Molecular Engineering of Multiple Weak Interactions for High Selectivity Bioseparations

1160039这项工作将利用几个最先进的新型配体和材料的文库与蛋白质文库相结合，详细研究在FPM存在下MM配体-蛋白质的相互作用。将使用几个蛋白质文库进行高通量批处理和柱式实验，以根据配体和修饰剂上存在的不同相互作用部分筛选选择性趋势。然后，将使用核磁共振和分子动力学模拟来研究具有独特选择性的蛋白质对和MM配体/FPM组合。将对标记蛋白质和MM配体进行溶液核磁共振滴定实验，以确定在FPM存在下蛋白质上结合动力学、结合亲和力和配体结合位置的变化。氢-氚交换实验和SS-核磁共振将与树脂结合的蛋白质一起进行，以检查固相系统中蛋白质-配体结合的相互作用。将在日益复杂的系统上进行广泛的MD模拟，以开发一个新的框架，用于理解、操纵和预测存在FPM的MM系统中的多个弱相互作用。这将通过以下方式实现：(I)使用模型溶质开发FPM对基本水介导相互作用的影响的数据库；(Ii)确定MM配体-蛋白质相互作用的关键贡献者及其协同作用；(Iii)开发工具来预测FPM对蛋白质-配体MM相互作用的影响，并进行分子动力学模拟，以深入了解配体灵活性、空间效应和亲和力效应的重要性。然后，通过实验和理论研究建立的知识库将用于制定设计MM分离器的流程，并将使用FAB变体对流程进行验证。最后，精制过程将被应用于设计高效的分离条件，用于由我们的工业合作者提供的复杂工业混合物的纯化。拟议的项目将使我们从根本上了解MM配体在溶液和固体体系中与蛋白质结合的性质，这将对MM层析技术的开发和实施产生重大影响，用于下游纯化。这项工作将使下一代MM色谱分析系统的设计成为可能，这将对从大规模生物制药到复杂生物分析应用的分离具有重要意义。由于弱相互作用的平衡是凝聚型高介电介质的标准，也不是特例，本提案中描述的系统研究有可能提供对这一重要问题的新理解，这将很容易转化为许多相关的生物学(分子识别、结合、聚集)、生物加工(配方)和生物医学系统(骨再生、生物材料设计)。这项拟议的研究也将对化学工程研究生和本科生的教育产生重要影响。克雷默实验室在培养化学工程师方面有着长期的记录，他们在色谱生物处理方面接受过一流的培训。该培训将继续对参与该项目的研究生和本科生进行。拟议的研究将在生物技术和跨学科研究中心进行，该中心为研究生和本科生提供绝佳的多学科培训机会。学生将接触到细胞培养、溶液核磁共振波谱、层析和分子模拟。该项目开发的研究还将被纳入最近开发的PI的化学工程高级实验室层析实验以及一门关于色谱分离过程的课程。最后，在这个项目中进行的模拟，以及分子相互作用的概念部分，将激发分子库项目的新方面，该项目使用动画电影来教授和启发各级学生关于迷人的分子世界。

1160039CramerThis work will employ several state of the art libraries of novel ligands and materials in concert with protein libraries in a detailed study of MM ligand--protein interactions in the presence of FPMs. High throughput batch and column experiments will be performed using several protein libraries to screen for selectivity trends based on the different interaction moieties present on the ligands and modifiers. Protein pairs and MM ligand/FPM combinations exhibiting unique selectivities will then be investigated using NMR and MD simulations. Solution NMR titration experiments will be carried out with labeled proteins and MM ligands to determine changes in binding kinetics, association affinities, and ligand binding sites on the proteins in the presence of FPMs. Hyrodgen-deuterium exchange experiments and SS-NMR will be performed with resin bound proteins to examine protein-ligand binding interactions in solid phase systems. Extensive MD simulations will be performed on increasingly complex systems to develop a new framework for the understanding, manipulation, and prediction of multiple weak interactions in MM systems in the presence of FPMs. This will be achieved by (i) developing a database of the effects of FPMs on the fundamental water-mediated interactions using model solutes; (ii) identifying the key contributors to MM ligand-protein interactions and their synergies; (iii) developing tools to predict the effects of FPMs on protein-ligand MM interactions and carrying out MD simulations to provide insights into the importance of ligand flexibility, steric effects, and avidity effects. The knowledge base created by the experimental and theoretical studies will then be used to formulate a process for designing MM separations and validation of process will be carried out using Fab variants. Finally, the refined process will be applied to engineer efficient separation conditions for the purification of complex industrial mixtures provided by our industrial collaborators The proposed project will provide a fundamental understanding of the nature of MM ligand binding to proteins in both solution and solid systems in the presence of FPMs which will have a significant impact on the development and implementation of MM chromatographic technology for downstream purification. This work will enable the design of next generation MM chromatographic systems which will have important implications for separations ranging from large scale biopharmaceuticals to complex bioanalytical applications. Since the balance of weak interactions is the norm and not the exception in condensed high dielectric media, the systematic studies described in this proposal have the potential to provide new understanding of this important problem which will be readily translated to many related biological (molecular recognition, binding, aggregation), bioprocessing (formulation) and biomedical systems (bone regeneration, biomaterial design). The proposed research will also have an important impact on the education of both graduate and undergraduate chemical engineering students. The Cramer laboratory has a long track record of producing chemical engineers with a first rate training in chromatographic bioprocessing. This training will continue with the graduate students and undergraduates involved in this project. The proposed research will be carried out in the Center for Biotechnology and Interdisciplinary Studies which provides excellent multidisciplinary training opportunities for graduate and undergraduate students. Students will gain exposure to cell culture, solution NMR spectroscopy, chromatography and molecular simulations. The research developed in this project will also be incorporated into a chemical engineering senior laboratory chromatography experiment recently developed the PI as well as a course on Chromatographic Separation Processes. Finally, simulations performed in this project, as well as conceptual parts of molecular interactions will motivate new aspects of the Molecularium project, which uses animation movies to teach and inspire students at all levels about the fascinating world of molecules.