Differential Enzyme Flexibility Probed Using Solid-State Nanopores.
Differential Enzyme Flexibility Probed Using Solid-State Nanopores.
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使用固态纳米孔探测了差异酶柔韧性。
DOI：
10.1021/acsnano.8b00734
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发表时间：
2018-05-22
期刊：
影响因子：
17.1
通讯作者：
Wanunu, Meni
中科院分区：
文献类型：
--
作者：
Hu, Rui;Rodrigues, Joao V.;Waduge, Pradeep;Yamazaki, Hirohito;Cressiot, Benjamin;Chishti, Yasmin;Makowski, Lee;Yu, Dapeng;Shakhnovich, Eugene;Zhao, Qing;Wanunu, Meni
酶和马达蛋白是动态的大分子，它们以许多能量相似的构象共存。蛋白质功能通常伴随着结构和柔性的变化，通常在与配体结合时诱导。然而，虽然测量活性和静息状态之间的蛋白质柔性变化具有治疗意义，但它仍然是一个挑战。最近，我们的研究小组已经证明，作为单个蛋白质分子的集合体，测量的电特征中的信号幅度的宽度通过固态纳米孔驱动，并与蛋白质构象动力学相关。在这里，我们扩展我们的研究，以解决微妙的灵活性变化，在二氢叶酸还原酶突变体从未标记的单分子在溶液中。我们首先证明了使用一个典型的蛋白质系统，腺苷酸激酶，大小和灵活性的变化，可以观察到结合到一个底物，锁定在一个封闭的构象的蛋白质。接下来，我们调查的电压偏置和孔的几何形状上测得的电脉冲统计在蛋白质运输的影响。最后，使用最佳的实验条件下，我们系统地研究了一系列的野生型和突变二氢叶酸还原酶蛋白，发现纳米孔测量的蛋白质构象动力学和平衡散装荧光探针测量之间的良好的相关性。我们的研究结果明确表明，基于纳米孔的测量可靠地探测天然蛋白质集合的构象多样性。
Enzymes and motor proteins are dynamic macromolecules that coexist in a number of conformations of similar energies. Protein function is usually accompanied by a change in structure and flexibility, often induced upon binding to ligands. However, while measuring protein flexibility changes between active and resting states is of therapeutic significance, it remains a challenge. Recently, our group has demonstrated that breadth of signal amplitudes in measured electrical signatures as an ensemble of individual protein molecules is driven through solid-state nanopores and correlates with protein conformational dynamics. Here, we extend our study to resolve subtle flexibility variation in dihydrofolate reductase mutants from unlabeled single molecules in solution. We first demonstrate using a canonical protein system, adenylate kinase, that both size and flexibility changes can be observed upon binding to a substrate that locks the protein in a closed conformation. Next, we investigate the influence of voltage bias and pore geometry on the measured electrical pulse statistics during protein transport. Finally, using the optimal experimental conditions, we systematically study a series of wild-type and mutant dihydrofolate reductase proteins, finding a good correlation between nanopore-measured protein conformational dynamics and equilibrium bulk fluorescence probe measurements. Our results unequivocally demonstrate that nanopore-based measurements reliably probe conformational diversity in native protein ensembles.
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影响因子：
1.6
作者：
DEBLOIS, RW;BEAN, CP
通讯作者：
BEAN, CP
影响因子：
3.5
作者：
GOLDBERG, ME;SEMISOTNOV, GV;SUGAI, S
通讯作者：
SUGAI, S
影响因子：
10.8
作者：
Kowalczyk, Stefan W.;Hall, Adam R.;Dekker, Cees
通讯作者：
Dekker, Cees
DOI：
10.1073/pnas.93.24.13770
发表时间：
1996-11-26
影响因子：
11.1
作者：
Kasianowicz, JJ;Brandin, E;Deamer, DW
通讯作者：
Deamer, DW
影响因子：
17.1
作者：
Cressiot, Benjamin;Oukhaled, Abdelghani;Pelta, Juan
通讯作者：
Pelta, Juan