to the nucleotide binding domain, the interdomain hinge, and regions involved in intersubunit interaction. Use of different cleaving agents in protein footprinting seems to give a more detailed picture of structural changes than the use of a single protease alone.