Internal Dynamics of the Tryptophan Repressor (TrpR) and Two Functionally Distinct TrpR Variants, L75F-TrpR and A77V-TrpR, in Their L-Trp-Bound Forms
Internal Dynamics of the Tryptophan Repressor (TrpR) and Two Functionally Distinct TrpR Variants, L75F-TrpR and A77V-TrpR, in Their L-Trp-Bound Forms
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DOI：
10.1021/bi200389k
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发表时间：
2011-06-14
期刊：
影响因子：
2.9
通讯作者：
Copie, Valerie
中科院分区：
文献类型：
--
作者：
Tripet, Brian P.;Goel, Anupam;Copie, Valerie
使用 N-15 核磁共振 (NMR) 弛豫对大肠杆菌色氨酸阻遏蛋白 (WT-TrpR) 和两种功能不同的变体 L75F-TrpR 和 A77V-TrpR 的全息（L-色氨酸辅阻遏物结合）形式的骨架酰胺动力学进行了表征。这三种蛋白质具有非常相似的结构，排除了主要构象差异作为其功能差异的根源，并表明蛋白质灵活性的变化是其独特功能特性的根源。位点特异性 N-15-T-1、N-15-T-2、N-15-{H-1} 核奥弗豪塞效应、降低的谱密度和广义序 (S-2) 参数的比较表明，三个全息抑制子的骨架动力学总体上非常相似，但存在于蛋白质 DNA 结合域内的骨架原子有一些值得注意和重要的例外。我们发现核心α螺旋（即螺旋A-C和F）中的酰胺的灵活性受到高度限制，并且对于三个全阻遏物的螺旋D-转角-螺旋E（HTH）DNA结合结构域的DNA识别螺旋（螺旋E）中的残基观察到类似的“硬化”。出乎意料的是，位于螺旋 D 和相邻转角区域的酰胺保持柔性。这些数据支持这样的概念：TrpR 的残余灵活性对于阻遏物功能、DNA 结合和目标操纵子的分子识别至关重要。 Holo-TrpR 与 apo-TrpR 的 N-15 NMR 弛豫参数的比较表明，单点氨基酸取代 L75F 和 A77V 以非常不同的方式扰乱 TrpR 骨架酰胺的灵活性，并且在三个阻遏蛋白的 apo 形式中最为明显。最后，我们在其他 DNA 结合蛋白以及蛋白质柔性在分子识别中的作用的背景下展示了这些发现。
Backbone amide dynamics of the Escherichia colt tryptophan repressor protein (WT-TrpR) and two functionally distinct variants, L75F-TrpR and A77V-TrpR, in their holo (L-trytophan corepressor-bound) form have been characterized using N-15 nuclear magnetic resonance (NMR) relaxation. The three proteins possess very similar structures, ruling out major conformational differences as the source of their functional differences, and suggest that changes in protein flexibility are at the origin of their distinct functional properties. Comparison of site specific N-15-T-1, N-15-T-2, N-15-{H-1} nuclear Overhauser effect, reduced spectral density, and generalized order (S-2) parameters indicates that backbone dynamics in the three holo-repressors are overall very similar with a few notable and significant exceptions for backbone atoms residing within the proteins' DNA-binding domain. We find that flexibility is highly restricted for amides in core alpha-helices (i.e., helices A-C and F), and a comparable "stiffening" is observed for residues in the DNA recognition helix (helix E) of the helix D-turn-helix E (HTH) DNA-binding domain of the three holo-repressors. Unexpectedly, amides located in helix D and in adjacent turn regions remain flexible. These data support the concept that residual flexibility in TrpR is essential for repressor function, DNA binding, and molecular recognition of target operators. Comparison of the N-15 NMR relaxation parameters of the holo-TrpRs with those of the apo-TrpRs indicates that the single-point amino acid substitutions, L75F and A77V, perturb the flexibility of backbone amides of TrpR in very different ways and are most pronounced in the apo forms of the three repressors. Finally, we present these findings in the context of other DNA-binding proteins and the role of protein flexibility in molecular recognition.