XUE Zhenyong; LI Xiangyun; HOU Zhiqi; QI Xingyu; LIU Yanhui; CHEN Hu

doi:10.7498/aps.74.20250504

Abstract

Cold shock proteins (Csps) are a class of highly conserved nucleic acid-binding protein composed of 65−70 amino acids that form a compact β-barrel structure with five antiparallel β-strands. As nucleic acid-binding proteins, Csps play an important role in bacterial response to cold shock, yet their precise working mechanism is still unclear. As is well known, DNA hairpin undergoes folding-unfolding transitions under small constant forces. Magnetic tweezers technique has obvious advantages in this kind of research, especially its capacity for extended-duration constant-force measurements at pico-Newton force level, which makes it very suitable for characterizing the conformational transition dynamics of DNA hairpin at low forces of several pico-Newton. In this study, we first stretch DNA hairpin from its N- and C-termini by using magnetic tweezers. Then, we sequentially introduce Csp buffer solutions with increasing concentrations into the flow chamber and measure the folding and unfolding rates of the DNA hairpin at different Csp concentrations. It is found that within a certain concentration range, increasing Csp concentration can significantly reduce the DNA hairpin folding rate while keeping the unfolding rate almost unchanged. This behavior occurs because Csp only binds to single-stranded DNA (ssDNA), and interacts with the ssDNA region of the unfolded DNA hairpin, thereby hindering the folding process. As Csp does not interact with double-stranded DNA (dsDNA), the above-mentioned effect on the unfolding process is negligible. Furthermore, the critical force of DNA hairpin progressively decreases with the increase of Csp concentration, demonstrating that Csp effectively destabilizes the hairpin structure. When the Csp concentration reaches sufficiently high levels, the DNA hairpin’s unfolding rate increases considerably. This phenomenon may be caused by the rapid binding of Csp to newly exposed ssDNA regions of partially unfolded DNA hairpins, which prevents refolding and accelerates the unfolding pathway. In force-jump experiments using Csp-containing buffers, the binding preference of Csp for either ssDNA or dsDNA can be directly determined by analyzing whether the delayed response of DNA hairpin extension occurs. In force-increasing jump experiments, no extension delay is observed in the DNA hairpin unfolding process. In contrast, force-decreasing jump experiments shows significant extension delay in the folding process. These single-molecule measurements provide direct evidence that Csp only specifically binds to ssDNA, further demonstrating that its binding kinetics occur very rapidly. This study delves into the molecular mechanisms by which Csps maintain normal cellular functions in cold chock conditions.

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Corresponding author: XUE Zhenyong, 19820200156654@stu.xmu.edu.cn ; LIU Yanhui, yhliu1@gzu.edu.cn ; CHEN Hu, chenhu@xmu.edu.cn

Funds: Project supported by the National Natural Science Foundation of China (Grant Nos. 12474200, 12174322) and the 111 Project, China (Grant No. B16029).

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Vol. 74, Issue 12 - 2025-06-20

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Corresponding author: XUE Zhenyong, 19820200156654@stu.xmu.edu.cn ; LIU Yanhui, yhliu1@gzu.edu.cn ; CHEN Hu, chenhu@xmu.edu.cn

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