Verity Jocelyne E, Chhabra Neetu, Sinnathamby Koneswaran, Yip Christopher M
Institute of Biomaterials and Biomedical Engineering, Terrence Donnelly Centre for Cellular and Biomolecular Research, University of Toronto, Toronto, Canada.
Biophys J. 2009 Aug 19;97(4):1225-31. doi: 10.1016/j.bpj.2009.06.013.
In situ atomic force microscopy (AFM) is an exceedingly powerful and useful technique for characterizing the structure and assembly of proteins in real-time, in situ, and especially at model membrane interfaces, such as supported planar lipid bilayers. There remains, however, a fundamental challenge with AFM-based imaging. Conclusions are inferred based on morphological or topographical features. It is conventionally very difficult to use AFM to confirm specific molecular conformation, especially in the case of protein-membrane interactions. In this case, a protein may undergo subtle conformational changes upon insertion in the membrane that may be critical to its function. AFM lacks the ability to directly measure such conformational changes and can, arguably, only resolve features that are topographically distinct. To address these issues, we have developed a platform that integrates in situ AFM with attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy. This combination of tools provides a unique means of tracking, simultaneously, conformational changes, not resolvable by in situ AFM, with topographical details that are not readily identified by conventional spectroscopy. Preliminary studies of thermal transitions in supported lipid bilayers and direct evidence of lipid-induced conformational changes in adsorbed proteins illustrates the potential of this coupled in situ functional imaging strategy.
原位原子力显微镜（AFM）是一种极其强大且有用的技术，可用于实时、原位地表征蛋白质的结构和组装，特别是在模型膜界面，如支撑的平面脂质双层。然而，基于AFM的成像仍存在一个根本性挑战。结论是基于形态学或地形学特征推断出来的。传统上，使用AFM很难确认特定的分子构象，尤其是在蛋白质-膜相互作用的情况下。在这种情况下，蛋白质插入膜中时可能会发生细微的构象变化，而这些变化可能对其功能至关重要。AFM缺乏直接测量此类构象变化的能力，可以说，它只能分辨地形上不同的特征。为了解决这些问题，我们开发了一个将原位AFM与衰减全反射傅里叶变换红外（ATR-FTIR）光谱相结合的平台。这种工具组合提供了一种独特的方法，可同时跟踪原位AFM无法分辨的构象变化以及传统光谱不易识别的地形细节。对支撑脂质双层热转变的初步研究以及吸附蛋白质中脂质诱导构象变化的直接证据说明了这种原位耦合功能成像策略的潜力。
