Center for Photochemical Sciences, Department of Chemistry, Bowling Green State University, Bowling Green, Ohio 43403, United States.
ACS Nano. 2012 Feb 28;6(2):1221-9. doi: 10.1021/nn2038669. Epub 2012 Feb 1.
Combining atomic force microscopy and fluorescence resonance energy transfer spectroscopy (AFM-FRET), we have developed a single-molecule AFM-FRET nanoscopy approach capable of effectively pinpointing and mechanically manipulating a targeted dye-labeled single protein in a large sampling area and simultaneously monitoring the conformational changes of the targeted protein by recording single-molecule FRET time trajectories. We have further demonstrated an application of using this nanoscopy on manipulation of single-molecule protein conformation and simultaneous single-molecule FRET measurement of a Cy3-Cy5-labeled kinase enzyme, HPPK (6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase). By analyzing time-resolved FRET trajectories and correlated AFM force pulling curves of the targeted single-molecule enzyme, we are able to observe the protein conformational changes of a specific coordination by AFM mechanic force pulling.
我们结合原子力显微镜和荧光共振能量转移光谱（AFM-FRET），开发了一种单分子 AFM-FRET 纳米技术方法，能够在大采样区域内有效地定位和机械操作靶向染料标记的单个蛋白质，并通过记录单分子 FRET 时间轨迹来同时监测靶向蛋白质的构象变化。我们进一步证明了这种纳米技术在单分子蛋白质构象操作和同时对 Cy3-Cy5 标记的激酶酶 HPPK（6-羟甲基-7,8-二氢蝶呤磷酸激酶）进行单分子 FRET 测量的应用。通过分析靶向单分子酶的时间分辨 FRET 轨迹和相关的 AFM 力拉伸曲线，我们能够通过 AFM 机械力拉伸观察到特定配位的蛋白质构象变化。
