Morrison Christopher J, Godawat Rahul, McCallum Scott A, Garde Shekhar, Cramer Steven M
Department of Chemical and Biological Engineering, Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, 110 8th Street, Troy, New York 12180, USA.
Biotechnol Bioeng. 2009 Apr 1;102(5):1428-37. doi: 10.1002/bit.22170.
A parallel batch screening technique was employed to identify chemically selective displacers which exhibited exclusive separation behavior for the protein pair alpha-chymotrypsin/ribonuclease A on a strong cation exchange resin. Two selective displacers, 1-(4-chlorobenzyl)piperidin-3-aminesulfate and N'1'-(4-methyl-quinolin-2-yl)-ethane-1,2-diamine dinitrate, and one non-selective displacer, spermidine, were selected as model systems to investigate the mechanism of chemically selective displacement chromatography. Saturation transfer difference (STD) NMR was used to directly evaluate displacer-protein binding. The results indicated that while binding occurred between the two chemically selective displacers and the more hydrophobic protein, alpha-chymotrypsin, no binding was observed with ribonuclease A. Further, the non-selective displacer, spermidine, was not observed to bind to either protein. Importantly, the binding event was observed to occur primarily on the aromatic portion of the selective displacers. Extensive molecular dynamic simulations of protein-displacer-water solution were also carried out. The MD results corroborated the NMR findings demonstrating that the binding of selective displacers occurred primarily on hydrophobic surface patches of alpha-chymotrypsin, while no significant long term binding to ribonuclease A was observed. The non-selective displacer did not show significant binding to either of the proteins. MD simulations also indicated that the charged amine group of the selective displacers in the bound state was primarily oriented towards the solvent, potentially facilitating their interaction with a resin surface. These results directly confirm that selective binding between a protein and displacer is the mechanism by which chemically selective displacement occurs. This opens up many possibilities for future molecular design of selective displacers for a range of applications.
采用平行批量筛选技术来鉴定化学选择性置换剂，这些置换剂在强阳离子交换树脂上对α-胰凝乳蛋白酶/核糖核酸酶A蛋白对表现出独特的分离行为。选择两种选择性置换剂1-(4-氯苄基)哌啶-3-胺硫酸盐和N'1'-(4-甲基喹啉-2-基)乙烷-1,2-二胺二硝酸盐，以及一种非选择性置换剂亚精胺作为模型系统，以研究化学选择性置换色谱的机制。利用饱和转移差(STD)核磁共振直接评估置换剂与蛋白质的结合。结果表明，虽然两种化学选择性置换剂与疏水性更强的蛋白质α-胰凝乳蛋白酶之间发生了结合，但未观察到与核糖核酸酶A的结合。此外，未观察到非选择性置换剂亚精胺与任何一种蛋白质结合。重要的是，观察到结合事件主要发生在选择性置换剂的芳香部分。还对蛋白质-置换剂-水溶液进行了广泛的分子动力学模拟。分子动力学结果证实了核磁共振结果，表明选择性置换剂的结合主要发生在α-胰凝乳蛋白酶的疏水表面斑块上，而未观察到与核糖核酸酶A的显著长期结合。非选择性置换剂未显示与任何一种蛋白质有显著结合。分子动力学模拟还表明，处于结合状态的选择性置换剂的带电胺基团主要朝向溶剂，这可能促进它们与树脂表面的相互作用。这些结果直接证实了蛋白质与置换剂之间的选择性结合是化学选择性置换发生的机制。这为未来一系列应用中选择性置换剂的分子设计开辟了许多可能性。
