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首页> 外文期刊>Biochemistry >Myb-DNA recognition: role of tryptophan residues and structural changes of the minimal DNA binding domain of c-Myb.
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Myb-DNA recognition: role of tryptophan residues and structural changes of the minimal DNA binding domain of c-Myb.

机译：Myb-DNA识别：色氨酸残基的作用和c-Myb最小DNA结合域的结构变化。

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摘要

The Myb oncoprotein specifically binds DNA by a domain composed of three imperfect repeats, R1, R2, and R3, each containing 3 tryptophans. The tryptophan fluorescence of the minimal binding domain, R2R3, of c-Myb was used to monitor structural flexibility changes occurring upon DNA binding to R2R3. The quenching of the Trp fluorescence by DNA titration shows that four out of the six tryptophans are involved in the formation of the specific R2R3-DNA complex and the environment of the tryptophan residues becomes more hydrophobic in the complex. The fluorescence intensity quenching of the tryptophans by binding of R2R3 to DNA is consistent with the decrease of the decay time: 1.46 ns for free R2R3 to 0.71 ns for the complexed protein. In the free R2R3, the six tryptophans are equally accessible to the iodide and acrylamide quenchers with a high collisional rate constant (4 x 10(9) and 3 x 10(9) M-1 s-1, respectively), indicating that R2R3 in solution is very flexible. In the R2R3-DNA complex, no Trp fluorescence quenching is observed with iodide whereas all tryptophan residues remain accessible to acrylamide with a collisional rate constant slightly slower than that in the free state. These results indicate that (i) a protein structural change occurs and (ii) the R2R3 molecule keeps a high mobility in the complex.The complex formation presents a two-step kinetics: a fast step corresponding to the R2R3-DNA association (7 x 10(5) M-1 s-1) and a slower one (0.004 s-1), which should correspond to a structural reorganization of the protein including a reordering of the water molecules at the protein-DNA interface.
机译：Myb癌蛋白通过由三个不完整重复序列R1，R2和R3组成的结构域特异性结合DNA，每个重复序列均包含3个色氨酸。 c-Myb的最小结合域R2R3的色氨酸荧光用于监测DNA与R2R3结合后发生的结构柔性变化。通过DNA滴定法对Trp荧光的淬灭表明，六个色氨酸中有四个参与了特定的R2R3-DNA复合物的形成，色氨酸残基的环境在该复合物中变得更加疏水。通过R2R3与DNA结合，色氨酸的荧光强度猝灭与衰减时间的减少是一致的：游离R2R3为1.46 ns，复合蛋白为0.71 ns。在游离的R2R3中，碘化物和丙烯酰胺猝灭剂具有较高的碰撞速率常数（分别为4 x 10（9）和3 x 10（9）M-1 s-1），这六个色氨酸同样可访问，表明R2R3在解决方案中非常灵活。在R2R3-DNA络合物中，碘化物未观察到Trp荧光猝灭，而所有色氨酸残基仍可与丙烯酰胺接触，其碰撞速率常数比在游离状态下慢一些。这些结果表明（i）发生蛋白质结构变化，并且（ii）R2R3分子在复合物中保持高迁移率。复合物的形成表现出两步动力学：与R2R3-DNA缔合相对应的快速步骤（7 x 10（5）M-1 s-1）和较慢的一个（0.004 s-1），这应对应于蛋白质的结构重组，包括水分子在蛋白质-DNA界面处的重新排序。

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