Edwards Devin T, Faulk Jaevyn K, LeBlanc Marc-André, Perkins Thomas T
JILA, National Institute of Standards and Technology and University of Colorado, Boulder, Colorado.
Department of Chemistry and Biochemistry, University of Colorado, Boulder, Colorado.
Biophys J. 2017 Dec 19;113(12):2595-2600. doi: 10.1016/j.bpj.2017.10.023. Epub 2017 Nov 11.
Atomic force microscopy (AFM)-based single-molecule force spectroscopy (SMFS) is a powerful yet accessible means to characterize the unfolding/refolding dynamics of individual molecules and resolve closely spaced, transiently occupied folding intermediates. On a modern commercial AFM, these applications and others are now limited by the mechanical properties of the cantilever. Specifically, AFM-based SMFS data quality is degraded by a commercial cantilever's limited combination of temporal resolution, force precision, and force stability. Recently, we modified commercial cantilevers with a focused ion beam to optimize their properties for SMFS. Here, we extend this capability by modifying a 40 × 18 μm cantilever into one terminated with a gold-coated, 4 × 4 μm reflective region connected to an uncoated 2-μm-wide central shaft. This "Warhammer" geometry achieved 8.5-μs resolution coupled with improved force precision and sub-pN stability over 100 s when measured on a commercial AFM. We highlighted this cantilever's biological utility by first resolving a calmodulin unfolding intermediate previously undetected by AFM and then measuring the stabilization of calmodulin by myosin light chain kinase at dramatically higher unfolding velocities than in previous AFM studies. More generally, enhancing data quality via an improved combination of time resolution, force precision, and force stability will broadly benefit biological applications of AFM.
基于原子力显微镜（AFM）的单分子力谱（SMFS）是一种强大且易于使用的手段，可用于表征单个分子的展开/重折叠动力学，并解析紧密间隔、短暂占据的折叠中间体。在现代商用AFM上，这些应用及其他应用目前受到悬臂梁机械性能的限制。具体而言，基于AFM的SMFS数据质量会因商用悬臂梁在时间分辨率、力精度和力稳定性方面的有限组合而下降。最近，我们使用聚焦离子束对商用悬臂梁进行了改性，以优化其用于SMFS的性能。在此，我们通过将一个40×18μm的悬臂梁改造成一个末端带有与未涂层的2μm宽中心轴相连的4×4μm镀金反射区域的悬臂梁，扩展了这一能力。这种“战锤”几何结构在商用AFM上测量时，实现了8.5μs的分辨率，同时在100秒内提高了力精度并实现了亚皮牛的稳定性。我们通过首先解析出AFM之前未检测到的钙调蛋白展开中间体，然后以比之前AFM研究中显著更高的展开速度测量肌球蛋白轻链激酶对钙调蛋白的稳定作用，突出了这种悬臂梁的生物学用途。更普遍地说，通过改进时间分辨率、力精度和力稳定性的组合来提高数据质量将广泛惠及AFM的生物学应用。
