Evans P D, Jaseja M, Jeeves M, Hyde E I
School of Biochemistry, University of Birmingham, Edgbaston, UK.
Eur J Biochem. 1996 Dec 15;242(3):567-75. doi: 10.1111/j.1432-1033.1996.0567r.x.
To understand the specificity of the Escherichia coli Trp repressor for its operators, we have begun to study complexes of the protein with alternative DNA sequences, using 1H-NMR spectroscopy. We report here the 1H-NMR chemical shifts of a 20-bp oligodeoxynucleotide containing the sequence of a symmetrised form of the trpR operator in the presence and absence of the holorepressor. Deuterated protein was used to assign the spectrum of the oligodeoxynucleotide in a 37-kDa complex with the Trp holorepressor. Many of the resonances of the DNA shift on binding to the protein, which suggests changes in conformation throughout the sequence. The largest changes in shifts for the aromatic protons in the major groove are for A15 and G16, which are thought to hydrogen bond to the protein, possibly via water molecules. We have also examined the effect of DNA binding on the corepressor, tryptophan, in this complex. The indole proton resonance of the tryptophan undergoes a downfield shift of 1.2 ppm upon binding of DNA. This large shift is consistent with hydrogen bonding of the tryptophan to the phosphate backbone of the trpR operator DNA, as in the crystal structure of the holoprotein with the trp operator.
为了解大肠杆菌色氨酸阻遏物对其操纵基因的特异性，我们已开始利用1H-NMR光谱研究该蛋白质与其他DNA序列形成的复合物。我们在此报告了一种20个碱基对的寡脱氧核苷酸在存在和不存在全阻遏物的情况下的1H-NMR化学位移，该寡脱氧核苷酸包含色氨酸操纵基因阻遏物（trpR）对称形式的序列。使用氘代蛋白来确定该寡脱氧核苷酸与色氨酸全阻遏物形成的37-kDa复合物的光谱。DNA与蛋白质结合时，许多共振峰发生位移，这表明整个序列的构象发生了变化。在大沟中，芳香族质子位移变化最大的是A15和G16，它们可能通过水分子与蛋白质形成氢键。我们还研究了该复合物中DNA结合对辅阻遏物色氨酸的影响。色氨酸的吲哚质子共振在DNA结合后发生了1.2 ppm的向低场位移。这种大的位移与色氨酸与trpR操纵基因DNA的磷酸骨架形成氢键一致，如同全蛋白与trp操纵基因的晶体结构那样。
