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[Preprint]. 2024 Jun 6:2024.06.06.597759.
doi: 10.1101/2024.06.06.597759.

A DNA-based molecular clamp for probing protein interactions and structure under force

Minhwan Chung  1 , Kun Zhou  2   3 , John Powell  2   3 , Chenxiang Lin  2   3   4 , Martin A Schwartz  1   2   4
Affiliations

A DNA-based molecular clamp for probing protein interactions and structure under force

Minhwan Chung et al. bioRxiv. .

Abstract

Cellular mechanotransduction, a process central to cell biology, embryogenesis, adult physiology and multiple diseases, is thought to be mediated by force-driven changes in protein conformation that control protein function. However, methods to study proteins under defined mechanical loads on a biochemical scale are lacking. We report the development of a DNA based device in which the transition between single-stranded and double-stranded DNA applies tension to an attached protein. Using a fragment of the talin rod domain as a test case, negative-stain electron microscopy reveals programmable extension while pull down assays show tension-induced binding to two ligands, ARPC5L and vinculin, known to bind to cryptic sites inside the talin structure. These results demonstrate the utility of the DNA clamp for biochemical studies and potential structural analysis.

Keywords: integrin; mechanotransduction; talin1; tension.

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Figures

Figure 1.
Figure 1.. DNA clamp-induced protein conformational change.
a, Diagram of the DNA clamp design and operation. b, Schematics depicting the conjugation reactants: benzylguanine- and chloroalkane-labeled DNA handles and the target protein, talin R1-R2 with terminal SNAP- and Halo-Tags (S-R1-R2-H). c, SDS-PAGE showing purified S-R1-R2-H before and after conjugation with DNA handles, with and without purification by size exclusion chromatography (SEC). d, Native-PAGE visualizing the handles with fluorescently tagged complementary strands. e, Native-PAGE demonstrating band shift after each step of DNA clamp assembly and extension. The 30-nm bridge is fluorescently labeled with Fluorescein (FAM). The fully extended protein band is indicated with a yellow arrowhead. f, Band shift observed with DNA clamps of different lengths on native-PAGE, visualized with sliver stain (top) and fluorescence (bottom). Extended protein bands are indicated with yellow arrowheads. g, TEM images of the protein with varying DNA clamp lengths. Selected particles represent extended protein for each clamp. Scale bar: 20 nm. h, Quantification of the end-to-end distance of the imaged particles by TEM. The means of each group were compared using a one-way ANOVA followed by Tukey’s multiple comparison test (*, P ≤0.05; **, P≤0.01; ns, not significant). i, Frequency distribution of the protein end-to-end length for each DNA clamp length, fitted by Gaussian distribution. The histograms of bridge lengths over 19 nm were fitted by the sum of two Gaussian distributions. The total number of measurements and the mean of each Gaussian distribution are indicated in each histogram. Each histogram bar represents a bin range of 2 μm; clamp length is indicated in each histogram. P: protein without the bridge. 30C: protein with a 30 nm bridge lacking the complementary sequence to bind the DNA handle on SNAP-tag side.
Figure 2.
Figure 2.. Vinculin and ARPC5L initiate binding to talin at different talin extension lengths.
a, Schematic of the pulldown assay. b and c, Western blots of S-R1-R2-H with varying clamp lengths pulled down by immobilized VinD1 and ARPC5L, respectively. d and e, Quantification of Western blot results. The ratio of S-R1-R2-H to VinD1 or ARPC5L was calculated and normalized to 30 nm condition for each repeat. Group means were compared using a one-way ANOVA (***, P ≤0.001; ****, P≤0.0001; ns, not significant).
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