多光谱法、分子对接模拟和生物膜干涉研究槲皮素与小窝蛋白-1的相互作用

doi:10.3964/j.issn.1000-0593(2023)03-0890-07

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摘要：小窝蛋白-1（CAV-1）在动脉粥样硬化等心血管疾病的发生发展中发挥关键作用。为了解槲皮素与CAV-1的相互作用，在模拟生理环境和不同温度条件下，采用多光谱法、同源模建、分子对接模拟和生物膜（BLI）技术进行研究。荧光猝灭数据结果显示，猝灭速率常数K_q值远大于2.0×10¹⁰ L·mol^-1·s^-1，且猝灭常数K_SV随温度升高而降低，证明槲皮素和CAV-1相互作用的猝灭过程为静态猝灭；而热力学参数，焓变ΔH＜0、熵变ΔS＜0且ΔG＜0，表明二者的结合过程是自发、焓驱动的，其相互作用的主要类型为范德华力和氢键作用。通过对槲皮素与CAV-1相互作用的同步荧光光谱和三维荧光光谱分析，随着槲皮素的加入，CAV-1的荧光强度逐渐降低，证明二者之间发生了相互作用。进一步分析发现，同步荧光光谱中槲皮素使CAV-1中的芳香族氨基酸残基的最大发射波长发生了轻微红移，周围的微环境极性增强，亲水性增加，表明槲皮素的加入使CAV-1的蛋白质构象发生了改变。紫外-可见光谱结果显示，CAV-1与槲皮素之间形成了一个基态复合物，进一步证实了CAV-1与槲皮素之间的静态猝灭机制。采用同源模建技术建立CAV-1的X射线晶体结构模板。分子对接模拟结果显示两者结合力为-7.372 kcal·mol^-1。对接结果表明槲皮素结合点位于由GLU20，ASP70，VAL16和ARG19等氨基酸形成的活性口袋中，与GLN21，VAL16和ARG19位点产生范德华力作用，与GLU20，ASP70位点存在氢键作用力。各种作用力影响了CAV-1的微环境变化，导致其荧光猝灭，是参与复合物形成的关键因素。最后，利用BLI技术对槲皮素和CAV-1的结合进行定量研究，研究结果显示，二者具有良好的的结合活性，结合解离平衡常数K_D值为2.50×10^-5 mol·L^-1；其响应信号值随槲皮素浓度的升高而增强，表明CAV-1与槲皮素之间存在特异性结合。该研究有助于了解槲皮素与CAV-1的相互作用机制，为槲皮素治疗动脉粥样硬化的作用靶点研究提供参考。

关键词：槲皮素；小窝蛋白-1；相互作用；多光谱法；同源模建；分子对接；生物膜干涉技术

Abstract：Caveolin-1 (CAV-1) plays a key role in developing cardiovascular diseases such as atherosclerosis. The interaction between quercetin and CAV-1 was studied by multispectral, homology modeling, molecular docking simulation and bio-layer interferometry (BLI) in the simulated physiological environment and different temperatures. The fluorescence quenching data showed that the K_q value (the quenching rate constant) were all much larger than 2.0×10¹⁰ L·mol^-1·s^-1, and the fluorescence quenching constant (K_SV) decreased with the increase of temperature, which proves that the quenching process of the interaction between quercetin and CAV-1 is static quenching. Furthermore, the thermodynamic parameters, enthalpy change ΔH＜0, entropy change ΔS＜0 and ΔG＜0 indicated that the bonding process is spontaneous and enthalpy driven, indicating that the main types of interaction are van der Waals force and hydrogen bonding. Through the synchronous fluorescence and three-dimensional fluorescence spectrums analysis of the interaction between quercetin and CAV-1, it was found that the fluorescence intensity of CAV-1 was progressively decreased upon the addition of quercetin, indicating that quercetin interacted with CAV-1. Further analysis showed that quercetin caused the redshift of the maximum emission wavelength of the aromatic amino acid residues in CAV-1, enhanced the polarity of the microenvironment around the CAV-1, enhanced its hydrophilicity, indicating that the addition of quercetin changed the protein conformation of CAV-1. The UV-Vis absorption spectrum showed that a ground-state complex was formed between CAV-1 and quercetin, which further confirmed the static quenching mechanism between CAV-1 and quercetin. The X-ray crystal structure template of CAV-1 was constructed using homology modeling. The molecular docking simulation results showed that the binding force of quercetin and CAV-1 was -7.372 kcal·mol^-1. The docking results showed that quercetin could bind to the active pocket composed of amino acids such as Glu20, ASP70, VAL16 and ARG19. There were the van der Waals forces between quercetin and residues GLN21, VAL16 and ARG19 of CAV-1, and hydrogen bonds between quercetin and GLU20 and ASP70. Various forces affected the micro-environmental changes of CAV-1 and led to its fluorescence quenching, which is a key factor involved in the formation of the complex. Finally, the binding of quercetin and CAV-1 was quantitatively studied by the BLI technique. The results showed that quercetin had a good binding activity with CAV-1 with an equilibrium constant (K_D) value of 2.50×10^-5 mol·L^-1. The response signal value increased with quercetin concentration, evidencing the specific binding between CAV-1 and quercetin. This research was helpful in understanding the mechanism of interaction between quercetin and CAV-1 and provide references for research on the therapeutic targets of quercetin in atherosclerosis.

Key words：Quercetin; Caveolin-1; Interaction; Multi-spectroscopy; Homology modeling; Molecular docking; Bio-layer Interferometry

收稿日期: 2022-01-07 修订日期: 2022-05-12

中图分类号:

R28

基金资助: 国家自然科学基金项目(82003881)，山西省自然科学基金项目(201801D121231)，山西省高校科技创新基金项目(2019L0718)，人才培养专项计划山西中医药大学青年科学家基金项目(2021PY-QN-05)资助

通讯作者: 原红霞，李青山 E-mail: yuanhongxia609@163.com; sxlqs0501@sxtcm.edu.cn

作者简介: 侯倩宜，1994年生，山西中医药大学中药与食品工程学院硕士研究生 e-mail: 1216480606@qq.com

引用本文:

侯倩宜，董壮壮，原红霞，李青山. 多光谱法、分子对接模拟和生物膜干涉研究槲皮素与小窝蛋白-1的相互作用[J]. 光谱学与光谱分析, 2023, 43(03): 890-896.
HOU Qian-yi, DONG Zhuang-zhuang, YUAN Hong-xia, LI Qing-shan. A Study of the Mechanism of Binding Between Quercetin and CAV-1 Based on Molecular Simulation, Bio-Layer Interferometry and Multi-Spectroscopy Methods. SPECTROSCOPY AND SPECTRAL ANALYSIS, 2023, 43(03): 890-896.

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