Molecular Mechanism Underlying the Non-covalent Interaction between Sinapic Acid and Rice Bran Glutelin
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摘要: 为探究芥子酸（sinapic acid，SA）与米糠谷蛋白（rice bran glutelin，RBG）的非共价相互作用的动态过程及其分子机制，本文利用荧光光谱法表征了荧光猝灭机制、结合位点数及热力学参数，并进一步通过分子对接结合分子动力学模拟解析了SA与RBG相互作用的动态过程和分子机制。结果表明，SA以静态猝灭的方式猝灭RBG蛋白的内源荧光形成复合物，结合位点数约为1。两者的结合是自发行为，疏水相互作用是主要驱动力。分子对接发现RBG上存在5个潜在结合位点。进一步的分子动力学模拟表明SA不仅稳定结合在C2位点，而且表现出最低的结合自由能，是最可能的结合位点。蛋白质回旋半径、均方根位移和均方根偏差分析进一步证实了SA与RBG结合的稳定性。结合自由能分解和相互作用分析从蛋白和小分子结构两个角度揭示了6个关键氨基酸（Ile131、Ile90、Gln261、Trp149、Tyr151及Tyr102）和SA的甲氧基对SA与RBG的结合具有重要作用。研究结果为SA-RBG复合物作为功能性食品配料的应用开发提供了理论基础。Abstract: The current study aimed to explore the dynamic binding process and molecular mechanism for the non-covalent interaction between sinapic acid (SA) and rice bran glutelin (RBG). Fluorescence spectroscopy was used to investigate the mechanism of fluorescence quenching, the number of binding sites and thermodynamic parameters. Further, the binding process of complex formation and the underlying molecular mechanism were revealed by the combination of homology modeling, molecular docking and molecular dynamic simulations. Fluorescence study showed that SA quenched the intrinsic fluorescence of RBG via static mode, indicating the formation of SA-RBG complex. The number of binding site was about 1. Thermodynamic parameters suggested that the SA could bind with RBG spontaneously, which was predominately driven by hydrophobic interactions. Molecular docking revealed that there were five potent binding sites on RBG for SA. Further molecular dynamic simulation revealed that SA could not only bind stably at binding site C2 but also exhibited the lowest binding free energy, suggesting that C2 was the most favorable binding cavity among the five predicted binding sites. Furthermore, molecular dynamic simulation results including the radius of gyrate, root mean square deviation and root mean square fluctuation further validated the binding stable between SA and RBG. The decomposition of binding free energy to per amino acid residue combined with binding mode analysis indicated that six key residues (including Ile131, Ile90, Trp149, Gln261, Tyr151 and Tyr102) of RBG and two methoxy groups of SA played critical roles in the binding process between SA an RBG. The above results would provide theoretical basis for the application and development of SA-RBG complex as functional ingredients.
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Key words:
- rice bran /
- glutelin /
- sinapic acid /
- interaction /
- molecular simulation
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图 2 SA对RBG内源荧光的猝灭机制
Figure 2. Quenching mechanism of SA on RBG endogenous fluorescence
注：A：298 K下不同浓度SA对RBG的荧光猝灭；B：302 K下不同浓度SA对RBG的荧光猝灭；C：306 K下不同浓度SA对RBG的荧光猝灭；D：Stern-Volmer拟合方程；E：双对数方程；图中a、b、c、d、e、f、g、h、i表示SA浓度分别为0、2.5、5.0、7.5、10.0、12.5、15.0、17.5、20.0 μmol/L。
图 3 SA对RBG同步荧光光谱的影响
Figure 3. Effect of SA on the synchronous fluorescence spectra of RBG
注：A：Δλ=15 nm，B：Δλ=60 nm；a、b、c、d、e、f、g、h、i表示SA浓度分别为0、2.5、5.0、7.5、10.0、12.5、15.0、17.5、20.0 μmol/L。
图 4 RBG三维结构及其与SA的分子对接结果
Figure 4. Three-dimensional structure of RBG and the molecular docking result between it and SA
注：A：同源模建获得的RBG三维结构；B：拉氏图分析；C：SA与RBG 5个可能结合位点及构象。
图 5 RMSD曲线及结合自由能
Figure 5. RMSD plots and binding free energy
注：A：MD过程中RBG的RMSD变化；B~F：5个结合位点配体SA的RMSD变化；插图表示MD模拟过程中SA在RBG结合口袋中的运动轨迹；G：C1-C4四个结合位点处SA与RBG的结合自由能；图中不同字母表示差异显著（P<0.05）。
图 6 结合稳定性及相互作用分子机制
Figure 6. Binding stability analysis and the interaction molecular mechanism
注：A：Rg变化情况；B：RMSF变化情况；C：结合自由能分解到RBG的每个氨基酸残基；D：MD最后一帧SA和RBG的2D相互作用模式。
表 1 SA与RBG相互作用的猝灭常数及热力学参数
Table 1. Quenching parameters and thermodynamic parameters for SA-RBG interaction
温度（K） Ksv
（×104 L/mol）n Ka
（×104 L/mol）ΔH
（kJ/mol）ΔG
（kJ/mol）ΔS
（J/mol/K）298 1.55±0.02 1.06 3.00±0.77 64.79 ?25.34 302.44 302 1.82±0.09 1.06 3.28±0.27 ?26.55 306 2.77±0.19 1.07 5.96±0.29 ?27.78 
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表 2 SA在RBG 5个潜在结合位点的分子对接打分
Table 2. Docking scores for SA at five potential binding sites of RBG
位点 C1 C2 C3 C4 C5 打分结果 ?6.6 ?6.1 ?6.7 ?6.0 ?5.1 
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