The high density of macromolecules makes the intracellular environment markedly different from bulk solution. It was recently shown that the high density, also described as macromolecular crowding, can be quantified with protein-based sensors inside cells. These sensors are based on Förster resonance energy transfer (FRET). In this thesis, I reveal the crowding sensing mechanism, showing that the sensors behave as polymers and their compression scales with their size and crowder concentration. In cells, compression also depends on the structure of the sensor linker region, which shows an important difference between experiments in buffer versus in the cell. I identify an artifact caused by slow maturation of the fluorescent proteins, and resolve this by sensor expression under a constitutive promoter. By tracking the crowding changes in Escherichia coli, we reveal how crowding changes in cells. Finally, we developed the first sensors to determine the ionic strength in living cells. These sensors for the crowding and ionic strength will allow us to deepen our understanding of the physicochemical aspects in the cell and serve as a stepping-stone to elucidate the role of physical chemical parameters on the biochemical organization and well-being of the living cell.
|Translated title of the contribution
|Kwantificering van macromoleculaire crowding en ionsterkte in levende cellen
|Doctor of Philosophy
|Place of Publication
|Published - 2018