Department of Chemical, Biological & Macromolecular Sciences, S.N. Bose National Centre for Basic Sciences, Block JD, Sector III, Salt Lake, Kolkata 700 098, India.
Biochimie. 2013 Jun;95(6):1127-35. doi: 10.1016/j.biochi.2012.12.018. Epub 2013 Jan 31.
Water molecules in hydrophobic biological cleft/cavities are of contemporary interest for the biomolecular structure and molecular recognition of hydrophobic ligands/drugs. Here, we have explored picosecond-resolved solvation dynamics of water molecules and associated polar amino acids in the hydrophobic cleft around Cys-34 position of Endogenous Serum Albumin (ESA). While site selective acrylodan labeling to Cys-34 allows us to probe solvation in the cleft, Förster resonance energy transfer (FRET) from intrinsic fluorescent amino acid Trp 214 to the extrinsic acrylodan probes structural integrity of the protein in our experimental condition. Temperature dependent solvation in the cleft clearly shows that the dynamics follows Arrhenius type behavior up to 60 °C, after which a major structural perturbation of the protein is evident. We have also monitored polarization gated dynamics of the acrylodan probe and FRET from Trp 214 to acrylodan at various temperatures. The dynamical behavior of the immediate environments around the probe acrylodan in the cleft has been compared with a model biomimetic cavity of a reverse micelle (w0 = 5). Using same fluorescent probe of acrylodan, we have checked the structural integrity of the model cavity at various temperatures using picosecond-resolved FRET from Trp to acrylodan in the cavity. We have also estimated possible distribution of donor-acceptor distances in the protein and reverse micelles. Our studies reveal that the energetics of the water molecules in the biological cleft is comparable to that in the model cavity indicating a transition from bound state to quasibound state, closely consistent with a recent MD simulation study.
水分子在疏水生物裂隙/腔中是当前研究疏水配体/药物生物分子结构和分子识别的热点。在这里，我们研究了内源性血清白蛋白（ESA）Cys-34 位置周围疏水裂隙中水分子和相关极性氨基酸的皮秒分辨溶剂化动力学。虽然选择性丙烯酰胺标记到 Cys-34 可以探测裂隙中的溶剂化，但荧光氨基酸色氨酸 214 到外源性丙烯酰胺探针的Förster 共振能量转移（FRET）在我们的实验条件下保持蛋白质的结构完整性。依赖于温度的裂隙溶剂化清楚地表明，动力学遵循 Arrhenius 类型行为，直到 60°C，之后蛋白质的主要结构扰动是明显的。我们还监测了各种温度下丙烯酰胺探针的极化门控动力学和色氨酸 214 到丙烯酰胺的 FRET。裂隙中探针丙烯酰胺周围的瞬时环境的动力学行为与反向胶束（w0 = 5）的模型仿生腔进行了比较。使用相同的丙烯酰胺荧光探针，我们使用皮秒分辨 FRET 从腔中的色氨酸到丙烯酰胺检查了各种温度下模型腔的结构完整性。我们还估计了蛋白质和反向胶束中供体-受体距离的可能分布。我们的研究表明，生物裂隙中水分子的能量与模型腔中的能量相当，表明从束缚态到准束缚态的转变，与最近的 MD 模拟研究密切一致。
