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.
The overarching goal of our research is to understand the chemistry of proteins as it occurs in living cells and in crowded conditions in vitro. The intracellular environment is vastly different from the simple buffered solutions used in most in vitro explorations of biomolecular structure and function. In cells, macromolecules reach concentrations of 300 g/L, occupying up to 30% of the cellular volume. Two types of interactions between macromolecules dominate the effects of this highly crowded environment: hard-core repulsions and weak chemical interactions. Hard-core repulsions reflect the impenetrable nature of atoms. These interactions reduce the conformational space available to proteins, thus favoring compact states. For many years hard-core repulsions were thought to be the most important component of crowding effects. Our most important accomplishment was showing the importance of weak, nonspecific attractive interactions in living cells and under physiologically relevant crowded conditions in vitro. Our first indication that these interactions could dominate hard-core repulsions came from our experiments on a protein whose stability had been deliberately compromised such that it was mostly unfolded in buffer. The protein remained unfolded in living cells. This behavior indicates the hard-core repulsions in the crowded cytoplasm could not overcome the individually weak, but collectively strong attractive interactions between the unfolded protein and the surrounding macromolecules. We then quantified this effect in several other small globular proteins. We showed that crowding by proteins can be destabilizing and showed that theories that purported to explain the effects of crowding were much too simple. These efforts culminated in a review about weak interactions. We also showed that weak attractive interactions affect protein diffusion and separated the effects of weak interactions from those of cytoplasmic viscosity in living cells.
