School of Science, Monash University Malaysia, Bandar Sunway, 47500, Selangor, Malaysia.
Materials Science and Nanotechnology Department, Faculty of Postgraduate Studies for Advanced Sciences (PSAS), Beni-Suef University, Beni-Suef, Egypt; Department of Chemistry, Faculty of Science, Sultan Qaboos University, P.O. Box 36, Postal Code 123 Muscat, Oman.
Spectrochim Acta A Mol Biomol Spectrosc. 2018 Apr 5;194:36-44. doi: 10.1016/j.saa.2018.01.005. Epub 2018 Jan 4.
The ability of human serum albumin (HSA) to bind medium-sized hydrophobic molecules is important for the distribution, metabolism, and efficacy of many drugs. Herein, the interaction between pyrene, a hydrophobic fluorescent probe, and HSA was thoroughly investigated using steady-state and time-resolved fluorescence techniques, ligand docking, and molecular dynamics (MD) simulations. A slight quenching of the fluorescence signal from Trp214 (the sole tryptophan residue in the protein) in the presence of pyrene was used to determine the ligand binding site in the protein, using Förster's resonance energy transfer (FRET) theory. The estimated FRET apparent distance between pyrene and Trp214 was 27Å, which was closely reproduced by the docking analysis (29Å) and MD simulation (32Å). The highest affinity site for pyrene was found to be in subdomain IB from the docking results. The calculated equilibrium structure of the complex using MD simulation shows that the ligand is largely stabilized by hydrophobic interaction with Phe165, Phe127, and the nonpolar moieties of Tyr138 and Tyr161. The fluorescence vibronic peak ratio I/I of bound pyrene inside HSA indicates the presence of polar effect in the local environment of pyrene which is less than that of free pyrene in buffer. This was clarified by the MD simulation results in which an average of 5.7 water molecules were found within 0.5nm of pyrene in the binding site. Comparing the fluorescence signals and lifetimes of pyrene inside HSA to that free in buffer, the high tendency of pyrene to form dimer was almost completely suppressed inside HSA, indicating a high selectivity of the binding pocket toward pyrene monomer. The current results emphasize the ability of HSA, as a major carrier of several drugs and ligands in blood, to bind hydrophobic molecules in cavities other than subdomain IIA which is known to bind most hydrophobic drugs. This ability stems from the nature of the amino acids forming the binding sites of the protein that can easily adapt their shape to accommodate a variety of molecular structures.
人血清白蛋白（HSA）结合中等大小疏水分子的能力对于许多药物的分布、代谢和疗效非常重要。本文使用稳态和时间分辨荧光技术、配体对接和分子动力学（MD）模拟，深入研究了疏水性荧光探针芘与 HSA 之间的相互作用。在存在芘的情况下，通过测定蛋白质中唯一色氨酸残基 Trp214 的荧光信号的轻微猝灭，利用福斯特共振能量转移（FRET）理论确定了蛋白质中的配体结合位点。用对接分析（29Å）和 MD 模拟（32Å）很好地再现了估计的芘与 Trp214 之间的 FRET 表观距离 27Å。从对接结果来看，芘的最高亲和力位点位于亚结构域 IB。使用 MD 模拟计算复合物的平衡结构表明，配体主要通过与 Phe165、Phe127 以及 Tyr138 和 Tyr161 的非极性部分的疏水相互作用得到稳定。结合 HSA 内的芘的荧光振子峰比 I/I 表明，在芘的局部环境中存在极性效应，这比在缓冲液中的自由芘要小。MD 模拟结果表明，在结合部位内，平均有 5.7 个水分子存在于芘的 0.5nm 范围内，这阐明了这一点。将 HSA 内芘的荧光信号和寿命与游离在缓冲液中的芘进行比较，发现 HSA 内芘形成二聚体的高趋势几乎完全被抑制，这表明结合口袋对芘单体具有很高的选择性。目前的结果强调了 HSA 作为血液中几种药物和配体的主要载体的能力，它能够在亚结构域 IIA 以外的腔体内结合疏水分子，而亚结构域 IIA 已知是结合大多数疏水性药物的部位。这种能力源于形成蛋白质结合位点的氨基酸的性质，这些氨基酸可以很容易地改变其形状以适应各种分子结构。
