Applied and Engineering Physics, Cornell University, Ithaca, NY, 14853, USA.
Broad Institute, Cambridge, MA, 02142, USA.
Sci Rep. 2020 Sep 10;10(1):14866. doi: 10.1038/s41598-020-71630-6.
The composition, stoichiometry and interactions of supramolecular protein complexes are a critical determinant of biological function. Several techniques have been developed to study molecular interactions and quantify subunit stoichiometry at the single molecule level. However, these typically require artificially low expression levels or detergent isolation to achieve the low fluorophore concentrations required for single molecule imaging, both of which may bias native subunit interactions. Here we present an alternative approach where protein complexes are assembled at physiological concentrations and subsequently diluted in situ for single-molecule level observations while preserving them in a near-native cellular environment. We show that coupling this dilution strategy with fluorescence correlation spectroscopy permits quantitative assessment of cytoplasmic oligomerization, while stepwise photobleaching and single molecule colocalization may be used to study the subunit stoichiometry of membrane receptors. Single protein recovery after dilution (SPReAD) is a simple and versatile means of extending the concentration range of single molecule measurements into the cellular regime while minimizing potential artifacts and perturbations of protein complex stoichiometry.
蛋白质超分子复合物的组成、化学计量和相互作用是决定其生物学功能的关键因素。已经开发了几种技术来研究分子相互作用，并在单分子水平上定量测定亚基化学计量。然而，这些技术通常需要人为地降低表达水平或使用去污剂分离来实现单分子成像所需的低荧光浓度，这两者都可能使天然亚基相互作用产生偏差。在这里，我们提出了一种替代方法，即在生理浓度下组装蛋白质复合物，然后在原位稀释进行单分子水平观察，同时保持其在接近天然的细胞环境中。我们表明，将这种稀释策略与荧光相关光谱法相结合，可以定量评估细胞质寡聚化，而逐步光漂白和单分子共定位可用于研究膜受体的亚基化学计量。稀释后的单蛋白回收（SPReAD）是一种简单而通用的方法，可以将单分子测量的浓度范围扩展到细胞范围内，同时最小化蛋白质复合物化学计量的潜在人为因素和干扰。
