Measuring the affinity of protein-protein interactions on a single-molecule level by mass photometry
Measuring the affinity of protein-protein interactions on a single-molecule level by mass photometry
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DOI：
10.1016/j.ab.2020.113575
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发表时间：
2020-03-01
影响因子：
2.9
通讯作者：
Piszczek, Grzegorz
中科院分区：
文献类型：
--
作者：
Wu, Di;Piszczek, Grzegorz
生物分子相互作用的测量对于理解它们促进的生物过程的机制至关重要。基于体积的方法，如ITC和SPR，提供了关于结合亲和力、化学计量学和相互作用动力学的重要信息。然而，集合平均方法不能探测生物系统通常表现出的内在异质性。涉及协同作用或导致形成多组分复合物的相互作用带来了额外的实验挑战。单分子技术以前曾被用于解决这些问题。然而，单分子实验通常在技术上要求很高，并且需要对所研究的分子进行标记或固定。最近开发的一种单分子方法，质谱光度法（MP），克服了这些限制。在这里，我们应用MP来测量生物分子相互作用的亲和力。我们已经演示了MP如何允许用户研究多价复合物，并在一次测量中量化不同结合位点的亲和力。所得结果经ITC和BLI验证。MP数据的质量和信息量，加上简单快速的测量和低样品消耗，使MP成为测量强蛋白质相互作用的新首选方法。
Measurements of biomolecular interactions are crucial to understand the mechanisms of the biological processes they facilitate. Bulk-based methods such as ITC and SPR provide important information on binding affinities, stoichiometry, and kinetics of interactions. However, the ensemble averaging approaches are not able to probe the intrinsic heterogeneity often displayed by biological systems. Interactions that involve cooperativity or result in the formation of multicomponent complexes pose additional experimental challenges. Single-molecule techniques have previously been applied to solve these problems. However, single-molecule experiments are often technically demanding and require labeling or immobilization of the molecules under study. A recently developed single-molecule method, mass photometry (MP), overcomes these limitations. Here we applied MP to measure the affinities of biomolecular interactions. We have demonstrated how MP allows the user to study multivalent complexes and quantify the affinities of different binding sites in a single measurement. Results obtained from this single-molecule technique have been validated by ITC and BLI. The quality and information content of the MP data, combined with simple and fast measurements and low sample consumption makes MP a new preferred method for measuring strong protein-protein interactions.