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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >The energetic cost of domain reorientation in maltose-binding protein as studied by NMR and fluorescence spectroscopy
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The energetic cost of domain reorientation in maltose-binding protein as studied by NMR and fluorescence spectroscopy

机译：核糖核酸和荧光光谱法研究麦芽糖结合蛋白中域重新定向的能量成本

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摘要

Maltose-binding protein (MBP) is a two-domain protein that undergoes a ligand-mediated conformational rearrangement from an "open" to a "closed" structure on binding to maltooligosacchar-ides. To characterize the energy landscape associated with this transition, we have generated five variants of MBP with mutations located in the hinge region of the molecule. Residual dipolar couplings, measured in the presence of a weak alignment medium, have been used to establish that the average structures of the mutant proteins are related to each other by domain rotation about an invariant axis, with the rotation angle varying from 5° to 28°. Additionally, the domain orientations observed in the wild-type apo and ligand-bound (maltose, maltotriose, etc.) structures are related through a rotation of 35° about the same axis. Remarkably, the free energy of unfolding, measured by equilibrium denaturation experiments and monitored by fluorescence spectroscopy, shows a linear correlation with the rotation angle, with the stability of the (apo)protein decreasing with domain closure by 212 +- 16 cal·mol~(-1) per degree of rotation. The apparent binding energy for maltose also shows a similar correlation with the interdomain angle, suggesting that the mutations, as they relate to binding, affect predominantly the ligand-free structure. The linearity of the energy change is interpreted in terms of an increase in the extent of hydrophobic surface that becomes solvent accessible on closure. The combination of structural, stability, and binding data allows separation of the energetics of domain reorientation from ligand binding. This work presents a near quantitative structure-energy-binding relationship for a series of mutants of MBP, illustrating the power of combined studies involving protein engineering and solution NMR spectroscopy.
机译：麦芽糖结合蛋白（MBP）是一个两域蛋白，在结合到麦芽寡糖-糖基上时，会经历从“开放”到“封闭”结构的配体介导的构象重排。为了表征与此过渡相关的能量格局，我们生成了MBP的五种变体，其突变位于分子的铰链区。在弱比对介质存在下测得的残留偶极偶合已用于确定突变蛋白的平均结构通过围绕恒定轴的结构域旋转而彼此相关，旋转角度从5°到28°不等°。另外，在野生型载脂蛋白和配体结合的（麦芽糖，麦芽三糖等）结构中观察到的结构域方向是通过围绕同一轴旋转35°来实现的。值得注意的是，通过平衡变性实验测量并通过荧光光谱监测的解折叠自由能显示出与旋转角度的线性相关，（apo）蛋白的稳定性随着域关闭而降低了212 +-16 cal·mol〜。 （-1）每旋转一度。麦芽糖的表观结合能与畴间角也显示出相似的相关性，表明与结合有关的突变主要影响无配体的结构。能量变化的线性可解释为疏水表面的程度增加，该疏水表面在封闭时变为溶剂可及的。结构，稳定性和结合数据的组合允许将域重新定向的能量与配体结合分离。这项工作提出了一系列的MBP突变体接近定量的结构-能量-结合关系，说明了涉及蛋白质工程和溶液NMR光谱学的组合研究的力量。

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