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冷休克蛋白(Cold shock protein,Csp)是一类高度保守的核酸结合蛋白,由65-70个氨基酸组成的5条反向平行β链,形成结构紧凑的β桶状结构。冷休克蛋白在细菌应对冷刺激过程中起重要作用,但其具体工作机制尚未完全阐明。本研究利用磁镊技术系统研究了不同浓度冷休克蛋白对DNA发夹结构折叠和去折叠动力学的影响,定量测定了相应条件下DNA发夹的折叠和去折叠速率。实验结果表明,在一定浓度范围内,随着冷休克蛋白浓度增加,DNA发夹的折叠速率显著降低;而去折叠速率保持不变。当冷休克蛋白达到一定浓度阈值时,去折叠速率也呈现明显上升趋势。进一步研究发现,冷休克蛋白浓度增加使DNA发夹的临界力减小,从而降低了发夹的结构稳定性。通过力跳变实验,更直观地表现出冷休克蛋白只与单链DNA结合,而不与双链DNA相互作用。这些单分子水平的研究结果揭示了冷休克蛋白通过调控核酸双螺旋结构稳定性来维持细菌低温适应性的分子机制。Cold shock proteins (Csps) are a class of highly conserved nucleic acid-binding proteins 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 remains unclear. It is known that DNA hairpin undergoes folding-unfolding transitions at small constant forces. Magnetic tweezers technique offers distinct advantages for such investigations, particularly its capacity for extended-duration constant-force measurements at pico-Newton force levels, making it ideally suited for characterizing the conformational transition dynamics of DNA hairpin at the low forces of several pico-Newton. In this study, we first stretch DNA hairpin from its N- and C-termini 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. We find that within a certain concentration range, increasing Csp concentration significantly reduces the DNA hairpin folding rate while leaving the unfolding rate virtually unchanged. This behavior arises because Csp exclusively binds to single-stranded DNA (ssDNA), interacting with the ssDNA regions of the unfolded DNA hairpin and thereby hindering the folding process. As Csp does not interact with double-stranded DNA (dsDNA), it has negligible effect on the unfolding process. Furthermore, the critical force of DNA hairpin progressively decreased with elevated Csp concentration, demonstrating that Csp effectively destabilizes the hairpin structure. When the Csp concentration reaches sufficiently high levels, we also detect a notable increase in the DNA hairpin's unfolding rate. This phenomenon likely arises from Csp's rapid binding to the newly exposed ssDNA regions of the partially unfolded DNA Hairpin, which prevents refolding and consequently accelerates the unfolding pathway. In force-jump experiments performed with Csp-containing buffers, the binding preference of Csp for either ssDNA or dsDNA can be directly determined by analyzing whether delayed response of DNA hairpin extension occur. In force-increasing jump experiments, we observe no extension delay during the DNA hairpin unfolding process. In contrast, force-decreasing jump experiments revealed significant extension delay during the folding process. These single-molecule measurements provide direct evidence that Csp specifically binds only to ssDNA, and further demonstrate that its binding kinetics occur with remarkable rapidity. This study provides insights to the molecular mechanism of Csps to maintain normal cellular functions in cold chock conditions.
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Keywords:
- Cold shock protein /
- DNA hairpin /
- Magnetic tweezers /
- ssDNA
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