基于氢氘交换核磁共振技术研究蛋白质与阳离子交换介质的相互作用

Interactions between proteins and cation exchange adsorbents analyzed by NMR and hydrogen/deuterium exchange technique

摘要:

原位实时捕捉多孔介质内的蛋白结构变化信息是蛋白质层析失活机理研究中的难点。为此，本文发展了蛋白质氢氘交换与核磁共振 (Nuclear magnetic resonance, NMR) 相结合的新型蛋白质液固界面表征路线。研究了溶菌酶在溶液态以及在阳离子交换介质内部吸附态时的去折叠行为，并揭示了蛋白与阳离子交换介质 (SP Sepharose FF) 相互作用机理。溶液态溶菌酶的一维核磁共振氢谱动力学显示蛋白质去折叠可导致残基暴露进而加快氢氘交换速率。吸附态溶菌酶的二维氢-氢全相关谱图 (Total correlation spectroscopy，TOCSY) 以及残基峰强度显示，溶菌酶在吸附态时的去折叠呈区域性，无规则卷曲 (Coil，bend，and turn) 片段的酰胺氢信号更容易失去，而二级结构域 (α-helix，β-sheet) 对酰胺氢信号保护更好。最终，利用蛋白表面静电势模拟计算结合氢氘标记的蛋白核核磁数据可确定出溶菌酶与阳离子交换介质的作用位点。这对于深刻理解层析过程中蛋白与层析介质微观作用机理以及层析过程中吸附剂的选择、设计具有重要意义，也为获取蛋白质与生物材料之间相互作用研究提供新的有效工具。

Abstract:

In silico acquirement of the accurate residue details of protein on chromatographic media is a bottleneck in protein chromatography separation and purification. Here we developed a novel approach by coupling with H/D exchange and nuclear magnetic resonance to observe hen egg white lysozyme (HEWL) unfolding behavior adsorbed on cation exchange media (SP Sepharose FF). Analysis of 1D 1H NMR shows that protein unfolding accelerated H/D exchange rate, leading to more loss of signal of amide hydrogen owing to exposure of residues and the more unfolding of protein. Analysis of two-dimensional hydrogen-hydrogen total correlation spectroscopy shows that lysozyme lost more signals and experienced great unfolding during its adsorption on media surface. However, for several distinct fragments, the protection degrees varied, the adsorbed lysozyme lost more signal intensity and was less protected at disorder structures (coil, bend, and turn), but was comparatively more protected against exchange at secondary structure domains (α-helix, β-sheet). Finally, the binding site was determined by electrostatic calculations using computer simulation methods in conjunction with hydrogen deuterium labeled protein and NMR. This study would help deeply understand the microscopic mechanism of protein chromatography and guide the purposely design of chromatographic process and media. Moreover, it also provide an effective tool to study the protein and biomaterials interaction in other applications.