| The cytoplasm is crowded, and the concentration of macromolecules can reach ∼ 300 g/L, an environment vastly different from the dilute, idealized conditions usually used in biophysical studies. Macromolecular crowding arise from two phenomena, excluded volume and nonspecific chemical interactions, until recently, only excluded volume effect has been considered. Theory predicts that this macromolecular crowding can have large effects. Most proteins, however, are studied outside cells in dilute solution with macromolecule concentrations of 10 g/L or less. In-cell NMR provides a means to assess protein biophysics at atomic resolution in living cells, but it remains in its infancy, and several potential challenges need to be addressed. One challenge is the inability to observe 15N-1H NMR spectra from many small globular proteins.;19F NMR was used to expand the application of in-cell NMR. This work suggests that high viscosity and weak interactions in the cytoplasm can make routine 15N enrichment a poor choice for in-cell NMR studies of globular proteins in Escherichia coli. To gain insight into this problem, I turned to in vitro experiments where conditions can be controlled with precision. Using both synthetic polymers and globular proteins, I studied the effects of crowding on the diffusion of the test protein, chymotrypsin inhibitor 2. The results not only pinpoint the source of the problem -- nonspecific chemical interactions -- but also suggest that proteins are more suitable mimics of the intracellular environment.;I also measured the stability of ubiquitin in solutions crowded with synthetic polymers or globular proteins to further elucidate the role of nonspecific chemical interactions under crowded conditions. The increased stability observed in synthetic crowders was consistent with a dominant entropic role for excluded volume, but the effect of protein crowders depended on charge. Protein-induced crowding increased stability when the sign of the net charge of the crowder was the same as that of ubiquitin, but decreased stability when the proteins were oppositely charged. The results indicate that synthetic polymers do not provide physiologically relevant insights and that the overall effect of macromolecular crowding depends on the winner of the near stalemate between excluded volume and nonspecific interactions. |
