Modulation of protein properties in living cells using nanobodies
Modulation of protein properties in living cells using nanobodies
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DOI：
10.1038/nsmb.1727
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发表时间：
2010-01-01
影响因子：
16.8
通讯作者：
Rothbauer, Ulrich
中科院分区：
文献类型：
--
作者：
Kirchhofer, Axel;Helma, Jonas;Rothbauer, Ulrich
蛋白质构象与功能密切相关，通常受与调节因子相互作用的控制。在这里，我们报告骆驼源性单域抗体（纳米抗体）的选择，调节的绿色荧光蛋白（GFP）的构象和光谱特性。一个纳米抗体可以可逆地将GFP荧光降低5倍，而其被第二个纳米抗体置换导致增加10倍。GFP-纳米抗体复合物的结构分析显示，这两种纳米抗体诱导发色团环境的微妙相反变化，导致吸收特性改变。与传统的抗体不同，这种小而稳定的纳米抗体在活细胞中发挥作用。通过比率成像检测纳米抗体诱导的变化，并用于监测蛋白质表达和亚细胞定位以及易位事件，如他莫昔芬诱导的雌激素受体的核定位。这项工作表明，蛋白质构象可以在活细胞中用纳米抗体进行操纵和研究。
Protein conformation is critically linked to function and often controlled by interactions with regulatory factors. Here we report the selection of camelid-derived single-domain antibodies (nanobodies) that modulate the conformation and spectral properties of the green fluorescent protein (GFP). One nanobody could reversibly reduce GFP fluorescence by a factor of 5, whereas its displacement by a second nanobody caused an increase by a factor of 10. Structural analysis of GFP-nanobody complexes revealed that the two nanobodies induce subtle opposing changes in the chromophore environment, leading to altered absorption properties. Unlike conventional antibodies, the small, stable nanobodies are functional in living cells. Nanobody-induced changes were detected by ratio imaging and used to monitor protein expression and subcellular localization as well as translocation events such as the tamoxifen-induced nuclear localization of estrogen receptor. This work demonstrates that protein conformations can be manipulated and studied with nanobodies in living cells.