Best R B, Li B, Steward A, Daggett V, Clarke J
Department of Chemistry, University of Cambridge, MRC Centre for Protein Engineering, Cambridge CB2 1EW, United Kingdom.
Biophys J. 2001 Oct;81(4):2344-56. doi: 10.1016/S0006-3495(01)75881-X.
Atomic force microscopy (AFM) experiments have provided intriguing insights into the mechanical unfolding of proteins such as titin I27 from muscle, but will the same be possible for proteins that are not physiologically required to resist force? We report the results of AFM experiments on the forced unfolding of barnase in a chimeric construct with I27. Both modules are independently folded and stable in this construct and have the same thermodynamic and kinetic properties as the isolated proteins. I27 can be identified in the AFM traces based on its previous characterization, and distinct, irregular low-force peaks are observed for barnase. Molecular dynamics simulations of barnase unfolding also show that it unfolds at lower forces than proteins with mechanical function. The unfolding pathway involves the unraveling of the protein from the termini, with much more native-like secondary and tertiary structure being retained in the transition state than is observed in simulations of thermal unfolding or experimentally, using chemical denaturant. Our results suggest that proteins that are not selected for tensile strength may not resist force in the same way as those that are, and that proteins with similar unfolding rates in solution need not have comparable unfolding properties under force.
原子力显微镜（AFM）实验为诸如肌肉中的肌联蛋白I27等蛋白质的机械展开提供了有趣的见解，但对于那些在生理上不需要抵抗外力的蛋白质，情况是否相同呢？我们报告了在与I27形成的嵌合构建体中对芽孢杆菌RNA酶进行强制展开的AFM实验结果。在这个构建体中，两个模块都是独立折叠且稳定的，并且具有与分离的蛋白质相同的热力学和动力学性质。基于之前的特征，可以在AFM轨迹中识别出I27，并且观察到芽孢杆菌RNA酶有明显的、不规则的低力峰。芽孢杆菌RNA酶展开的分子动力学模拟还表明，它在比具有机械功能的蛋白质更低的力下展开。展开途径涉及蛋白质从末端解开，与热展开模拟或使用化学变性剂进行实验观察相比，在过渡态中保留了更多类似天然的二级和三级结构。我们的结果表明，未被选择用于抗张强度的蛋白质可能不会以与那些被选择的蛋白质相同的方式抵抗外力，并且在溶液中具有相似展开速率的蛋白质在受力情况下不一定具有可比的展开特性。
