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.

Aggregate formation of superoxide dismutase 1 (SOD1) inside motor neurons is known as a major factor in onset of amyotrophic lateral sclerosis. The thermodynamic stability of the SOD1 beta-barrel has been shown to decrease in crowded environments such as inside a cell, but it remains unclear how the thermodynamics of crowding-induced protein destabilization relate to SOD1 aggregation. Here we have examined the effects of a protein crowder, lysozyme, on fibril aggregate formation of the SOD1 beta-barrel. We found that aggregate formation of SOD1 is decelerated even in mildly crowded solutions. Intriguingly, transient diffusive interactions with lysozyme do not significantly affect the static structure of the SOD1 beta-barrel but stabilize an alternative excited invisible state. The net effect of crowding is to favor species off the aggregation pathway, thereby explaining the decelerated aggregation in the crowded environment. Our observations suggest that the intracellular environment may have a similar negative (inhibitory) effect on fibril formation of other amyloidogenic proteins in living cells. Deciphering how crowded intracellular environments affect aggregation and fibril formation of such disease-associated proteins will probably become central in understanding the exact role of aggregation in the etiology of these enigmatic diseases.

In the cytosolic environment, protein crowding and Brownian motions result in numerous transient encounters. Each such encounter event increases the apparent size of the interacting molecules, leading to slower rotational tumbling. The extent of transient protein complexes formed in live cells can conveniently be quantified by an apparent viscosity, based on NMR-detected spin-relaxation measurements, that is, the longitudinal (T-1) and transverse (T-2) relaxation. From combined analysis of three different proteins and surface mutations thereof, we find that T-2 implies significantly higher apparent viscosity than T-1. At first sight, the effect on T-1 and T-2 seems thus nonunifiable, consistent with previous reports on other proteins. We show here that the T-1 and T-2 deviation is actually not a inconsistency but an expected feature of a system with fast exchange between free monomers and transient complexes. In this case, the deviation is basically reconciled by a model with fast exchange between the free-tumbling reporter protein and a transient complex with a uniform 143 kDa partner. The analysis is then taken one step further by accounting for the fact that the cytosolic content is by no means uniform but comprises a wide range of molecular sizes. Integrating over the complete size distribution of the cytosolic interaction ensemble enables us to predict both T-1 and T-2 from a single binding model. The result yields a bound population for each protein variant and provides a quantification of the transient interactions. We finally extend the approach to obtain a correction term for the shape of a database-derived mass distribution of the interactome in the mammalian cytosol, in good accord with the existing data of the cellular composition.

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.

Most biochemical reactions have evolved in crowded intracellular environments. However, the complexity of intracellular environments is often neglected in structural or functional studies of proteins. In these cases, reactions involving proteins are deliberately separated from the perturbations of co-solutes in order to simplify data acquisition and interpretation. Having acquired an enormous body of knowledge under these simplified dilute-buffer conditions, methodological progress of the past two decades has made the study of proteins inside living cells increasingly accessible and concomitantly kindled an interest to investigate proteins in their native habitat. Naturally, major questions that arose were to what extent ubiquitous transient interactions alter protein structure, function and thermodynamics and, not least, what role protein surfaces and their physicochemical properties play in determining the frequency and duration of these diffusive encounters.

By looking at the rotational-tumbling rates of three structurally well-characterized proteins in live cells with nuclear magnetic resonance (NMR) relaxation, we expand on previous research performed in the bacterium Escherichia coli and establish the physicochemical principles that determine diffusive interactions in the mammalian cytosol of the human ovarian cancer cell line A2780. Just as in E. coli, net charge is the dominating factor in regulating protein interactivity, albeit with the impact on rotational retardation greatly diminished. We ascribe this to the generally lower macromolecular concentrations in the eukaryotic cytosol, and put forward a hypothesis in which less stringent rules regarding protein surface decoration in eukaryotes could have facilitated the development of multi-cellular organisms. Furthermore, by developing a model where a distribution of differently sized interaction partners is taken into account when examining rotational retardation, we reconcile transverse and longitudinal in-cell relaxation with theory, and are able to estimate the populations of the bound and free form of a set of reporter proteins. Looking at the populations of bound protein instead of a mean-field rotational retardation finally allows us to re-assess the guiding rules behind diffusive cytosolic interactions. Last, we outline a putative mechanism behind the in-cell destabilization of a variant of Superoxide dismutase 1 (SOD1^barrel). By mimicking generic poly-anionic intracellular co-solutes with poly-acetic acid (NaPAc1200), we identify the positively charged N-terminal portion of the unfolded form of the protein as the interaction site with the highest affinity. Further examining the unfolded ensemble of SOD1^barrel with a mutationally destabilized variant reveals a compact state, that remains almost unchanged upon binding to NaPAc1200. This suggests that NaPAc1200-mediated destabilization occurs mainly through mass action, in full accord with the postulated mechanism for in-cell protein destabilization.

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.