Yip C M, Ward M D
Department of Chemical Engineering and Materials Science, University of Minnesota, Minneapolis 55455, USA.
Biophys J. 1996 Aug;71(2):1071-8. doi: 10.1016/S0006-3495(96)79307-4.
Atomic force microscopy performed on single crystals of three different polymorphs of bovine insulin revealed molecularly smooth (001) layers separated by steps whose heights reflect the dimensions of a single insulin hexamer. Whereas contact mode imaging caused etching that prevented molecular-scale resolution, tapping mode imaging in solution provided molecular-scale contrast that enabled determination of lattice parameters and polymorph identification while simultaneously enabling real-time examination of growth modes and assessment of crystal quality. Crystallization proceeds layer by layer, a process in which the protein molecules assemble homoepitaxially with nearly perfect orientational and translational commensurism. Tapping mode imaging also revealed insulin aggregates attached to the (001) faces, their incorporation into growing terraces, and their role in defect formation. These observations demonstrate that tapping mode imaging is ideal for real-time in situ investigation of the crystallization of soft protein crystals of relatively small proteins such as insulin, which cannot withstand the lateral shear forces exerted by the scanning probe in conventional imaging modes.
对牛胰岛素三种不同多晶型的单晶进行的原子力显微镜观察显示，分子光滑的(001)层被台阶隔开，台阶的高度反映了单个胰岛素六聚体的尺寸。接触模式成像会导致蚀刻，从而无法实现分子尺度的分辨率，而溶液中的轻敲模式成像提供了分子尺度的对比度，能够确定晶格参数并识别多晶型，同时还能实时检查生长模式并评估晶体质量。结晶过程是逐层进行的，在此过程中蛋白质分子以几乎完美的取向和平移共格性进行同外延组装。轻敲模式成像还揭示了附着在(001)面上的胰岛素聚集体、它们并入生长平台的过程以及它们在缺陷形成中的作用。这些观察结果表明，轻敲模式成像非常适合对相对较小的蛋白质（如胰岛素）的软蛋白晶体结晶进行实时原位研究，这类蛋白质无法承受传统成像模式下扫描探针施加的横向剪切力。
