School of Physics and Astronomy, University of Leeds, Leeds, United Kingdom.
Biophys J. 2011 Apr 6;100(7):1800-9. doi: 10.1016/j.bpj.2011.02.021.
Force spectroscopy using the atomic force microscope (AFM) can yield important information on the strength and lifetimes of the folded states of single proteins and their complexes when they are loaded with force. For example, by mechanically unfolding concatenated proteins at different velocities, a dynamic force spectrum can be built up that allows reconstruction of the energy landscape that the protein traverses during unfolding. To characterize fully the unfolding landscape, however, it is necessary both to explore the entire force spectrum and to characterize each species populated during unfolding. In the conventional AFM apparatus, force is applied to the protein construct through a compliant cantilever. This limits the dynamic range of the force spectrum that can be probed, and the cantilever recoil after unfolding may mask the presence of metastable intermediates. Here, we describe to our knowledge a new technique-constant-deflection AFM-in which the compliance of the AFM cantilever is removed. Using this technique, we show that protein L exhibits a more complex unfolding energy landscape than previously detected using the conventional technique. This technique is also able to detect the presence of a refolding intermediate whose formation is otherwise prevented by cantilever recoil.
利用原子力显微镜（AFM）进行力谱学分析可以提供有关单蛋白及其复合物在受力时折叠状态的强度和寿命的重要信息。例如，通过以不同速度机械地展开串联蛋白，可以构建动态力谱，从而可以重建蛋白在展开过程中所经历的能量景观。然而，要全面描述展开景观，不仅需要探索整个力谱，还需要对展开过程中出现的每个物种进行特征描述。在传统的 AFM 仪器中，力通过柔顺的悬臂施加到蛋白结构上。这限制了可以探测的力谱的动态范围，并且展开后悬臂的回弹可能会掩盖亚稳态中间体的存在。在这里，我们描述了一种新的技术——恒偏置 AFM，其中去除了 AFM 悬臂的柔顺性。使用这种技术，我们表明蛋白 L 表现出比使用传统技术检测到的更复杂的展开能量景观。该技术还能够检测到折叠中间体的存在，否则由于悬臂回弹而阻止了其形成。
