Distant residues mediate picomolar binding affinity of a protein cofactor.
Distant residues mediate picomolar binding affinity of a protein cofactor.
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DOI：
10.1038/ncomms2010
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发表时间：
2012
影响因子：
16.6
通讯作者：
中科院分区：
文献类型：
--
作者：
许多蛋白质需要辅因子才能活跃。计算机模拟表明，协作交互网络实现了最佳的辅因子绑定。有必要对稳定这些网络和辅因子结合至关重要的残基进行实验鉴定。本文研究了含有黄素单核苷酸作为非共价结合辅因子的电子转运体黄氧素。我们发现，在以纳摩尔亲和力结合黄素单核苷酸后，蛋白质弛豫非常缓慢（时间常数~5天）到具有皮摩尔亲和力的能量更有利的状态。通过黄素N(3)H的H/D交换，揭示了这种状态的罕见的小尺度开口。我们发现H/D交换可以精确定位导致紧密辅因子结合的氨基酸。这些迄今为止未知的残基分散在整个结构中，其中许多离黄素很远，似乎与黄氧还蛋白的功能无关。配体结合热力学的量化对于理解、工程、设计和进化配体结合蛋白具有重要意义。黄氧还蛋白需要与其FMN辅因子紧密结合才能发挥活性，但涉及的残基尚不清楚。在这项生物物理研究中，FMN结合被证明在极慢的蛋白质松弛过程中从纳摩尔亲和力转变为皮摩尔亲和力，并确定了负责辅因子结合的残基。
Numerous proteins require cofactors to be active. Computer simulations suggest that cooperative interaction networks achieve optimal cofactor binding. There is a need for the experimental identification of the residues crucial for stabilizing these networks and thus for cofactor binding. Here we investigate the electron transporter flavodoxin, which contains flavin mononucleotide as non-covalently bound cofactor. We show that after binding flavin mononucleotide with nanomolar affinity, the protein relaxes extremely slowly (time constant ~5 days) to an energetically more favourable state with picomolar-binding affinity. Rare small-scale openings of this state are revealed through H/D exchange of N(3)H of flavin. We find that H/D exchange can pinpoint amino acids that cause tight cofactor binding. These hitherto unknown residues are dispersed throughout the structure, and many are located distantly from the flavin and seem irrelevant to flavodoxin's function. Quantification of the thermodynamics of ligand binding is important for understanding, engineering, designing and evolving ligand-binding proteins. Flavodoxin requires tight binding of its FMN cofactor to be active, but the residues involved are unknown. In this biophysical study, FMN binding is shown to change from nanomolar to picomolar affinity on extremely slow protein relaxation and the residues responsible for cofactor binding are identified.
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DOI：
10.1073/pnas.0509133103
发表时间：
2006-03-14
影响因子：
11.1
作者：
Bollen, YJM;Kamphuis, MB;van Mierlo, CPM
通讯作者：
van Mierlo, CPM
DOI：
10.1073/pnas.96.18.10118
发表时间：
1999-08-31
影响因子：
11.1
作者：
Freire, E
通讯作者：
Freire, E
影响因子：
56.9
作者：
Boehr, David D.;McElheny, Dan;Wright, Peter E.
通讯作者：
Wright, Peter E.
影响因子：
5.6
作者：
KRAGELUND, BB;KNUDSEN, J;POULSEN, FM
通讯作者：
POULSEN, FM
影响因子：
2.9
作者：
LOH, SN;PREHODA, KE;MARKLEY, JL
通讯作者：
MARKLEY, JL