A striking feature of nucleic acids and lipid membranes is that they all carry net negative charge and so is true for the majority of intracellular proteins. It is suggested that the role of this negative charge is to assure a basal intermolecular repulsion that keeps the cytosolic content suitably ‘fluid’ for function. We focus in this review on the experimental, theoretical and genetic findings which serve to underpin this idea and the new questions they raise. Unlike the situation in test tubes, any functional protein-protein interaction in the cytosol is subject to competition from the densely crowded background, i.e. surrounding stickiness. At the nonspecific limit of this stickiness is the ‘random’ protein-protein association, maintaining profuse populations of transient and constantly interconverting complexes at physiological protein concentrations. The phenomenon is readily quantified in studies of the protein rotational diffusion, showing that the more net negatively charged a protein is the less it is retarded by clustering. It is further evident that this dynamic protein-protein interplay is under evolutionary control and finely tuned across organisms to maintain optimal physicochemical conditions for the cellular processes. The emerging picture is then that specific cellular function relies on close competition between numerous weak and strong interactions, and where all parts of the protein surfaces are involved. The outstanding challenge is now to decipher the very basics of this many-body system: how the detailed patterns of charged, polar and hydrophobic side chains not only control protein-protein interactions at close- and long-range but also the collective properties of the cellular interior as a whole.

Mutations in the human Superoxide dismutase 1 (hSOD1) gene are well-established cause of the motor neuron disease ALS. Patients and transgenic (Tg) ALS model mice carrying mutant variants develop hSOD1 aggregates in the CNS. We have identified two hSOD1 aggregate strains, which both transmit spreading template-directed aggregation and premature fatal paralysis when inoculated into adult transgenic mice. This prion-like spread of aggregation could be a primary disease mechanism in SOD1-induced ALS. Human SOD1 aggregation has been studied extensively both in cultured cells and under various conditions in vitro. To determine how the structure of aggregates formed in these model systems related to disease-associated aggregates in the CNS, we used a binary epitope-mapping assay to examine aggregates of hSOD1 variants G93A, G85R, A4V, D90A, and G127X formed in vitro, in four different cell lines and in the CNS of Tg mice. We found considerable variability between replicate sets of in vitro-generated aggregates. In contrast, there was a high similarity between replicates of a given hSOD1 mutant in a given cell line, but pronounced variations between different hSOD1 mutants and different cell lines in both structures and amounts of aggregates formed. The aggregates formed in vitro or in cultured cells did not replicate the aggregate strains that arise in the CNS. Our findings suggest that the distinct aggregate morphologies in the CNS could result from a micro-environment with stringent quality control combined with second-order selection by spreading ability. Explorations of pathogenesis and development of therapeutics should be conducted in models that replicate aggregate structures forming in the CNS.

In solution, the charge of a protein is intricately linked to its stability, but electrospray ionization distorts this connection, potentially limiting the ability of native mass spectrometry to inform about protein structure and dynamics. How the behavior of intact proteins in the gas phase depends on the presence and distribution of ionizable surface residues has been difficult to answer because multiple chargeable sites are present in virtually all proteins. Turning to protein engineering, we show that ionizable side chains are completely dispensable for charging under native conditions, but if present, they are preferential protonation sites. The absence of ionizable side chains results in identical charge state distributions under native-like and denaturing conditions, while coexisting conformers can be distinguished using ion mobility separation. An excess of ionizable side chains, on the other hand, effectively modulates protein ion stability. In fact, moving a single ionizable group can dramatically alter the gas-phase conformation of a protein ion. We conclude that although the sum of the charges is governed solely by Coulombic terms, their locations affect the stability of the protein in the gas phase.

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.

Cellular function is generally depicted at the level of functional pathways and detailed structural mechanisms, based on the identification of specific protein-protein interactions. For an individual protein searching for its partner, however, the perspective is quite different: The functional task is challenged by a dense crowd of nonpartners obstructing the way. Adding to the challenge, there is little information about how to navigate the search, since the encountered surrounding is composed of protein surfaces that are predominantly nonconserved or, at least, highly variable across organisms. In this study, we demonstrate from a colloidal standpoint that such a blindfolded intracellular search is indeed favored and has more fundamental impact on the cellular organization than previously anticipated. Basically, the unique polyion composition of cellular systems renders the electrostatic interactions different from those in physiological buffer, leading to a situation where the protein net-charge density balances the attractive dispersion force and surface heterogeneity at close range. Inspection of naturally occurring proteomes and in-cell NMR data show further that the nonconserved protein surfaces are by no means passive but chemically biased to varying degree of net-negative repulsion across organisms. Finally, this electrostatic control explains how protein crowding is spontaneously maintained at a constant level through the intracellular osmotic pressure and leads to the prediction that the extreme in halophilic adaptation is not the ionic-liquid conditions per se but the evolutionary barrier of crossing its physicochemical boundaries.

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.

Although folded proteins are commonly depicted as simplistic combinations of β-strands and α-helices, the actual properties and functions of these secondary-structure elements in their native contexts are just partly understood. The principal reason is that the behavior of individual β- and α-elements is obscured by the global folding cooperativity. In this study, we have circumvented this problem by designing frustrated variants of the mixed α/β-protein S6, which allow the structural behavior of individual β-strands and α-helices to be targeted selectively by stopped-flow kinetics, X-ray crystallography, and solution-state NMR. Essentially, our approach is based on provoking intramolecular "domain swap." The results show that the α- and β-elements have quite different characteristics: The swaps of β-strands proceed via global unfolding, whereas the α-helices are free to swap locally in the native basin. Moreover, the α-helices tend to hybridize and to promote protein association by gliding over to neighboring molecules. This difference in structural behavior follows directly from hydrogen-bonding restrictions and suggests that the protein secondary structure defines not only tertiary geometry, but also maintains control in function and structural evolution. Finally, our alternative approach to protein folding and native-state dynamics presents a generally applicable strategy for in silico design of protein models that are computationally testable in the microsecond–millisecond regime.

A detailed understanding of the molecular pathways for amyloid-β (Aβ) peptide aggregation from monomers into amyloid fibrils, a hallmark of Alzheimer’s disease, is crucial for the development of diagnostic and therapeutic strategies. We investigate the molecular details of peptide fibrillization in vitro by perturbing this process through addition of differently charged metal ions. Here, we used a monovalent probe, the silver ion, that, similarly to divalent metal ions, binds to monomeric Aβ peptide and efficiently modulates Aβ fibrillization. On the basis of our findings, combined with our previous results on divalent zinc ions, we propose a model that links the microscopic metal ion binding to Aβ monomers to its macroscopic impact on the peptide self-assembly observed in bulk experiments. We found that sub-stoichiometric concentrations of the investigated metal ions bind specifically to the N-terminal region of Aβ, forming a dynamic, partially compact complex. The metal ion bound state appears to be incapable of aggregation, effectively reducing the available monomeric Aβ pool for incorporation into fibrils. This is especially reflected in a decreased fibril-end elongation rate. However, since the bound state is significantly less stable than the amyloid state, Aβ peptides are only transiently redirected from fibril formation and eventually almost all Aβ monomers are integrated into fibrils. Taken together, these findings unravel the mechanistic consequences of delaying Aβ aggregation via weak metal ion binding, quantitatively linking the contributions of specific interactions of metal ions with monomeric Aβ to their effects on bulk aggregation.