Bertz Morten, Rief Matthias
Physik Department E22, Technische Universität München, James-Franck-Strasse, 85748 Garching, Germany.
J Mol Biol. 2009 Nov 13;393(5):1097-105. doi: 10.1016/j.jmb.2009.08.066. Epub 2009 Sep 3.
In the past decade, single-molecule force spectroscopy has provided new insights into the key interactions stabilizing folded proteins. A few recent studies probing the effects of ligand binding on mechanical protein stability have come to quite different conclusions. While some proteins seem to be stabilized considerably by a bound ligand, others appear to be unaffected. Since force acts as a vector in space, it is conceivable that mechanical stabilization by ligand binding is dependent on the direction of force application. In this study, we vary the direction of the force to investigate the effect of ligand binding on the stability of maltose binding protein (MBP). MBP consists of two lobes connected by a hinge region that move from an open to a closed conformation when the ligand maltose binds. Previous mechanical experiments, where load was applied to the N and C termini, have demonstrated that MBP is built up of four building blocks (unfoldons) that sequentially detach from the folded structure. In this study, we design the pulling direction so that force application moves the two MBP lobes apart along the hinge axis. Mechanical unfolding in this geometry proceeds via an intermediate state whose boundaries coincide with previously reported MBP unfoldons. We find that in contrast to N-C-terminal pulling experiments, the mechanical stability of MBP is increased by ligand binding when load is applied to the two lobes and force breaks the protein-ligand interactions directly. Contour length measurements indicate that MBP is forced into an open conformation before unfolding even if ligand is bound. Using mutagenesis experiments, we demonstrate that the mechanical stabilization effect is due to only a few key interactions of the protein with its ligand. This work illustrates how varying the direction of the applied force allows revealing important details about the ligand binding mechanics of a large protein.
在过去十年中，单分子力谱为稳定折叠蛋白的关键相互作用提供了新的见解。最近一些探究配体结合对蛋白机械稳定性影响的研究得出了截然不同的结论。虽然一些蛋白似乎因结合的配体而显著稳定，但另一些则似乎不受影响。由于力在空间中是矢量，因此可以想象配体结合导致的机械稳定作用取决于力的施加方向。在本研究中，我们改变力的方向以研究配体结合对麦芽糖结合蛋白（MBP）稳定性的影响。MBP由两个叶通过一个铰链区连接而成，当配体麦芽糖结合时，这两个叶从开放构象转变为封闭构象。先前在N端和C端施加负载的机械实验表明，MBP由四个构建模块（解折叠单元）组成，它们依次从折叠结构中脱离。在本研究中，我们设计拉力方向，使力的施加沿着铰链轴将两个MBP叶拉开。在这种几何结构中的机械解折叠通过一个中间状态进行，该中间状态的边界与先前报道的MBP解折叠单元一致。我们发现，与N - C端拉力实验不同，当在两个叶上施加负载且力直接破坏蛋白 - 配体相互作用时，配体结合会增加MBP的机械稳定性。轮廓长度测量表明，即使结合了配体，MBP在解折叠之前也会被迫进入开放构象。通过诱变实验，我们证明机械稳定作用仅归因于蛋白与其配体的少数关键相互作用。这项工作说明了改变施加力的方向如何能够揭示关于一种大型蛋白的配体结合机制的重要细节。
