Collaborative Research: The Thermodynamics of Protein Separations

Johns Hopkins University, Baltimore MD

Investigators

Abstract

ABSTRACT CTS-0078491 Michael Paulaitis Johns Hopkins U. The objective of the proposed research is to develop quantitative tools for the analysis and design of protein separation processes. These tools will be compared to those now used routinely to design conventional separations in the chemical process industries. This development is based on combining theory and experiment to enable a new generation of thermodynamic models capable of describing mixtures of proteins. The approach builds on the paradigm that has so far emerged from our work, namely that protein thermodynamic properties are dominated by specific biological interactions in the form of geometric complementarity. It is believed that these specific interactions can be understood and accounted for in terms of electrostatic, dispersion, and hydrophobic interactions. This proposal is centered on extending our understanding of these interactions and finding optimal ways in which to manipulate and exploit them in designing effective separation strategies. Specifically, we will: 1. complete our, model formulation to allow efficient mechanistic calculation of protein-protein interactions for both like and unlike protein pairs; 2. measure the model interactions between like and unlike protein pairs, and correlate results with measure separation performance. 3. use interactions between unlike protein pairs as the basis for developing strategies for optimizing protein separation trains. The molecular theories will be based on recently developed computational methods to account for interactions, and will be combined into an accurate potential of mean force (PMF) describing the interactions between both like and unlike proteins. This PMF is the foundation for the foundation for the description of both the physical properties of the protein solution thermodynamic function that determine the phase behavior. Several computational and molecular simulation methods will be explored for calculating of the phase behavior of the protein solutions. Our working hypothesis is that is the relative magnitudes of like vs, unlike protein interactions that determine how a particular separation can best be accomplished. An array of experimental measurements will complement and guide theory. The experiments first involve characterization of structural and functional protein properties using light scattering and small angle neutron scattering (SANS). Next, measurements of thermodynamic and physical properties and mixtures of proteins will be made to test the validity of the thermodynamic descriptions. One thermodynamic variable of particular interest will be pressure. Specifically the investigator will investigate the effect of pressure on protein stability and how protein folding can be manipulated with pressure to control protein separations and purification. Finally, separation experiments will be conducted to evaluate the predictions of performance based on the thermodynamic models. These measurements and calculations will guide efforts to find conditions under which adverse interactions impair separation effectiveness, and to design and test suitable remedies. Several proteins and protein mixtures will be examined, including eglin C and staphylococcal nuclease. This combination of experimental and theoretical tools will identify the properties of proteins important in the design of separation processes, and will allow separation performance to be predicted on the basis of protein properties. The project is part of a group proposal with investigators at the University of Delaware (0078844).