Temperature-dependent simultaneous ligand binding in human serum albumin.
Temperature-dependent simultaneous ligand binding in human serum albumin.
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DOI：
10.1021/jp709809b
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发表时间：
2008-04
期刊：
影响因子：
--
通讯作者：
S. S. Sinha-S.;R. K. Mitra;S. Pal
中科院分区：
文献类型：
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作者：
S. S. Sinha-S.;R. K. Mitra;S. Pal
人血清白蛋白(HSA)是循环系统中的一种可溶性蛋白质，可与多种药物和配体结合。自从苏德洛在配体结合位点上的开创性工作以来，生物物理/生化研究的主要目的是表征该蛋白质的结构、功能和动力学性质。对人血清白蛋白的结构研究揭示了不同的温度诱导折叠状态。尽管知道配体结合的性质和对结合很重要的残基，但对蛋白质的依赖于温度的分子识别了解较少。在这里，我们制备了热诱导蛋白质的未折叠状态，并用圆二色谱(CD)和差热分析(DTA)技术对其进行了表征。动态光散射(DLS)测量也表征了蛋白质热展开引起的球状结构的变化。我们使用了两种不同性质的荧光配体(4-(dicyanomethylene)-2-methyl-6-(p-dimethylaminostyryl)4H-吡喃(DCM；疏水；中性)和尼罗蓝(NB；阳离子)来表征蛋白质在自然和热未折叠状态下的配体结合特性。配体可能的结合部位与其他在人血清白蛋白中有明确结合部位的药物分子竞争结合。皮秒分辨Förster共振能量转移(FRET)研究以及蛋白质中配基的稳态和偏振门控光谱揭示了不同温度下结合位置的动态变化。通过FRET研究，试图表征HSA在不同温度依赖的折叠状态下两个配体的同时结合。
Human serum albumin (HSA) is a soluble protein in our circulatory system, which is known to bind a variety of drugs and ligands. Since Sudlow's pioneering works on the ligand-binding sites, a major effort of the biophysical/biochemical research has been directed to characterize the structural, functional, and dynamical properties of this protein. Structural studies on HSA have revealed distinct temperature-induced folded states. Despite knowing about the ligand-binding properties and residues important for the binding, less is understood about the temperature-dependent molecular recognition of the protein. Here, we have prepared thermally induced unfolded states of the protein and characterized those by circular dichroism (CD) and differential thermal analysis (DTA) techniques. The change in the globular structure of the protein as a consequence of thermal unfolding has also been characterized by dynamic light scattering (DLS) measurements. We have used two fluorescent ligands (4-(dicyanomethylene)-2-methyl-6-(p-dimethylaminostyryl) 4H-pyran) (DCM; hydrophobic; neutral) and Nile blue (NB; cationic) of different natures to characterize the ligand-binding properties of the protein in the native and thermally unfolded states. The possible binding sites of the ligands have been characterized by competitive binding with other drug molecules having definite binding sites in HSA. Picosecond-resolved Förster resonance energy transfer (FRET) studies along with steady-state and polarization-gated spectroscopies on the ligands in the protein reveal the dynamics of the binding sites at various temperatures. From the FRET studies, an attempt has been made to characterize the simultaneous binding of the two ligands in various temperature-dependent folded states of HSA.