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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Evaluation of protein adsorption and preferred binding regions in multimodal chromatography using NMR
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Evaluation of protein adsorption and preferred binding regions in multimodal chromatography using NMR

机译：使用NMR评估多峰色谱中蛋白质的吸附和优选的结合区域

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摘要

NMR titration experiments with labeled human ubiquitin were employed in concert with chromatographic data obtained with a library of ubiquitin mutants to study the nature of protein adsorption in multimodal (MM) chromatography. The elution order of the mutants on the MM resin was significantly different from that obtained by ion-exchange chromatography. Further, the chromatographic results with the protein library indicated that mutations in a defined region induced greater changes in protein affinity to the solid support. Chemical shift mapping and determination of dissociation constants from NMR titration experiments with the MM ligand and isotopically enriched ubiquitin were used to determine and rank the relative binding affinities of interaction sites on the protein surface. The results with NMR confirmed that the protein possessed a distinct preferred binding region for the MM ligand in agreement with the chromatographic results. Finally, coarsegrained ligand docking simulations were employed to study the modes of interaction between the MM ligand and ubiquitin. The use of NMR titration experiments in concert with chromatographic data obtained with protein libraries represents a previously unde-scribed approach for elucidating the structural basis of protein binding affinity in MM chromatographic systems.
机译：结合标记的人泛素的NMR滴定实验与通过泛素突变体文库获得的色谱数据一起使用，以研究多峰（MM）色谱法中蛋白质吸附的性质。 MM树脂上突变体的洗脱顺序与通过离子交换层析获得的洗脱顺序显着不同。此外，蛋白质文库的色谱结果表明，定义区域中的突变诱导了蛋白质对固相支持体的更大变化。使用MM配体和同位素富集的泛素，通过NMR滴定实验的化学位移图谱和解离常数的测定，用于确定蛋白质表面相互作用位点的相对结合亲和力并对其进行排名。 NMR的结果证实了该蛋白质具有与色谱结果一致的MM配体的独特的优选结合区。最后，使用粗粒配体对接模拟来研究MM配体与泛素之间的相互作用模式。 NMR滴定实验与蛋白质文库获得的色谱数据结合使用，代表了以前未描述的方法，用于阐明MM色谱系统中蛋白质结合亲和力的结构基础。

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  • 作者

    Wai Keen Chung; Alexander S. Freed; Melissa A. Holstein; Scott A. McCallum; Steven M. Cramer;

  • 作者单位

    Isermann Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180 Department of Biology, Rensselaer Polytechnic Institute, Troy, NY 12180;

    rnIsermann Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180 Department of Biology, Rensselaer Polytechnic Institute, Troy, NY 12180;

    rnIsermann Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180 Department of Biology, Rensselaer Polytechnic Institute, Troy, NY 12180;

    rnDepartment of Biology, Rensselaer Polytechnic Institute, Troy, NY 12180 Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, Troy, NY 12180;

    rnIsermann Department of Chemical and Biological Engineering, Rensselaer Polytechnic Institute, Troy, NY 12180 Department of Biology, Rensselaer Polytechnic Institute, Troy, NY 12180 3211 Center for Biotechnology and Interdisciplinary Studies, Rensselaer Polytechnic Institute, 110 8th Street, Troy, NY 12180;

  • 收录信息 美国《科学引文索引》(SCI);美国《生物学医学文摘》(MEDLINE);美国《化学文摘》(CA);
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

    ligand binding site; mixed mode chromatography; protein-ligand interactions; binding site mapping; pseudoaffinity;

    机译：配体结合位点;混合模式色谱蛋白质-配体相互作用;结合位点图;伪亲和力;

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