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. 2024 Jan 4;15:249. doi: 10.1038/s41467-023-44607-y

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© The Author(s) 2024

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — a The reaction scheme of introducing a vinyl group to Thr-S and Hir-S with BS ligand. b Schematic illustration of AFM-based single-molecule force measurements. Thr-S and Hir-S were linked to the BS-modified probe and substrate, respectively. c Typical force–retract curve for the rupture of the Thr-S and Hir-S at a pulling speed of 200 nm/s (gray lines). Worm-like chain fitting of the force–extension curves (black lines) confirmed that the peak at an extension range of 10–30 nm corresponds to the rupture of the Thr-S/Hir-S interaction. d Rupture force histograms at a pulling speed of 200 nm/s. The Gaussian fitting shows an average rupture force of 47.20 ± 15.34 pN. e The rupture forces of the interactions between Thr-S and Hir-S at different pulling speeds. The rupture forces increased gradually as the pulling rates became larger. f ITC characterization of the Thr-S/Hir-S binding. The tests were conducted in PBS buffer (pH = 7.4), the Thr-S concentration in the bath was 26.2 μM, and the Hir-S concentration in the syringe was 108 μM. g Tests of the catalytic activities of Thr-S in the presence or absence of Hir-S using a fluorogenic substrate. In the absence of an inhibitor, Thr-S catalyzed the cleavage of the substrate, producing strong fluorescence, as shown in the inset picture. Data are presented as mean values with error bars representing the standard deviation of three independent replicates (n = 3 in each group). Source data are provided as a Source Data file. Fluoro, Fluorogenic. Thr-S, thrombin-SNAP. Hir-S, Hirudin-SNAP.