Advanced R&D Center, Horiba, Ltd. , 2 Miyanohigashi, Kisshoin, Minami-ku, Kyoto 601-8510, Japan.
Department of Biomolecular Chemistry, Kyoto Prefectural University , Sakyo-ku, Kyoto 606-8522, Japan.
Langmuir. 2016 Jul 26;32(29):7372-82. doi: 10.1021/acs.langmuir.6b01228. Epub 2016 Jul 14.
The behavior of proteins in crowded environments is dominated by protein-crowder interactions (the entropic/excluded volume effect) and protein-protein interactions (the soft chemical effect). The details of these interactions, however, are not fully understood. In this study, the behavior of bovine serum albumin (BSA) in crowded environments, including high protein concentrations and in the presence of another protein, was investigated by Raman spectroscopy. A detailed analysis of the water, Tyr, and Phe Raman bands revealed that the excluded volume effect with an increase in the protein concentration changed the local environment of hydrophobic residues. In contrast, no specific changes to the secondary structure were observed from the analysis of the concentration dependence of the amide I band. BSA was experimentally shown to adopt a more compact state in the presence of the crowding agent. Moreover, H-D exchange experiments of the amide I band revealed that the intramolecular hydrogen bonds of BSA were strengthened in the presence of the protein crowder. Thus, the Raman spectroscopy results have revealed the molecular behavior of proteins in crowded environments by extracting information about the excluded volume effect, soft chemical interactions, and the hydration effect.
蛋白质在拥挤环境中的行为主要由蛋白质-拥挤剂相互作用（熵/排除体积效应）和蛋白质-蛋白质相互作用（软化学效应）决定。然而，这些相互作用的细节尚不完全清楚。在这项研究中，通过拉曼光谱研究了牛血清白蛋白（BSA）在拥挤环境中的行为，包括高蛋白质浓度和存在另一种蛋白质的情况。对水、Tyr 和 Phe 拉曼带的详细分析表明，随着蛋白质浓度的增加，排除体积效应改变了疏水性残基的局部环境。相比之下，从酰胺 I 带的浓度依赖性分析中观察到二级结构没有特定变化。实验表明，在拥挤剂存在的情况下，BSA 会采取更紧凑的状态。此外，酰胺 I 带的 H-D 交换实验表明，在蛋白质拥挤剂存在的情况下，BSA 的分子内氢键增强。因此，拉曼光谱结果通过提取关于排除体积效应、软化学相互作用和水合作用的信息，揭示了蛋白质在拥挤环境中的分子行为。
