Macromolecular crowding in vitro and in cells

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Cells contain a complex and crowded collection of macromolecules whose concentration can exceed 300 g/L. Thus, the most relevant environment for studying a biological macromolecule is a crowded one. Most of what is known about biological macromolecules, however, comes from solutions where the concentration of macromolecules is less than 10 g/L. Furthermore, many theories predict that crowding will have large affects on the properties of biological macromolecules. This project will investigate how crowding affects the physical properties of proteins. The goal of moving quantitative biophysics from simple solutions to crowded environments, including the inside of living cells, is a major challenge with important outcomes. The work will facilitate the training of undergraduate and graduate students in the practice of cutting-edge research. In addition, the knowledge gained will both add to the fundamental understanding of biology and inform efforts to produce designer enzymes and stabilize protein-based reagents. These efforts are key to building the US bioeconomy.The overarching objective of the proposed research is to understand the molecular biophysics of proteins in living cells and under crowded conditions in vitro. The principal investigator and his laboratory have developed nuclear magnetic resonance-based tools to make these measurements. The protein properties to be assessed include equilibrium thermodynamic stability and solvation. This work has already resulted in the discovery that chemical interactions between macromolecules play a much larger role in crowding than previously thought. The principal investigator and his student colleagues will now identify the source of these chemical interactions, examine the impact of the intracellular environment on disordered proteins and enhance the biological and biotechnological relevance of their efforts by studying the multidomain protein enzyme, dihydrofolate reductase.