Adsorption of protein onto dye-ligand affinity adsorbent: Molecular dynamics simulation and experimental study

亲和色谱法是一种基于高度特异性分子识别的鉴定、纯化和分离大分子的成熟方法。染料配体亲和层析法已广泛应用于蛋白质纯化。固定化纺织三嗪染料，特别是Cibacron蓝3GA (CB)，长期以来被用作亲和层析纯化蛋白质的工具。CB能够结合多种蛋白质。然而，它与大量看似不相关的蛋白质相互作用，不可避免地降低了其蛋白质结合特异性，使这些分子具有严重的缺陷。为了提高纺织染料在蛋白质分离中的应用，了解染料配体与蛋白质的结合及其特异性机制是非常重要的。在本项目中，我们将进一步研究我们之前研究过的血清白蛋白和血红蛋白作为模型蛋白，研究它们在染料配体亲和层析介质上的吸附。由于缺乏可以检测吸附剂孔隙内动态吸附过程的实验方法，本研究将首次使用粗粒度和全原子模型的分子动力学（MD）模拟来研究分子尺度上染料配体亲和层析对蛋白质的吸附。在MD模拟结果和前人实验研究的基础上，根据MD模拟得到的结合机理，在合理设计的条件下，对这些蛋白质在染料配体亲和吸附剂上进行单吸附和双吸附实验。将分子动力学模拟与吸附实验相结合，从微观和宏观的角度全面了解亲和吸附的结合和具体机理。这一认识为新的亲和性染料配体的设计和筛选，以及蛋白质染料配体亲和层析的设计、优化和控制提供了深刻的基础。

Affinity chromatography is a well-established method for the identification, purification, and separation of macromolecules based on highly specific molecular recognition. Dye ligand affinity chromatography has been widely utilized for protein purification. Immobilized textile triazine dyes, particularly Cibacron blue 3GA (CB) has been used as affinity chromatography tools for protein purification for a long time. CB is able to bind various proteins. However, its interaction with a large number of seemingly unrelated proteins inevitably compromises its protein binding specificity and endows these molecules with a serious drawback. For an improved application of textile dyes in the separation of proteins, it is very important to understand the binding and specific mechanisms between dye ligand and proteins. In this project, serum albumin and hemoglobin which have been investigated in our previous works will be further studied as model proteins to investigate their adsorption onto dye ligand (CB) affinity chromatographic media. Due to the lack of experimental method that can detect the dynamic adsorption process within adsorbent pores, molecular dynamics (MD) simulations with both coarse grained and all atom models will be used for the first time in this study to investigate the dye ligand affinity chromatographic adsorption of proteins on a molecular scale. On the basis of the results of MD simulations and the former experimental research, both single and binary adsorption experiments of these proteins onto dye ligand affinity adsorbent will be performed under conditions which are rationally designed according to the binding mechanism learned from the MD simulations. A combination of MD simulations and adsorption experiments will give a comprehensive understanding of the binding and specific mechanisms of the affinity adsorption from both microscopic and macroscopic point of view. This understanding is a profound basis for the design and screening of new affinity dye ligands, as well as the design, optimization and control of the dye ligand affinity chromatography of proteins.