Relating Protein Structure to Stability in the Solution and Adsorbed Phases

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PROPOSAL NUMBER: 0731055PRINCIPAL INVESTIGATOR: Erik FernandezINSTITUTION: University of VirginiaPROPOSAL TITLE: Relating Protein Structure to Stability in the Solution and Adsorbed PhasesThe goal of the project is to establish predictive relationships between the structural and molecular properties of proteins in solution and their unfolding behavior on hydrophobic interaction chromatography (HIC) surfaces. There are three elements of the research effort: experiment, computation, and modeling. The first aim is measuring batch adsorption, chromatography, and hydrogen-deuterium isotope exchange (HX) to establish the relevant equilibrium, kinetic, and structural aspects of a set of single and multidomain proteins on different adsorbent surfaces. Residue-level comparisons of solvent accessibilities in solutions and on surfaces will be made to reveal expected patterns of local stability propensities under the different environments. The second aim is to match these experimentally detected patterns with predicted local stabilities in solution computed by the COREX software based on statistical mechanical ensembles of protein conformation to confirm its capability for obtaining distributions of microstateenergies. The third aim is to formulate in ensemble terms a four-state modelthat has been successful in correlating static and dynamic stability behavior of such adsorbing systems, and use the experimental and simulated results to develop robust relationships for protein adsorption and stability. The intended outcome is to providetechniques for predicting when and how proteins unfold upon adsorption, includingelucidation of local regions of instability. The most direct and critical application of such a predictive capability is to therapeutic protein purification. In that context, removing misfolded, unfolded, and aggregated protein from therapeutic proteins is of growing importance. These predictive tools will ultimately enable process development engineers to minimize unfolding of native protein and maximize selectivity between folded and misfolded or unfolded proteins. Controlling the behavior of proteins on surfaces is central to drug delivery, biomaterials, biosensors, protein arrays, and nanoscale devices. In all of these situations, controlling the degree and rate of protein adsorption, the changes in protein conformation, and the appearance of aggregation are critical. Undergraduates recruited through the University of Virginia Center for Diversity in Engineering will participate in the research. A multidisciplinary collaboration with Prof. Vincent Hilser (U. Texas Medical Branch) will enable both graduate and undergraduate students to apply important biophysical and statistical mechanical approaches to problems of fundamental and practical importance. Collaborative interactions with supporting biopharmaceutical companies will provide relevant multidomain proteins for the work, disseminate the results to practitioners, and expose students to protein adsorption and stability issues in a commercially relevant context. Finally, a new module for biochemical engineering courses will be developed to incorporate aspects of this research, including computational and modeling activities. This module will be disseminated to other chemical engineering faculty via an electronic journal website being set up jointly with San Jose State University.