Macromolecular Crowding and Protein Stability In Vitro and in Cells

University Of North Carolina At Chapel Hill, Chapel Hill NC

Investigators

Abstract

Intellectual Merit The overall goal is to understand the effects of macromolecular crowding on protein chemistry. The focus is on equilibrium protein stability. Experiments will be performed both in vitro and in cells. Nuclear resonance spectroscopy will be used to assess both crowding induced changes in stability and protein-crowder interactions. Two unresolved questions will be addressed. Question 1: Which interactions between the crowder and the test protein determine the effects of crowding on stability? Crowding effects can be divided into two parts: volume exclusion and chemical interactions and their relative importance is the most critical unresolved question in crowding. Question 2: Which crowding agents provide the most physiologically relevant information? Synthetic polymers, like Poly Vinyl Pyrrolidone (PVP), Ficoll, Poly Ethylene Glycol (PEG), and Dextran, are often used in crowding studies, but their biological relevance has to be assessed. In an effort to address these questions three hypotheses will be tested. The first hypothesis involves synthetic polymers as crowding agents. It is now known that protein stability can be assessed in solutions crowded with PVP. The research project will explore other commonly used crowders: Ficoll, Dextran, and PEG. The hypothesis is that PVP, Ficoll, and Dextran work mostly by volume exclusion, and chemical interactions will be of minor importance. The results have implications for stabilizing proteins important to the chemical and pharmaceutical industries. The second hypothesis involves protein crowders. The hypothesis is that destabilizing non-specific chemical interactions between protein crowders and the test protein have been underestimated. The prediction is that crowding by proteins can lead to destabilization. The results have implications for understanding cellular processes. The third hypothesis focuses on the ultimate biological relevance. The hypothesis is that in the cytoplasm of Escherichia coli, the stabilizing excluded volume effect is offset by non-specific chemical interactions. Broader Impacts Many efforts to increase scientific diversity are designed to identify and attract individual high school students. The present award will be used to leverage these efforts by invigorating science teachers from schools with diverse student bodies and low college attendance. The plan is to bring one such North Carolina high school science teacher to the lab for seven weeks each summer. The mission of the National College Advising Corps, located at UNC, is to place recent college graduates in high schools with low college attendance in an attempt to reduce barriers to college access. The Corps interacts directly with the schools by visiting them. This resource will be used to identify the teachers, who would be encouraged to apply. The teacher will work within the research group on the project. The Principal Investigator will meet at least once a week with the teacher to develop a lesson plan to introduce his or her students to research. The PI will also set up meetings with professionals from the biotech and pharmaceutical industries. As an example, leaders from local biotech firms in Research Triangle Park could address the path a drug takes from the lab to the patient, and a representative from the UNC Office of Technology Development could address intellectual property issues.