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 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 to a few minutes, which prevents to completely characterize a protein individual and, hence, capture the heterogeneity among molecular populations. Here, we introduce a magnetic tweezers design, which showcases enhanced stability and resolution that allows us to measure the folding dynamics of a single protein during several uninterrupted days with a high temporal and spatial resolution. Thanks to this instrumental development, we do a complete characterization of two proteins with a very different force-response: the talin R3IVVI domain and protein L. Days-long recordings on the same single molecule accumulate several thousands of folding transitions sampled with sub-ms resolution, which allows 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. Our instrumental development offers a new tool for profiling individual molecules, opening the gates to the characterization of biomolecular heterogeneity.

中文翻译：

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相同的序列，不同的行为：在单分子水平上捕获的蛋白质多样性

经典的“一种序列，一种结构，一种功能”的范例已经塑造了我们对蛋白质如何在细胞内部发挥作用的许多直觉。部分由于批量生化分析提供的见解，假定单个生物分子的行为相同，并且其表征依赖于将任何构象多样性展平为独特表型的整体平均值。尽管单分子技术的出现打开了询问单个分子的大门，但技术缺陷通常将这些测量的持续时间限制在几分钟之内，这无法完全表征蛋白质个体，因此无法捕获分子种群之间的异质性。在这里，我们介绍一种磁性镊子设计，它展示了增强的稳定性和分辨率，使我们能够以高的时间和空间分辨率在数个不间断的时间内测量单个蛋白质的折叠动力学。由于仪器的发展，我们对两种具有不同力响应的蛋白质进行了完整表征：talin R3IVVI结构域和L蛋白。同一单个分子的为时数日的录音累积了数千个折叠亚基采样的过渡分辨率，这使我们能够重建它们的自由能态并描述它们如何在力的作用下演化。通过绘制许多不同蛋白质个体的纳米力学特性图，我们直接捕获了它们的分子多样性，作为对其力响应和折叠动力学的可量化分散。