Department of Neuroscience, University of Wisconsin-Madison, 1111 Highland Ave., Madison, WI, 53705, USA.
Department of Chemistry, University of Wisconsin-Madison, 1101 University Ave., Madison, WI, 53706, USA.
Angew Chem Int Ed Engl. 2017 Feb 20;56(9):2399-2402. doi: 10.1002/anie.201612050. Epub 2017 Jan 24.
Single-molecule fluorescence microscopy is a powerful tool for revealing chemical dynamics and molecular association mechanisms, but has been limited to low concentrations of fluorescent species and is only suitable for studying high affinity reactions. Here, we combine nanophotonic zero-mode waveguides (ZMWs) with fluorescence resonance energy transfer (FRET) to resolve single-molecule association dynamics at up to millimolar concentrations of fluorescent species. This approach extends the resolution of molecular dynamics to >100-fold higher concentrations, enabling observations at concentrations relevant to biological and chemical processes, and thus making single-molecule techniques applicable to a tremendous range of previously inaccessible molecular targets. We deploy this approach to show that the binding of cGMP to pacemaking ion channels is weakened by a slower internal conformational change.
单分子荧光显微镜是揭示化学动力学和分子结合机制的有力工具，但一直受到荧光物质浓度低的限制，并且仅适用于研究高亲和力反应。在这里，我们将纳米光子零模波导 (ZMW) 与荧光共振能量转移 (FRET) 结合使用，以在高达毫摩尔浓度的荧光物质下解析单分子结合动力学。这种方法将分子动力学的分辨率扩展到高出 100 倍的浓度，使在与生物和化学过程相关的浓度下进行观察成为可能，从而使单分子技术适用于以前无法企及的大量分子靶标。我们采用这种方法表明，cGMP 与起搏离子通道的结合因内部构象变化较慢而减弱。
