Direct Imaging of Protein Organization in an Intact Bacterial Organelle Using High-Resolution Atomic Force Microscopy.
Direct Imaging of Protein Organization in an Intact Bacterial Organelle Using High-Resolution Atomic Force Microscopy.
复制标题
使用高分辨率原子力显微镜在完整细菌细胞器中直接对蛋白质组织进行直接成像。
DOI：
10.1021/acsnano.6b05647
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发表时间：
2017-01-24
期刊：
影响因子：
17.1
通讯作者：
Hobbs JK
中科院分区：
文献类型：
--
作者：
Kumar S;Cartron ML;Mullin N;Qian P;Leggett GJ;Hunter CN;Hobbs JK
生物能量膜的功能受到其组成膜蛋白的空间排列的强烈影响。原子力显微镜（AFM）可用于高分辨率探测蛋白质组织，从而识别单个蛋白质。然而，以前的生物膜的AFM研究通常需要弯曲的膜在样品制备过程中破裂和变平，有可能破坏天然蛋白质的排列或蛋白质的损失。成像天然的，弯曲的膜需要最小的尖端样品在横向和垂直方向的相互作用。在这里，通过优化成像缓冲液来减少长距离尖端-样品相互作用。轻敲模式AFM与高谐振频率的小而软的杠杆，结合高速AFM，减少了由于反馈误差的力，并使应用的平均成像力的几十微微牛顿。使用这种方法，我们已经成像完整的囊泡细菌光合“细胞器”，色素细胞的膜组织。尽管高度弯曲的性质的色素细胞膜和缺乏直接的支持，分辨率是足够的，以确定光系统复合物和量化其在自然状态下的安排。连续的成像显示，蛋白质保持惊人的静态，在几分钟的时间尺度上具有最小的旋转或平移。观察到RC-LH 1-PufX复合物的高阶组装，并且成功地成像了完整的ATP酶。这里开发的方法可能适用于广泛的富含蛋白质的囊泡或弯曲的膜系统，这是一个几乎无处不在的天然细胞器的功能。
The function of bioenergetic membranes is strongly influenced by the spatial arrangement of their constituent membrane proteins. Atomic force microscopy (AFM) can be used to probe protein organization at high resolution, allowing individual proteins to be identified. However, previous AFM studies of biological membranes have typically required that curved membranes are ruptured and flattened during sample preparation, with the possibility of disruption of the native protein arrangement or loss of proteins. Imaging native, curved membranes requires minimal tip–sample interaction in both lateral and vertical directions. Here, long-range tip–sample interactions are reduced by optimizing the imaging buffer. Tapping mode AFM with high-resonance-frequency small and soft cantilevers, in combination with a high-speed AFM, reduces the forces due to feedback error and enables application of an average imaging force of tens of piconewtons. Using this approach, we have imaged the membrane organization of intact vesicular bacterial photosynthetic “organelles”, chromatophores. Despite the highly curved nature of the chromatophore membrane and lack of direct support, the resolution was sufficient to identify the photosystem complexes and quantify their arrangement in the native state. Successive imaging showed the proteins remain surprisingly static, with minimal rotation or translation over several-minute time scales. High-order assemblies of RC-LH1-PufX complexes are observed, and intact ATPases are successfully imaged. The methods developed here are likely to be applicable to a broad range of protein-rich vesicles or curved membrane systems, which are an almost ubiquitous feature of native organelles.