Baichoo N, Heyduk T
Edward A. Doisy Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, 1402 South Grand Boulevard, St. Louis, Missouri 63104, USA.
Biochemistry. 1997 Sep 9;36(36):10830-6. doi: 10.1021/bi970714v.
We have used protein footprinting [Heyduk, E., & Heyduk, T. (1994) Biochemistry 33, 9643] to detect and map ligand-induced conformational changes in cAMP receptor protein (CRP). The binding of cAMP to CRP dramatically increases the specific DNA binding activity of the protein and, as has been previously shown, induces conformational changes in the protein. Protein footprinting experiments with the free CRP, the CRP-cAMP complex, and the CRP-cGMP complex were analyzed quantitatively. Binding of cAMP produced measurable differences in the susceptibility of CRP to the cleavage by Fe-EDTA. Almost all of these changes occurred in the C-terminal domain (DNA binding domain) of the protein. Additional changes were observed at the ends of the C alpha-helix, which is involved in intersubunit contacts in the CRP dimer, and in the hinge peptide, connecting N-terminal and C-terminal domains of the protein. The boundaries of the regions in the C-terminal domain, which exhibited changes in susceptibility to Fe-EDTA cleavage, almost exactly corresponded to D, E, and F alpha-helices which are involved directly in the recognition of DNA. The F alpha-helix, which provides all base-specific contacts in the CRP-DNA complex, became hypersensitive to Fe-EDTA-mediated cleavage, whereas the solvent exposure of D and E alpha-helices was decreased upon binding of cAMP. These results suggest that a significant part of cAMP-induced conformational change in CRP involves a movement of secondary structure elements in the C-terminal domain of the protein so that the recognition F alpha-helix becomes exposed to the solvent. In contrast to cAMP, binding of cGMP produced insignificant changes in susceptibility to Fe-EDTA-mediated cleavage. This is consistent with the inability of cGMP to induce functional conformational changes in CRP. The protein footprinting technique appears to be sufficiently sensitive for detection and mapping of ligand-induced conformational changes in proteins.
我们已运用蛋白质足迹法[Heyduk, E., & Heyduk, T. (1994) Biochemistry 33, 9643]来检测和绘制环磷酸腺苷受体蛋白（CRP）中配体诱导的构象变化。环磷酸腺苷与CRP的结合显著增强了该蛋白的特异性DNA结合活性，并且如先前所示，会诱导蛋白发生构象变化。我们对游离CRP、CRP - 环磷酸腺苷复合物以及CRP - 环磷酸鸟苷复合物进行了蛋白质足迹实验的定量分析。环磷酸腺苷的结合使CRP对Fe - EDTA切割的敏感性产生了可测量的差异。几乎所有这些变化都发生在蛋白的C末端结构域（DNA结合结构域）。在参与CRP二聚体亚基间相互作用的Cα螺旋末端以及连接蛋白N末端和C末端结构域的铰链肽处也观察到了其他变化。C末端结构域中对Fe - EDTA切割敏感性发生变化的区域边界，几乎恰好对应于直接参与DNA识别的D、E和Fα螺旋。在CRP - DNA复合物中提供所有碱基特异性接触的Fα螺旋，对Fe - EDTA介导的切割变得高度敏感，而D和Eα螺旋在结合环磷酸腺苷后溶剂可及性降低。这些结果表明，环磷酸腺苷诱导的CRP构象变化的很大一部分涉及蛋白C末端结构域中二级结构元件的移动，从而使识别性的Fα螺旋暴露于溶剂中。与环磷酸腺苷不同，环磷酸鸟苷的结合对Fe - EDTA介导的切割敏感性产生的变化不显著。这与环磷酸鸟苷无法诱导CRP功能性构象变化一致。蛋白质足迹技术似乎对检测和绘制配体诱导的蛋白质构象变化足够敏感。
