Label-free tracking and mass measurement of single protein complexes on lipid bilayers
Label-free tracking and mass measurement of single protein complexes on lipid bilayers
复制标题
脂质双层上单一蛋白质复合物的无标记追踪和质量测量
DOI：
10.1016/j.bpj.2021.11.2810
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发表时间：
2022
影响因子：
3.4
通讯作者：
Foley E
中科院分区：
文献类型：
--
作者：
Foley E
脂双层内和脂双层上的生物分子相互作用对于许多细胞过程是关键的，但很难稳健地和化学计量地量化。在脂质双层上可视化和跟踪单个蛋白质的方法主要依赖于荧光标记，这在观察时间和分辨不同物种和低聚状态的能力方面有固有的限制，使得生物分子相互作用的定量和化学计量表征具有挑战性。在这里，我们演示了动态质量光度法，这是一种无标记成像、跟踪和质量测量单个膜相关蛋白的方法。我们将我们的方法应用于GTP酶Dynamin1，并展示了在330赫兹处<；50 kDa的质量分辨率和<；20 nm的定位精度。通过跟踪Dynamin1低聚物在支持的脂质双层上的运动，我们能够量化依赖于低聚物的迁移率、膜亲和力和单个复合体的(去结合)事件。我们的结果揭示了一种基于二聚体的低聚物的高度异质性混合物。此外，我们观察到随着低聚状态的增加，扩散减少和脂质双层亲和力增加，这表明接触面积增加，并且脂双层的存在诱导和/或稳定了基于二聚体的低聚物的组装。总体而言，动态质量光度法的这些能力，加上基于分析的研究完整膜蛋白的进展，可能会对脂双层内和脂双层上的生物分子机制的研究产生变革。
Biomolecular interactions in and on lipid bilayers are critical for a number of cellular processes, but are difficult to robustly and stoichiometrically quantify. Approaches to visualise and track single proteins on lipid bilayers mostly rely on fluorescent labelling, which has inherent limitations in observation times and the ability to resolve different species and oligomeric states, making the quantitative and stoichiometric characterisation of biomolecular interactions challenging. Here, we demonstrate dynamic mass photometry, a method for label-free imaging, tracking and mass measurement of individual membrane-associated proteins on supported lipid bilayers. We applied our method to the GTPase dynamin1 and demonstrate a mass resolution of< 50 kDa and< 20 nm localisation precision at 330 Hz. By tracking the movement of dynamin1 oligomers on the supported lipid bilayer, we were able to quantify oligomer-dependent mobilities, membrane affinities and (dis) association events of individual complexes. Our results revealed a highly heterogeneous mixture of dimer-based oligomers. Additionally, we observed decreasing diffusion and increasing lipid bilayer affinity with increasing oligomeric state, which suggests an increase in contact area and that the presence of the lipid bilayer induces and/or stabilises assembly of the dimer-based oligomers. Overall, these capabilities of dynamic mass photometry, together with assay-based advances for studying integral membrane proteins, could be transformative for studies of biomolecular mechanisms in and on lipid bilayers.