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.

The structural stability of proteins is found to markedly change upon their transfer to the crowded interior of live cells. For some proteins, the stability increases, while for others, it decreases, depending on both the sequence composition and the type of host cell. The mechanism seems to be linked to the strength and conformational bias of the diffusive in-cell interactions, where protein charge is found to play a decisive role. Because most proteins, nucleotides, and membranes carry a net-negative charge, the intracellular environment behaves like a polyanionic (Z:1) system with electrostatic interactions different from those of standard 1:1 ion solutes. To determine how such polyanion conditions influence protein stability, we use negatively charged polyacetate ions to mimic the net-negatively charged cellular environment. The results show that, per Na⁺ equivalent, polyacetate destabilizes the model protein SOD1^barrel significantly more than monoacetate or NaCl. At an equivalent of 100 mM Na⁺, the polyacetate destabilization of SOD1^barrel is similar to that observed in live cells. By the combined use of equilibrium thermal denaturation, folding kinetics, and high-resolution nuclear magnetic resonance, this destabilization is primarily assigned to preferential interaction between polyacetate and the globally unfolded protein. This interaction is relatively weak and involves mainly the outermost N-terminal region of unfolded SOD1^barrel. Our findings point thus to a generic influence of polyanions on protein stability, which adds to the sequence-specific contributions and needs to be considered in the evaluation of in vivo data.

Random encounters between proteins in crowded cells are by no means passive, but found to be under selective control. This control enables proteome solubility, helps to optimise the diffusive search for interaction partners, and allows for adaptation to environmental extremes. Interestingly, the residues that modulate the encounters act mesoscopically through protein surface hydrophobicity and net charge, meaning that their detailed signatures vary across organisms with different intracellular constraints. To examine such variations, we use in-cell NMR relaxation to compare the diffusive behaviour of bacterial and human proteins in both human and Escherichia coli cytosols. We find that proteins that ‘stick’ in E. coli are generally less restricted in mammalian cells. Furthermore, the rotational diffusion in the mammalian cytosol is less sensitive to surface-charge mutations. This implies that, in terms of protein motions, the mammalian cytosol is more forgiving to surface alterations than E. coli cells. The cellular differences seem not linked to the proteome properties per se, but rather to a 6-fold difference in protein concentrations. Our results outline a scenario in which the tolerant cytosol of mammalian cells, found in long-lived multicellular organisms, provides an enlarged evolutionary playground, where random protein-surface mutations are less deleterious than in short-generational bacteria.

The unfolded state of a globular protein in a physiologically relevant environment is by no means an inert random coil.  On the contrary, its structural and dynamic properties are crucial for e.g., protein folding and aggregation.  Despite its importance, it has been studied relatively sparsely, which is partly due to its low population which tend to obstruct detailed biophysical characterization.  Here, introduction of two destabilizing core mutations allow us to study the unfolded state of the central b-barrel of Superoxide Dismutase 1 under native conditions.  

In order to structurally characterise the unfolded state, we use high-resolution nuclear magnetic resonance (NMR), including paramagnetic relaxation enhancement, to obtain constraints for the generation of unfolded ensembles.  The results show that the unfolded state is more compact than the chemically denatured state of the same protein.  This compacted state seems to be stabilised by long-range hydrophobic contacts, out of which many coincide with those found in the native state.  We also investigated the previously observed destabilising effect on the unfolded state by a poly-anion, and find that; the interaction does not alter the overall ensemble dimensions, nor the pattern in native-like contacts.  On the other hand, addition of the chemical denaturant urea results in a more expanded state.  The varying compaction with different co-solutes was validated by pulsed-field gradient NMR diffusion measurements.  

Unlike helical proteins, b-proteins lack the ability to fulfil hydrogen bonds by local native interactions. This forces specific prerequisites on the collapsed pre-folding state.  Here, the compaction is enabled by both native-like and non-native long-range contacts in the unfolded ensemble, and we suggest that the average topology of the collapsed state is determined by the sequence distribution of hydrophobic patches, separated by non-interacting hydrophilic clusters.