Molecular Engineering of Multiple Weak Interactions for High Selectivity Bioseparations

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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.