Diffusive intracellular interactions: On the role of protein net charge and functional adaptation.

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.

Mutant SOD1 aggregates formed in vitro and in cultured cells are polymorphic and differ from those arising in the CNS.

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.

Physicochemical classification of organisms.

The hypervariable residues that compose the major part of proteins' surfaces are generally considered outside evolutionary control. Yet, these "nonconserved" residues determine the outcome of stochastic encounters in crowded cells. It has recently become apparent that these encounters are not as random as one might imagine, but carefully orchestrated by the intracellular electrostatics to optimize protein diffusion, interactivity, and partner search. The most influential factor here is the protein surface-charge density, which takes different optimal values across organisms with different intracellular conditions. In this study, we examine how far the net-charge density and other physicochemical properties of proteomes will take us in terms of distinguishing organisms in general. The results show that these global proteome properties not only follow the established taxonomical hierarchy, but also provide clues to functional adaptation. In many cases, the proteome­property divergence is even resolved at species level. Accordingly, the variable parts of the genes are not as free to drift as they seem in sequence alignment, but present a complementary tool for functional, taxonomic, and evolutionary assignment.

On the osmotic pressure of cells.

The chemical potential of water () provides an essential thermodynamic characterization of the environment of living organisms, and it is of equal significance as the temperature. For cells, is conventionally expressed in terms of the osmotic pressure (πosm). We have previously suggested that the main contribution to the intracellular πosm of the bacterium E. coli is from soluble negatively-charged proteins and their counter-ions. Here, we expand on this analysis by examining how evolutionary divergent cell types cope with the challenge of maintaining πosm within viable values. Complex organisms, like mammals, maintain constant internal πosm ≈ 0.285 osmol, matching that of 0.154 M NaCl. For bacteria it appears that optimal growth conditions are found for similar or slightly higher πosm (0.25-0.4 osmol), despite that they represent a much earlier stage in evolution. We argue that this value reflects a general adaptation for optimising metabolic function under crowded intracellular conditions. Environmental πosm that differ from this optimum require therefore special measures, as exemplified with gram-positive and gram-negative bacteria. To handle such situations, their membrane encapsulations allow for a compensating turgor pressure that can take both positive and negative values, where positive pressures allow increased frequency of metabolic events through increased intracellular protein concentrations. A remarkable exception to the rule of 0.25-0.4 osmol, is found for halophilic archaea with internal πosm ≈ 15 osmol. The internal organization of these archaea differs in that they utilize a repulsive electrostatic mechanism operating only in the ionic-liquid regime to avoid aggregation, and that they stand out from other organisms by having no turgor pressure.

Polyanions Cause Protein Destabilization Similar to That in Live Cells.

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 SOD1barrel significantly more than monoacetate or NaCl. At an equivalent of 100 mM Na+, the polyacetate destabilization of SOD1barrel 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 SOD1barrel. 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.

Charge Engineering Reveals the Roles of Ionizable Side Chains in Electrospray Ionization Mass Spectrometry.

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.

Connecting Longitudinal and Transverse Relaxation Rates in Live-Cell NMR.

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 (T1) and transverse (T2) relaxation. From combined analysis of three different proteins and surface mutations thereof, we find that T2 implies significantly higher apparent viscosity than T1. At first sight, the effect on T1 and T2 seems thus nonunifiable, consistent with previous reports on other proteins. We show here that the T1 and T2 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 T1 and T2 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.

Exposing the distinctive modular behavior of ß-strands and α-helices in folded proteins.

Although folded proteins are commonly depicted as simplistic combinations of ß-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 ß- 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/ß-protein S6, which allow the structural behavior of individual ß-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 ß-elements have quite different characteristics: The swaps of ß-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.

Colloidal stability of the living cell.

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.

The Pierced Lasso Topology Leptin has a Bolt on Dynamic Domain Composed by the Disordered Loops I and III.

Leptin is an important signaling hormone, mostly known for its role in energy expenditure and satiety. Furthermore, leptin plays a major role in other proteinopathies, such as cancer, marked hyperphagia, impaired immune function, and inflammation. In spite of its biological relevance in human health, there are no NMR resonance assignments of the human protein available, obscuring high-resolution characterization of the soluble protein and/or its conformational dynamics, suggested as being important for receptor interaction and biological activity. Here, we report the nearly complete backbone resonance assignments of human leptin. Chemical shift-based secondary structure prediction confirms that in solution leptin forms a four-helix bundle including a pierced lasso topology. The conformational dynamics, determined on several timescales, show that leptin is monomeric, has a rigid four-helix scaffold, and a dynamic domain, including a transiently formed helix. The dynamic domain is anchored to the helical scaffold by a secondary hydrophobic core, pinning down the long loops of leptin to the protein body, inducing motional restriction without a well-defined secondary or tertiary hydrogen bond stabilized structure. This dynamic region is well suited for and may be involved in functional allosteric dynamics upon receptor binding.

Diffusive protein interactions in human versus bacterial cells.

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.

Exposure of a cryptic Hsp70 binding site determines the cytotoxicity of the ALS-associated SOD1-mutant A4V.

The accumulation of toxic protein aggregates is thought to play a key role in a range of degenerative pathologies, but it remains unclear why aggregation of polypeptides into non-native assemblies is toxic and why cellular clearance pathways offer ineffective protection. We here study the A4V mutant of SOD1, which forms toxic aggregates in motor neurons of patients with familial amyotrophic lateral sclerosis (ALS). A comparison of the location of aggregation prone regions (APRs) and Hsp70 binding sites in the denatured state of SOD1 reveals that ALS-associated mutations promote exposure of the APRs more than the strongest Hsc/Hsp70 binding site that we could detect. Mutations designed to increase the exposure of this Hsp70 interaction site in the denatured state promote aggregation but also display an increased interaction with Hsp70 chaperones. Depending on the cell type, in vitro this resulted in cellular inclusion body formation or increased clearance, accompanied with a suppression of cytotoxicity. The latter was also observed in a zebrafish model in vivo. Our results suggest that the uncontrolled accumulation of toxic SOD1A4V aggregates results from insufficient detection by the cellular surveillance network.

The Cost of Long Catalytic Loops in Folding and Stability of the ALS-Associated Protein SOD1.

A conspicuous feature of the amyotrophic lateral sclerosis (ALS)-associated protein SOD1 is that its maturation into a functional enzyme relies on local folding of two disordered loops into a catalytic subdomain. To drive the disorder-to-order transition, the protein employs a single Zn2+ ion. The question is then if the entropic penalty of maintaining such disordered loops in the immature apoSOD1 monomer is large enough to explain its unusually low stability, slow folding, and pathological aggregation in ALS. To find out, we determined the effects of systematically altering the SOD1-loop lengths by protein redesign. The results show that the loops destabilize the apoSOD1 monomer by ∼3 kcal/mol, rendering the protein marginally stable and accounting for its aggregation behavior. Yet the effect on the global folding kinetics remains much smaller with a transition-state destabilization of <1 kcal/mol. Notably, this 1/3 transition-state to folded-state stability ratio provides a clear-cut example of the enigmatic disagreement between the Leffler α value from loop-length alterations (typically 1/3) and the "standard" reaction coordinates based on solvent perturbations (typically >2/3). Reconciling the issue, we demonstrate that the disagreement disappears when accounting for the progressive loop shortening that occurs along the folding pathway. The approach assumes a consistent Flory loop entropy scaling factor of c = 1.48 for both equilibrium and kinetic data and has the added benefit of verifying the tertiary interactions of the folding nucleus as determined by phi-value analysis. Thus, SOD1 not only represents a case where evolution of key catalytic function has come with the drawback of a destabilized apo state but also stands out as a well-suited model system for exploring the physicochemical details of protein self-organization.

A biosensor-based framework to measure latent proteostasis capacity.

The pool of quality control proteins (QC) that maintains protein-folding homeostasis (proteostasis) is dynamic but can become depleted in human disease. A challenge has been in quantitatively defining the depth of the QC pool. With a new biosensor, flow cytometry-based methods and mathematical modeling we measure the QC capacity to act as holdases and suppress biosensor aggregation. The biosensor system comprises a series of barnase kernels with differing folding stability that engage primarily with HSP70 and HSP90 family proteins. Conditions of proteostasis stimulation and stress alter QC holdase activity and aggregation rates. The method reveals the HSP70 chaperone cycle to be rate limited by HSP70 holdase activity under normal conditions, but this is overcome by increasing levels of the BAG1 nucleotide exchange factor to HSPA1A or activation of the heat shock gene cluster by HSF1 overexpression. This scheme opens new paths for biosensors of disease and proteostasis systems.

Physicochemical code for quinary protein interactions in Escherichia coli.

How proteins sense and navigate the cellular interior to find their functional partners remains poorly understood. An intriguing aspect of this search is that it relies on diffusive encounters with the crowded cellular background, made up of protein surfaces that are largely nonconserved. The question is then if/how this protein search is amenable to selection and biological control. To shed light on this issue, we examined the motions of three evolutionary divergent proteins in the Escherichia coli cytoplasm by in-cell NMR. The results show that the diffusive in-cell motions, after all, follow simplistic physical-chemical rules: The proteins reveal a common dependence on (i) net charge density, (ii) surface hydrophobicity, and (iii) the electric dipole moment. The bacterial protein is here biased to move relatively freely in the bacterial interior, whereas the human counterparts more easily stick. Even so, the in-cell motions respond predictably to surface mutation, allowing us to tune and intermix the protein's behavior at will. The findings show how evolution can swiftly optimize the diffuse background of protein encounter complexes by just single-point mutations, and provide a rational framework for adjusting the cytoplasmic motions of individual proteins, e.g., for rescuing poor in-cell NMR signals and for optimizing protein therapeutics.

Comparing protein behaviour in vitro and in vivo, what does the data really tell us?

The recent advancement in moving 'biophysical' analysis of proteins in vivo has finally brought us to a position where we can start to make quantitative comparisons with existing in-vitro data. A striking observation is that protein behaviour in live cells seems, after all, not that different from in test tubes, not even at the level of complex mechanisms like protein aggregation. The example examined in this review is the ALS associated protein SOD1 that apparently retains its in-vitro properties in vivo. Does this mean that the protocols for studying proteins in vivo are somehow oversimplified, or that the macromolecular properties and interplay - despite being intrinsically malleable - are evolutionary more 'streamlined' than previously anticipated? Whatever the answer may be the time is now right to put these data to critical biological test.

Tricking a Protein To Swap Strands.

Despite continuing interest in partly unfolded proteins as precursors for aggregation and adverse gain-of-function in human disease, there is yet little known about the local transitions of native structures that possibly lead to such intermediate states. To target this problem, we present here a protein-design strategy that allows real-time detection of rupture and swapping of complete secondary-structure elements in globular proteins-molecular events that have previously been inaccessible experimental analysis. The approach is applied to the dynamic ß-barrel of SOD1, associated with pathologic aggregation in the neurodegenerative disease ALS. Data show that rupture and re-insertion of individual ß-strands do not take place locally but require the SOD1 barrel to unfold globally. The finding questions the very existence of partly unfolded intermediates in the SOD1 aggregation process and presents new clues to the mechanism by which hydrogen bonding maintains global structural integrity.

Structural insights into HetR-PatS interaction involved in cyanobacterial pattern formation.

The one-dimensional pattern of heterocyst in the model cyanobacterium Anabaena sp. PCC 7120 is coordinated by the transcription factor HetR and PatS peptide. Here we report the complex structures of HetR binding to DNA, and its hood domain (HetRHood) binding to a PatS-derived hexapeptide (PatS6) at 2.80 and 2.10 Å, respectively. The intertwined HetR dimer possesses a couple of novel HTH motifs, each of which consists of two canonical α-helices in the DNA-binding domain and an auxiliary α-helix from the flap domain of the neighboring subunit. Two PatS6 peptides bind to the lateral clefts of HetRHood, and trigger significant conformational changes of the flap domain, resulting in dissociation of the auxiliary α-helix and eventually release of HetR from the DNA major grove. These findings provide the structural insights into a prokaryotic example of Turing model.

Thermodynamics of protein destabilization in live cells.

Although protein folding and stability have been well explored under simplified conditions in vitro, it is yet unclear how these basic self-organization events are modulated by the crowded interior of live cells. To find out, we use here in-cell NMR to follow at atomic resolution the thermal unfolding of a ß-barrel protein inside mammalian and bacterial cells. Challenging the view from in vitro crowding effects, we find that the cells destabilize the protein at 37 °C but with a conspicuous twist: While the melting temperature goes down the cold unfolding moves into the physiological regime, coupled to an augmented heat-capacity change. The effect seems induced by transient, sequence-specific, interactions with the cellular components, acting preferentially on the unfolded ensemble. This points to a model where the in vivo influence on protein behavior is case specific, determined by the individual protein's interplay with the functionally optimized "interaction landscape" of the cellular interior.

SOD1 aggregation in ALS mice shows simplistic test tube behavior.

A longstanding challenge in studies of neurodegenerative disease has been that the pathologic protein aggregates in live tissue are not amenable to structural and kinetic analysis by conventional methods. The situation is put in focus by the current progress in demarcating protein aggregation in vitro, exposing new mechanistic details that are now calling for quantitative in vivo comparison. In this study, we bridge this gap by presenting a direct comparison of the aggregation kinetics of the ALS-associated protein superoxide dismutase 1 (SOD1) in vitro and in transgenic mice. The results based on tissue sampling by quantitative antibody assays show that the SOD1 fibrillation kinetics in vitro mirror with remarkable accuracy the spinal cord aggregate buildup and disease progression in transgenic mice. This similarity between in vitro and in vivo data suggests that, despite the complexity of live tissue, SOD1 aggregation follows robust and simplistic rules, providing new mechanistic insights into the ALS pathology and organism-level manifestation of protein aggregation phenomena in general.