School of Science, RMIT University, Bundoora West Campus, Plenty Road, Melbourne, VIC 3083, Australia.
School of Science, RMIT University, Bundoora West Campus, Plenty Road, Melbourne, VIC 3083, Australia.
Food Chem. 2021 Oct 30;360:130059. doi: 10.1016/j.foodchem.2021.130059. Epub 2021 May 11.
Protein-phenolic compound interactions are commonly investigated with inappropriate linear equations for the analysis of binding strength and stoichiometry. This work utilises more appropriate protocols for the investigation of molecular interactions between vanillic acid and β-lactoglobulin at pH 2.4, where the protein predominately exists as a monomer. Non-linear binding and Job plot analysis were conducted on fluorescence data to effectively determine the interaction's dissociation constant (K, 2.93 × 10 M) and stoichiometry (1:1). Furthermore, spectroscopic techniques revealed statistically significant alterations to the conformational characteristics of β-lactoglobulin upon complexation. Molecular dynamics (MD) simulations support a 1:1 interaction stoichiometry and reveal that the stabilisation of vanillic acid was dynamic in nature but mainly supported by four π-alkyl interactions and one hydrogen bond, located within the β-barrel of the monomer. Water molecules, which are generally not accounted for in MD simulation analysis, were shown to be an important factor in the ligand stabilization via bridging interactions.
蛋白质-酚类化合物相互作用通常使用不合适的线性方程来分析结合强度和化学计量比。本工作利用更合适的方案来研究 pH 2.4 下香草酸与β-乳球蛋白之间的分子相互作用，在该 pH 下，蛋白质主要以单体形式存在。通过荧光数据进行非线性结合和工作曲线分析，有效地确定了相互作用的离解常数（K，2.93×10^-4 M）和化学计量比（1:1）。此外，光谱技术揭示了β-乳球蛋白构象特征在络合后发生了统计学上显著的变化。分子动力学（MD）模拟支持 1:1 的相互作用化学计量比，并表明香草酸的稳定性是动态的，但主要由四个π-烷基相互作用和一个氢键支持，位于单体的β-桶内。在 MD 模拟分析中通常不考虑的水分子，通过桥接相互作用被证明是配体稳定的一个重要因素。
