Fast and Angström-resolution AFM to visualise conformational change in biomolecules

原子力显微镜（AFM）基本上就像一个微型盲人的手杖（“悬臂”），沿着样品表面的轮廓，逐行重建表面形貌的三维表示。这种逐行扫描是与其他更常见的显微镜技术的根本区别，也是通常需要几分钟才能完成一张图像的主要原因。AFM的独特之处在于将亚分子分辨率成像与在液体中操作的能力相结合。对于生物样品的高分辨率成像，分子通常被吸附在坚硬的表面上，这是与生理条件相比唯一的妥协。膜蛋白是特别感兴趣的样本，因为它们代表了现代药物靶点的50%以上，因此具有重要的药学意义。它们作为分子纳米机器的功能是由发生在毫秒时间尺度上的埃级结构（“构象”）变化决定的。对于未来医疗保健和基础科学理解的应用，关键问题是分子结构和这种结构的变化如何与膜蛋白的生物学功能相关。该项目结合了高分辨率AFM技术（已经产生了原子分辨率！）和快速扫描，以埃空间和毫秒时间分辨率获得膜蛋白图像。这将使我们能够实时可视化构象变化，并观察生物分子的作用。这将在细菌视紫红质上得到证明，细菌视紫红质是一种光驱动的分子机器，可以将质子泵过细胞膜。

Atomic Force Microscopy (AFM) basically acts as a miniature blind man's stick ('cantilever') following the contours of a sample surface, and line by line reconstructing a three-dimensional representation of the surface topography. This line-by-line scanning is a fundamental difference from other, more common microscopy techniques and a main reason why it generally takes minutes to complete a single image. AFM is unique in combining sub-molecular resolution imaging with the ability to operate in liquids. For high-resolution imaging of biological samples, molecules are generally adsorbed on a hard surface, which is the only compromise compared to physiological conditions. Membrane proteins are samples of particular interest, since they represent more than 50% of modern drug targets and therefore are of major pharmaceutical importance. Their function as molecular nanomachines is determined by Angstrom-sized structural ('conformational') changes occurring at millisecond time scales. For applications in future healthcare and for basic scientific understanding, the crucial question is how molecular structure and changes in this structure relate to the biological function of membrane proteins. This project combines high-resolution AFM techniques (that have yielded atomic resolution!) with fast scanning, to obtain images of membrane proteins with Angstrom spatial and millisecond temporal resolution. This will enable us to visualise conformational changes in real time and observe biomolecules at work. This will be demonstrated on bacteriorhodopsin, a light-driven molecular machine that pumps protons through the cell membrane.