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  

A Study of the Mechanism of Binding Between Quercetin and CAV-1 Based on Molecular Simulation,Bio-Layer Interferometry and Multi-Spectroscopy Methods

作　　者：侯倩宜 董壮壮 原红霞 李青山 HOU Qian-yi;DONG Zhuang-zhuang;YUAN Hong-xia;LI Qing-shan(School of Traditional Chinese Medicine and Food Engineering,Shanxi University of Chinese Medicine,Jinzhong 030619,China;Shanxi Key Laboratory of Innovative Drug for the Treatment of Serious Diseases Basing on the Chronic Inflammation,Shanxi University of Chinese Medicine,Jinzhong 030619,China)

机构地区：[1]山西中医药大学中药与食品工程学院,山西晋中030619 [2]山西中医药大学,基于炎性反应的重大疾病创新药物山西省重点实验室,山西晋中030619

出　　处：《光谱学与光谱分析》2023年第3期890-896,共7页Spectroscopy and Spectral Analysis

基　　金：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。

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Kq值远大于2.0×10^(10) L·mol^(-1)·s^(-1),且猝灭常数KSV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�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 Kq value(the quenching rate constant)were all much larger than 2.0×10^(10) L·mol^(-1)·s^(-1),and the fluorescence quenching constant(KSV)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 changeΔH<0,entropy changeΔS<0 andΔG<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

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 

分 类 号：R28[医药卫生—中药学]