SGER:Developing New Methods Based on Folding and Unfolding Pathways

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Abstract Proposal Title: SGER Developing New Methods Based on Folding and Unfolding Pathways. Proposal Number: CTS-0621216 Principal Investigator: Anubhav Tripathi, Institution: Brown University Intellectual Merit: High resolution separation methodologies aim to resolve complex mixtures of macromolecules into individual components for subsequent identification of macromolecular expression patterns and, on a broader scale, gain a fundamental understanding of biological system regulation. Common separation strategies are: (1) electrophoresis in which macromolecules are separated based on electrophoretic mobility and isoelectric point, and (2) chromatography in which the macromolecules are separated based on mobility, hydrophobicity and/or affinity. Since these strategies use either fully folded or fully denatured macromolecules, it is extremely difficult to resolve macromolecules with similar mobilities, isoelectric points and/or hydrophobicities. Proteins and RNAs are two examples of macromolecules which adopt different folded-unfolded structures based on the quality of aqueous environment which can disrupt the hydrophobic interactions or alter the net charge on the molecule causing electrostatic repulsion or disruption of some hydrogen bonds. A new research direction in separation and purification science, in which macromolecules such as proteins and RNAs can be targeted to adopt different folding-unfolding pathways under specific buffer conditions, is needed. Under these conditions, two molecules can be separated based on the "enhanced" difference in their mobility or adsorption/desorption characteristics. The investigator has recently demonstrated that two isoforms of lactoglobulin possess conformationally different structures under certain buffer conditions. In this SGER proposal, the PI has requested funding to perform preliminary experiments to demonstrate that under specific buffer conditions, due to differences in conformations, the two isoforms have different mobilities and different adsorption isotherms. Furthermore, differences in mobilities and adsorption behavior can be used to separate the two isoforms using (a) electrophoresis and (b) adsorption on a packed bed of spheres in microchip geometry. The results from this SGER will validate the claim that the changes in protein properties that accompany partial unfolding might be utilized for separation. If successful, the PI anticipates that this SGER proposal will lead to a more detailed CAREER proposal where the intimate details of protein folding and unfolding pathways will be modeled and characterized within a carefully crafted experimental and teaching plan. The broader impact of research is the quantification of macromolecules with similar mobilities that has become important in light of their central role in pathological states of mutations, improper expression or function (e.g. prion and various cancers). In the future, a research program in this area might offer a quality control process to test purity and stability of recombinant human proteins. The program also might offer a screening method for the pharmaceutical industry where binding ability of thousands of ligand molecules can be tested to inhibit the activity of a relevant target enzyme or protein. The fundamental experiments to be performed in this SGER will explore these issues and will provide several host new topics for interdisciplinary research. The educational program couples core skills of thermodynamics, kinetics and transport phenomenon to separation and purification science.