Protein Biophysics in Cells

University Of North Carolina At Chapel Hill, Chapel Hill NC

Investigators

Abstract

The inside of a cell is a crowded place where macromolecular solutes, mostly proteins, reach concentrations of hundreds of grams per liter. However, almost all in vitro studies of protein biophysics are conducted at solute concentrations that rarely exceed a fraction of a gram per liter. This discrepancy poses an important question: do the observations made in dilute solution always coincide with what happens inside cells? Preliminary data show that the answer is no. Specifically, NMR data show that FlgM, a protein that is unfolded in dilute solution, gains structure inside living Escherichia coli cells. Additional data show that FlgM gains structure in vitro on adding high concentrations (400 g/L) of glucose, bovine serum albumin, or ovalbumin. Given these data proving the biological significance of crowding, the objective of the research is to understand the effects of physiologically relevant crowded environments on protein equilibria, particularly direct studies in living cells. The proteins to be studied are FlgM, alpha -synuclein, and cytochrome c. Like FlgM, alpha -synuclein is unfolded in dilute solution. The first hypothesis is that there are two classes of natively unfolded proteins. Class 1 (FlgM) collapses and folds under physiologically relevant crowded conditions. Class 2 (alpha -synuclein) collapses but does not fold under these conditions. The second hypothesis is that macromolecular crowding stabilizes globular proteins in cells. The globular protein cytochrome c will be used to test this hypothesis. The methods to be used include, heteronuclear multidimensional NMR, especially NMR detected amide proton exchange experiments, circular dichroism spectropolarimetry, and directed mutagenesis.