Protein behavior in living cells

The intracellular environment influences e.g. biomolecule aggregation, phase separation, transcription, translation, metabolic activity, and the structure of the cytoplasm due to its high crowding with biomacromolecules (concentrations ranging between 20-40 %w/w) and ions and small molecules (0.2-2 M) that are all enclosed in between lipid bilayers. Because these molecules interact continuously, intracellular conditions are thermodynamically far from ideal and the effect of crowding, surfaces and confinement depends on the concentration, dynamics, size, shape, and surface chemistry of the crowding molecules, which in turn depend on the growth condition and cell-type. To understand these physicochemical effects on the behaviour of a protein in a cell, we need to bridge overly simplified artificial systems and the complexity of the living cell.

To this end, complexity needs to increase in a stepwise manner from simpler model systems to living cells. This requires excellent probe proteins that provide a selective and understandable readout of the physicochemical parameters in both living cell and artificial systems. Then, observation of the same protein behaviour in living cells and artificial systems provides a better understanding. Thus, while macromolecular crowding is inherent to the living cell, predicting the effects and relevance of this excluded volume calls for physiologically relevant model systems.In this proposal, we will describe the behaviour of model proteins in living cells with model systems of stepwise increased complexity.We will construct genetically encoded probes as model proteins with different sensitivities towards biochemical organization, including a model protein that allows facile determination of the free energy change caused by crowding effects with high spatiotemporal resolution.

We will re-enact aspects of biochemical organization in buffer with increasing complexity and analyse its effects on model proteins. To this end, we present a platform that allows comparison of confinement and macromolecular crowding without changing the surface chemistry. Finally, we bridge in vitro and in vivo by reconstitution of cell lysates in combination with varying amount of crowder in artificial cells and comparison with living cells. With this, we determine which parameters in cell lysates determine behaviour of the probes, and which molecules allow bridging in vitro and in vivo behaviour. Together, we present a side-by-side comparison of stepwise increasingly complex in vitro crowding & confinement environments towards a living cell.