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冷休克蛋白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大, 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, 冷休克蛋白浓度增大使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 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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Keywords:
- cold shock protein /
- DNA hairpin /
- magnetic tweezers /
- single-stranded DNA
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图 1 三种DNA发夹结构 (a), (b)茎部为随机序列的发夹-15R60T7 (a)和发夹-15R60T3 (b); (c)茎部含特异性ATTGG基序(虚线框)的发夹-19R52T7
Fig. 1. Three DNA Hairpin structures: (a), (b) Hairpin-15R60T7 (a) and Hairpin 15R60T3 (b) with random stem sequences; (c) Hairpin -19R52T7 containing the specific ATTGG motif in the stem (marked by a dashed box).
图 2 DNA发夹构建物示意图. 限制性内切酶特异性切割DNA手柄(红线)后, 通过T4 DNA连接酶与发夹、侧链部分连接. 生物素标记的Handle-1的5’端与磁球表面的链霉亲和素相连, 巯基修饰的Handle-2的5’端与载玻片表面Sulfo-SMCC相连. 斜向虚线表示省略的碱基对, 未按标准比例绘制
Fig. 2. Schematic of DNA Hairpin construct. The DNA handles are specifically cleaved by restriction endonuclease (red lines), followed by T4 DNA ligase-mediated junction with the Hairpin and flank segments. The 5’ end of biotin-labeled Handle-1 is conjugated to streptavidin on the surface of magnetic beads, while the 5’-thiol-modified end of Handle-2 is conjugated with Sulfo-SMCC-coated glass substrates. Dashed diagonal lines represent omitted base pairs. Structural dimensions are not proportionally scaled.
图 3 DNA发夹-15R60T7代表性的力-延伸示意图. 当拉力增大时, DNA发夹在大约9.4 pN处以12.2 nm的步长去折叠, 紧接着又折叠回去; 在约10.2 pN处发生第二次去折叠, 步长为13.3 nm, 插图描述了DNA 发夹在力作用下发生折叠-去折叠转变
Fig. 3. Representative force-extension curve of DNA Hairpin-15R60T7. As the force increases, the DNA Hairpin unfolds at approximately 9.4 pN with a step size of 12.2 nm, followed by a refolding event. A second unfolding event occurs at around 10.2 pN with a step size of 13.3 nm. The inset illustrates the force-induced folding and unfolding transitions of the DNA Hairpin.
图 4 BcCsp浓度梯度下DNA发夹平衡态动力学表征 (a)—(c) DNA发夹-15R60T7在无BcCsp体系中的时间-延伸曲线, 分别对应10.73 pN, 9.88 pN和9.04 pN的恒力测量条件; (d)—(f)含梯度浓度BcCsp (50, 500和5000 nmol/L)时, 9.04 pN力场下DNA发夹的动力学响应; 右侧面板为对应平滑延伸的相对频率分布, 均呈现双峰分布特征(黑色原始数据采样率200 Hz, 红色曲线经0.1 s 时间窗口平滑处理)
Fig. 4. Equilibrium measurement of DNA Hairpin in solutions with different concentration of BcCsp: (a)–(c) Extension time course of DNA Hairpin-15R60T7 in the absence of BcCsp under constant force measurements at 10.73 pN, 9.88 pN, and 9.04 pN; (d)–(f) dynamic responses of the DNA Hairpin at 9.04 pN in solutions with BcCsp concentrations of 50, 500, and 5000 nmol/L. The right panels show relative frequency of the smoothed extensions, exhibiting two peaks. The raw data (black) is recorded at 200 Hz and smoothed over a 0.1 s time window (red).
图 5 在0—5000 nmol/L BcCsp范围内, DNA发夹-15R60T7在 9.04 pN下去折叠态(a)和折叠态(b)的存活概率, 其中实线表示指数拟合以确定kf和ku
Fig. 5. Survival probability of folded (a) and unfolded states (b) of DNA Hairpin-15R60T7 at 9.04 pN in solutions with 0–5000 nmol/L BcCsp. The solid curves represent the exponential fitting to determine kf and ku.
图 6 DNA发夹在Csp测量缓冲液中力依赖的折叠和去折叠速率 (a), (b) DNA发夹-15R60T7 (a)和发夹-15R60T3 (b)分别在0—5000 nmol/L BcCsp测量缓冲液中力依赖的折叠和去折叠速率; (c) DNA发夹-15R60T3在0—3000 nmol/L BsCsp测量缓冲液中力依赖的折叠和去折叠速率; 箭头表示的交叉点给出了DNA发夹在不同浓度Csp下的临界力
Fig. 6. Force-dependent folding and unfolding rates of DNA Hairpin in solutions with different concentration of Csp: (a), (b) Force-dependent folding and unfolding rates of DNA Hairpin-15R60T7 (a) and Hairpin-15R60T3 (b) in solutions with 0–5000 nmol/L BcCsp; (c) force-dependent folding and unfolding rates of DNA Hairpin-15R60T3 in solutions with 0–3000 nmol/L BsCsp. The intersection points indicated by the arrows give the critical forces of the DNA Hairpin at different concentrations of Csp.
图 7 DNA发夹-19R52T7典型的力-延伸示意图. DNA发夹在大约12.0 pN处以17.6 nm的步长发生去折叠, 当力加载到65 pN时, DNA双链手柄发生过渡拉伸转变
Fig. 7. Representative force-extension curve of DNA Hairpin-19R52T7. The DNA Hairpin unfolds at approximately 12.0 pN with a step size of 17.6 nm, and the DNA double-stranded handles overstretch when force is 65 pN.
图 8 力跳跃实验研究TmCsp与单双链DNA结合动力学. (a) 0 nmol/L, (b) 100 nmol/L和(c) 500 nmol/L TmCsp 存在时, DNA发夹-19R52T7 去折叠和折叠的典型力跳变测量; (a)—(c)左侧的第1个力跳变验证TmCsp是否与dsDNA结合, 左侧的第2个力跳变验证TmCsp是否与ssDNA相互作用
Fig. 8. Force-jump experiments to investigate the binding kinetics of TmCsp to ssDNA and dsDNA. Representative force-jump measurements of DNA Hairpin-19R52T7 in the presence of (a) 0 nmol/L, (b) 100 nmol/L, and (c) 500 nmol/L TmCsp. In panels (a)–(c), the first force jump (left) determines whether TmCsp binds to dsDNA, while the second force jump (right) assesses its interaction with ssDNA.
图 9 DNA发夹-19R52T7与TmCsp结合动力学的蛋白浓度和去折叠时间依赖性分析 (a) 100 nmol/L和500 nmol/L TmCsp溶液中, DNA发夹-19R52T7在8.0 pN下的去折叠态的存活概率, 其中实线为指数拟合, 用于确定折叠速率; (b), (c) 16.0 pN拉力条件下, 去折叠态停留时间对DNA发夹在8.0 pN下的折叠速率(b)和结合概率(c)的影响
Fig. 9. Analysis of protein concentration and unfolding time dependence in the binding kinetics of TmCsp with DNA Hairpin-19R52T7. (a) Survival probability of the unfolded state for DNA Hairpin-19R52T7 at 8.0 pN in the presence of 100 nmol/L and 500 nmol/L TmCsp. The solid lines represent exponential fitting to determine the folding rates. (b), (c) Effect of unfolded state dwell time of DNA Hairpin at 16.0 pN on the folding rate at 8.0 pN (b) and the binding probability (c).
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[1] Watson J D, Crick F H C 1953 Nature 171 737
Google Scholar
[2] Travers A, Muskhelishvili G 2015 FEBS. J. 282 2279
Google Scholar
[3] Bailly C, Waring M J, Travers A A 1995 J. Mol. Biol. 253 1
Google Scholar
[4] Virstedt J, Berge T, Henderson R M, Waring M J, Travers A A 2004 J. Struct. Biol. 148 66
Google Scholar
[5] Dessinges M N, Maier B, Zhang Y, Peliti M, Bensimon D, Croquette V 2002 Phys. Rev. Lett. 89 248102
Google Scholar
[6] Zhang C, Tian F J, Zuo H W, et al. 2025 Nat. Commun. 16 113
Google Scholar
[7] Hunter C A 1993 J. Mol. Biol. 230 1025
Google Scholar
[8] Bosco A, Camunas-Soler J, Ritort F 2014 Nucleic Acids Res. 42 2064
Google Scholar
[9] Budkina K S, Zlobin N E, Kononova S V, Ovchinnikov L P, Babakov A V 2020 Biochemistry (Mosc.) 85 1
Google Scholar
[10] Lopez M M, Yutani K, Makhatadze G I 1999 J. Biol. Chem. 274 33601
Google Scholar
[11] Graumann P, Marahiel M A 1994 FEBS Lett. 338 157
Google Scholar
[12] Bae W, Xia B, Inouye M, Severinov K 2000 Proc. Natl. Acad. Sci. 97 7784
Google Scholar
[13] Phadtare S, Inouye M, Severinov K 2002 J. Biol. Chem. 277 7239
Google Scholar
[14] Jiang W, Jones P, Inouye M 1993 J. Bacteriol. 175 5824
Google Scholar
[15] Brandi A, Pietroni P, Gualerzi C O, Pon C L 1996 Mol. Microbiol. 19 231
Google Scholar
[16] Goldenberg D, Azar I, Oppenheim A B 1996 Mol. Microbiol. 19 241
Google Scholar
[17] Jones P G, Inouye M 1994 Mol. Microbiol. 11 811
Google Scholar
[18] Mani A, Gupta D K 2015 J. Biomol. Struct. Dyn. 33 861
Google Scholar
[19] Caballero C J, Menendez-Gil P, Catalan-Moreno A, et al. 2018 Nucleic Acids Res. 46 1345
Google Scholar
[20] Zhang Y, Burkhardt D H, Rouskin S, Li G W, Weissman J S, Gross C A 2018 Mol. Cell 70 274
Google Scholar
[21] Horn G, Hofweber R, Kremer W, Kalbitzer H R 2007 Cell. Mol. Life Sci. 64 1457
Google Scholar
[22] Bustamante C, Alexander L, Maciuba K, Kaiser C M 2020 Annu. Rev. Biochem. 89 443
Google Scholar
[23] Ashkin A, Dziedzic J M, Bjorkholm J E, Chu S 1986 Opt. Lett. 11 288
Google Scholar
[24] Zlatanova J, Lindsay S M, Leuba S H 2000 Prog. Biophys. Mol. Bio. 74 37
Google Scholar
[25] Smith S B, Finzi L, Bustamante C 1992 Science 258 1122
Google Scholar
[26] Stirnemann G, Giganti D, Fernandez J M, Berne B J 2013 Proc. Natl. Acad. Sci. 110 3847
Google Scholar
[27] Xue Z Y, Sun H, Hong H Y, Zhang Z W, Zhang Y H, Guo Z L, Le S M, Chen H 2024 Phys. Rev. Res. 6 023170
Google Scholar
[28] Hong H Y, Guo Z L, Sun H, Yu P, Su H H, Ma X N, Chen H 2021 Commun. Chem. 4 156
Google Scholar
[29] Xue Z Y, Yu P, Zhang Y H, Zhang Z W, Sun H, Hou Z Q, Hong H Y, Le S M, Chen H 2025 Phys. Rev. E 111 014413
Google Scholar
[30] Petrosyan R, Narayan A, Woodside M T 2021 J. Mol. Biol. 433 167207
Google Scholar
[31] Liang T, Yang C, Song X Y, Feng Y Y, Liu Y H, Chen H 2023 Phys. Rev. E 108 014406
Google Scholar
[32] Zeeb M, Balbach J 2003 Protein Sci. 12 112
Google Scholar
[33] Lopez M M, Yutani K, Makhatadze G I 2001 J. Biol. Chem. 276 15511
Google Scholar
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