Cell wall damage increases macromolecular crowding effects in the Escherichia coli cytoplasm.

The intracellular milieu is crowded with biomacromolecules. Macromolecular crowding changes the interactions, diffusion, and conformations of biomacromolecules. Changes in intracellular crowding have been mostly ascribed to differences in biomacromolecule concentration. However, spatial organization of these molecules should play a significant role in crowding effects. Here, we find that cell wall damage causes increased crowding effects in the Escherichia coli cytoplasm. Using a genetically encoded macromolecular crowding sensor, we see that crowding effects in spheroplasts and penicillin-treated cells well surpass crowding effects obtained using hyperosmotic stress. The crowding increase is not because of osmotic pressure, cell shape, or volume changes and therefore not crowder concentration. Instead, a genetically encoded nucleic acid stain and a DNA stain show cytoplasmic mixing and nucleoid expansion, which could cause these increased crowding effects. Our data demonstrate that cell wall damage alters the biochemical organization in the cytoplasm and induces significant conformational changes in a probe protein.

Engineering crowding sensitivity into protein linkers.

The intracellular environment contains a high concentration of biomacromolecules that present steric barriers and ample surface area for weak chemical interactions. Consequently, these forces influence protein conformations and protein self-assembly, with an outcome that depends on the sum of the effects resulting from crowding. Linkers are disordered domains that lack tertiary structure, and this flexible nature would render them susceptible to compression or extension under crowded conditions, compared to the equilibrium conformation in a dilute buffer. The change in distance between the linked proteins can become essential where it attenuates protein activity. In this chapter, we first discuss the experimental findings in vitro and in the cell on how linkers and other relevant macromolecules are affected by crowding. We focus on the dependence on the linker's size, flexibility, and the intra- and intermolecular interactions. Although the experimental data on the systematic variation of proteins in a buffer and cells is limited, extrapolating the available insights allows us to propose a protocol on how to engineer the directionality of crowding effects in the linker. Finally, we describe a straightforward experimental protocol on the determination of crowding sensitivity in a buffer and cell.