Proteins are crucial for all cellular life. Every signal received by a cell, and every response to it, is mediated by proteins. Inside cells, these proteins diffusively sample each other’s surfaces, as they travel through the cytoplasm in search of their specific interaction partners. In order to carry out their function, proteins need to navigate this net negatively charged and highly crowded milieu without getting stuck with undesirable partners. How do they achieve this?

Previously published data has shown that the bacterial cytoplasm is governed by physicochemical restrictions: There is a net charge interval within which proteins remain soluble. Meaning, if a protein is too positively charged, it will get stuck to the surrounding molecules. If it is too negatively charged, the intracellular mobility approaches that in water, potentially reducing the chance of the protein finding its functional partner. Using in-cell NMR, we have shown that similar charge-based rules govern the molecular mobility inside human cells. The less crowded human cytoplasm does, however, seem more forgiving than the bacterial counterpart, as proteins that experience restricted mobility inside bacteria seem to move freely inside human cells.

The human and bacterial cytoplasm both have a destabilising effect on the ALS-associated ROS scavenger Superoxide Dismutase 1 (SOD1). Our results show that: Stabilised by electrostatic interactions between the positively charged N-terminal and the negatively charged contents of the cytoplasm, the folding equilibrium is shifted towards the unfolded state. Additionally, in the absence of metals, native metal-coordinating surface-exposed histidine residues also contribute to the intracellular destabilisation of SOD1.

Finally, the unfolded state of SOD1 has been characterised in the absence of chemical denaturants. We show that the unfolded state is more compact than previously anticipated. We hypothesise that the increased compactness is caused by the pre-formation of long-range native-like contacts. This implies that: Not only does the primary structure contain the information required for folding, it also contains information on how the unfolded state needs to organise itself to increase the probability of successful folding.