The classical “one sequence, one structure, one function” paradigm has shaped much of our intuition of how proteins work inside the cell. Partially due to the insight provided by bulk biochemical assays, individual biomolecules are often assumed to behave as identical entities, and their characterization relies on ensemble averages that flatten any conformational diversity into a unique phenotype. While the emergence of single-molecule techniques opened the gates to interrogating individual molecules, technical shortcomings typically limit the duration of these measurements, which precludes a complete characterization of an individual protein and, hence, capturing the heterogeneity among molecular populations. Here, we introduce an ultrastable magnetic tweezers design, which enables us to measure the folding dynamics of a single protein during several uninterrupted days with high temporal and spatial resolution. Thanks to this instrumental development, we fully characterize the nanomechanics of two proteins with a very distinct force response, the talin R3 domain and protein L. Days-long recordings on the same protein individual accumulate thousands of folding transitions with submicrosecond resolution, allowing us to reconstruct their free energy landscapes and describe how they evolve with force. By mapping the nanomechanical identity of many different protein individuals, we directly capture their molecular diversity as a quantifiable dispersion on their force response and folding kinetics. By significantly expanding the measurable timescales, our instrumental development offers a tool for profiling individual molecules, opening the gates to directly characterizing biomolecular heterogeneity.

中文翻译：

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相同的序列，不同的行为：在单分子水平捕获蛋白质多样性

经典的“一个序列、一种结构、一种功能”范式塑造了我们对蛋白质如何在细胞内发挥作用的大部分直觉。部分由于大量生化测定提供的洞察力，单个生物分子通常被认为表现得相同的实体，并且它们的表征依赖于将任何构象多样性扁平化为独特表型的整体平均值。虽然单分子技术的出现打开了询问单个分子的大门，但技术缺陷通常限制了这些测量的持续时间，这妨碍了对单个蛋白质的完整表征，从而无法捕获分子群体之间的异质性。在这里，我们介绍了一种超稳定的磁性镊子设计，它使我们能够在几天不间断的时间内以高时间和空间分辨率测量单个蛋白质的折叠动力学。得益于这一仪器开发，我们充分表征了两种具有非常独特的力响应的蛋白质（talin R3 结构域和蛋白质 L）的纳米力学特征。对同一蛋白质个体的长达数天的记录积累了数千个亚微秒分辨率的折叠转变，使我们能够重建它们的自由能景观并描述它们如何随力演化。通过绘制许多不同蛋白质个体的纳米力学特性，我们直接捕捉它们的分子多样性，作为它们的力响应和折叠动力学的可量化的分散。通过显着扩展可测量的时间尺度，我们的仪器开发提供了一种分析单个分子的工具，为直接表征生物分子异质性打开了大门。