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.

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.

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.

Metal ion coordination delays amyloid-ß peptide self-assembly by forming an aggregation-inert complex.

A detailed understanding of the molecular pathways for amyloid-ß (Aß) 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 Aß peptide and efficiently modulates Aß 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 Aß monomers to its macroscopic impact on the peptide self-assembly observed in bulk experiments. We found that substoichiometric concentrations of the investigated metal ions bind specifically to the N-terminal region of Aß, forming a dynamic, partially compact complex. The metal-ion bound state appears to be incapable of aggregation, effectively reducing the available monomeric Aß pool for incorporation into fibrils. This is especially reflected in a decreased fibril-end elongation rate. However, because the bound state is significantly less stable than the amyloid state, Aß peptides are only transiently redirected from fibril formation, and eventually almost all Aß monomers are integrated into fibrils. Taken together, these findings unravel the mechanistic consequences of delaying Aß aggregation via weak metal-ion binding, quantitatively linking the contributions of specific interactions of metal ions with monomeric Aß to their effects on bulk aggregation.

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.

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.

Zinc as chaperone-mimicking agent for retardation of amyloid ß peptide fibril formation.

Metal ions have emerged to play a key role in the aggregation process of amyloid ß (Aß) peptide that is closely related to the pathogenesis of Alzheimer's disease. A detailed understanding of the underlying mechanistic process of peptide-metal interactions, however, has been challenging to obtain. By applying a combination of NMR relaxation dispersion and fluorescence kinetics methods we have investigated quantitatively the thermodynamic Aß-Zn(2+) binding features as well as how Zn(2+) modulates the nucleation mechanism of the aggregation process. Our results show that, under near-physiological conditions, substoichiometric amounts of Zn(2+) effectively retard the generation of amyloid fibrils. A global kinetic profile analysis reveals that in the absence of zinc Aß40 aggregation is driven by a monomer-dependent secondary nucleation process in addition to fibril-end elongation. In the presence of Zn(2+), the elongation rate is reduced, resulting in reduction of the aggregation rate, but not a complete inhibition of amyloid formation. We show that Zn(2+) transiently binds to residues in the N terminus of the monomeric peptide. A thermodynamic analysis supports a model where the N terminus is folded around the Zn(2+) ion, forming a marginally stable, short-lived folded Aß40 species. This conformation is highly dynamic and only a few percent of the peptide molecules adopt this structure at any given time point. Our findings suggest that the folded Aß40-Zn(2+) complex modulates the fibril ends, where elongation takes place, which efficiently retards fibril formation. In this conceptual framework we propose that zinc adopts the role of a minimal antiaggregation chaperone for Aß40.

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.

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.

Structural and kinetic analysis of protein-aggregate strains in vivo using binary epitope mapping.

Despite considerable progress in uncovering the molecular details of protein aggregation in vitro, the cause and mechanism of protein-aggregation disease remain poorly understood. One reason is that the amount of pathological aggregates in neural tissue is exceedingly low, precluding examination by conventional approaches. We present here a method for determination of the structure and quantity of aggregates in small tissue samples, circumventing the above problem. The method is based on binary epitope mapping using anti-peptide antibodies. We assessed the usefulness and versatility of the method in mice modeling the neurodegenerative disease amyotrophic lateral sclerosis, which accumulate intracellular aggregates of superoxide dismutase-1. Two strains of aggregates were identified with different structural architectures, molecular properties, and growth kinetics. Both were different from superoxide dismutase-1 aggregates generated in vitro under a variety of conditions. The strains, which seem kinetically under fragmentation control, are associated with different disease progressions, complying with and adding detail to the growing evidence that seeding, infectivity, and strain dependence are unifying principles of neurodegenerative disease.

Membrane-Induced Folding of the Plant Stress Dehydrin Lti30.

Dehydrins are disordered proteins that are expressed in plants as a response to embryogenesis and water-related stress. The molecular function and structural action of the dehydrins are yet elusive, but increasing evidence points to a role in protecting the structure and functional dynamics of cell membranes. An intriguing example is the cold-induced dehydrin Lti30 that binds to membranes by its conserved K segments. Moreover, this binding can be regulated by pH and phosphorylation and shifts the membrane phase transition to lower temperatures, consistent with the protein's postulated function in cold stress. In this study, we reveal how the Lti30-membrane interplay works structurally at atomic level resolution in Arabidopsis (Arabidopsis thaliana). Nuclear magnetic resonance analysis suggests that negatively charged lipid head groups electrostatically capture the protein's disordered K segments, which locally fold up into α-helical segments on the membrane surface. Thus, Lti30 conforms to the general theme of structure-function relationships by folding upon binding, in spite of its disordered, atypically hydrophilic and repetitive sequence signatures. Moreover, the fixed and well-defined structure of the membrane-bound K segments suggests that dehydrins have the molecular prerequisites for higher level binding specificity and regulation, raising new questions about the complexity of their biological function.

Ionic Strength Modulation of the Free Energy Landscape of Aß40 Peptide Fibril Formation.

Protein misfolding and formation of cross-ß structured amyloid fibrils are linked to many neurodegenerative disorders. Although recently developed quantitative approaches have started to reveal the molecular nature of self-assembly and fibril formation of proteins and peptides, it is yet unclear how these self-organization events are precisely modulated by microenvironmental factors, which are known to strongly affect the macroscopic aggregation properties. Here, we characterize the explicit effect of ionic strength on the microscopic aggregation rates of amyloid ß peptide (Aß40) self-association, implicated in Alzheimer's disease. We found that physiological ionic strength accelerates Aß40 aggregation kinetics by promoting surface-catalyzed secondary nucleation reactions. This promoted catalytic effect can be assigned to shielding of electrostatic repulsion between monomers on the fibril surface or between the fibril surface itself and monomeric peptides. Furthermore, we observe the formation of two different ß-structured states with similar but distinct spectroscopic features, which can be assigned to an off-pathway immature state (Fß*) and a mature stable state (Fß), where salt favors formation of the Fß fibril morphology. Addition of salt to preformed Fß* accelerates transition to Fß, underlining the dynamic nature of Aß40 fibrils in solution. On the basis of these results we suggest a model where salt decreases the free-energy barrier for Aß40 folding to the Fß state, favoring the buildup of the mature fibril morphology while omitting competing, energetically less favorable structural states.

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.

Global structural motions from the strain of a single hydrogen bond.

The origin and biological role of dynamic motions of folded enzymes is not yet fully understood. In this study, we examine the molecular determinants for the dynamic motions within the ß-barrel of superoxide dismutase 1 (SOD1), which previously were implicated in allosteric regulation of protein maturation and also pathological misfolding in the neurodegenerative disease amyotrophic lateral sclerosis. Relaxation-dispersion NMR, hydrogen/deuterium exchange, and crystallographic data show that the dynamic motions are induced by the buried H43 side chain, which connects the backbones of the Cu ligand H120 and T39 by a hydrogen-bond linkage through the hydrophobic core. The functional role of this highly conserved H120-H43-T39 linkage is to strain H120 into the correct geometry for Cu binding. Upon elimination of the strain by mutation H43F, the apo protein relaxes through hydrogen-bond swapping into a more stable structure and the dynamic motions freeze out completely. At the same time, the holo protein becomes energetically penalized because the twisting back of H120 into Cu-bound geometry leads to burial of an unmatched backbone carbonyl group. The question then is whether this coupling between metal binding and global structural motions in the SOD1 molecule is an adverse side effect of evolving viable Cu coordination or plays a key role in allosteric regulation of biological function, or both?

Folding without charges.

Surface charges of proteins have in several cases been found to function as "structural gatekeepers," which avoid unwanted interactions by negative design, for example, in the control of protein aggregation and binding. The question is then if side-chain charges, due to their desolvation penalties, play a corresponding role in protein folding by avoiding competing, misfolded traps? To find out, we removed all 32 side-chain charges from the 101-residue protein S6 from Thermus thermophilus. The results show that the charge-depleted S6 variant not only retains its native structure and cooperative folding transition, but folds also faster than the wild-type protein. In addition, charge removal unleashes pronounced aggregation on longer timescales. S6 provides thus an example where the bias toward native contacts of a naturally evolved protein sequence is independent of charges, and point at a fundamental difference in the codes for folding and intermolecular interaction: specificity in folding is governed primarily by hydrophobic packing and hydrogen bonding, whereas solubility and binding relies critically on the interplay of side-chain charges.

Fibrillation precursor of superoxide dismutase 1 revealed by gradual tuning of the protein-folding equilibrium.

Although superoxide dismutase 1 (SOD1) stands out as a relatively soluble protein in vitro, it can be made to fibrillate by mechanical agitation. The mechanism of this fibrillation process is yet poorly understood, but attains considerable interest due to SOD1's involvement in the neurodegenerative disease amyotrophic lateral sclerosis (ALS). In this study, we map out the apoSOD1 fibrillation process from how it competes with the global folding events at increasing concentrations of urea: We determine how the fibrillation lag time (τ(lag)) and maximum growth rate (ν(max)) depend on gradual titration of the folding equilibrium, from the native to the unfolded state. The results show that the agitation-induced fibrillation of apoSOD1 uses globally unfolded precursors and relies on fragmentation-assisted growth. Mutational screening and fibrillation m-values (∂ log τ(lag)/∂[urea] and ∂ log ν(max)/∂[urea]) indicate moreover that the fibrillation pathway proceeds via a diffusely bound transient complex that responds to the global physiochemical properties of the SOD1 sequence. Fibrillation of apoSOD1, as it bifurcates from the denatured ensemble, seems thus mechanistically analogous to that of disordered peptides, save the competing folding transition to the native state. Finally, we examine by comparison with in vivo data to what extent this mode of fibrillation, originating from selective amplification of mechanically brittle aggregates by sample agitation, captures the mechanism of pathological SOD1 aggregation in ALS.

Formation of dynamic soluble surfactant-induced amyloid ß peptide aggregation intermediates.

Intermediate amyloidogenic states along the amyloid ß peptide (Aß) aggregation pathway have been shown to be linked to neurotoxicity. To shed more light on the different structures that may arise during Aß aggregation, we here investigate surfactant-induced Aß aggregation. This process leads to co-aggregates featuring a ß-structure motif that is characteristic for mature amyloid-like structures. Surfactants induce secondary structure in Aß in a concentration-dependent manner, from predominantly random coil at low surfactant concentration, via ß-structure to the fully formed α-helical state at high surfactant concentration. The ß-rich state is the most aggregation-prone as monitored by thioflavin T fluorescence. Small angle x-ray scattering reveals initial globular structures of surfactant-Aß co-aggregated oligomers and formation of elongated fibrils during a slow aggregation process. Alongside this slow (minutes to hours time scale) fibrillation process, much faster dynamic exchange (k(ex) ∼1100 s(-1)) takes place between free and co-aggregate-bound peptide. The two hydrophobic segments of the peptide are directly involved in the chemical exchange and interact with the hydrophobic part of the co-aggregates. Our findings suggest a model for surfactant-induced aggregation where free peptide and surfactant initially co-aggregate to dynamic globular oligomers and eventually form elongated fibrils. When interacting with ß-structure promoting substances, such as surfactants, Aß is kinetically driven toward an aggregation-prone state.

The hairpin conformation of the amyloid ß peptide is an important structural motif along the aggregation pathway.

The amyloid ß (Aß) peptides are 39-42 residue-long peptides found in the senile plaques in the brains of Alzheimer's disease (AD) patients. These peptides self-aggregate in aqueous solution, going from soluble and mainly unstructured monomers to insoluble ordered fibrils. The aggregation process(es) are strongly influenced by environmental conditions. Several lines of evidence indicate that the neurotoxic species are the intermediate oligomeric states appearing along the aggregation pathways. This minireview summarizes recent findings, mainly based on solution and solid-state NMR experiments and electron microscopy, which investigate the molecular structures and characteristics of the Aß peptides at different stages along the aggregation pathways. We conclude that a hairpin-like conformation constitutes a common motif for the Aß peptides in most of the described structures. There are certain variations in different hairpin conformations, for example regarding H-bonding partners, which could be one reason for the molecular heterogeneity observed in the aggregated systems. Interacting hairpins are the building blocks of the insoluble fibrils, again with variations in how hairpins are organized in the cross-section of the fibril, perpendicular to the fibril axis. The secondary structure propensities can be seen already in peptide monomers in solution. Unfortunately, detailed structural information about the intermediate oligomeric states is presently not available. In the review, special attention is given to metal ion interactions, particularly the binding constants and ligand structures of Aß complexes with Cu(II) and Zn(II), since these ions affect the aggregation process(es) and are considered to be involved in the molecular mechanisms underlying AD pathology.

Solid-state NMR studies of metal-free SOD1 fibrillar structures.

Copper-zinc superoxide dismutase 1 (SOD1) is present in the protein aggregates deposited in motor neurons of amyotrophic lateral sclerosis (ALS) patients. ALS is a neurodegenerative disease that can be either sporadic (ca. 90%) or familial (fALS). The most widely studied forms of fALS are caused by mutations in the sequence of SOD1. Ex mortuo SOD1 aggregates are usually found to be amorphous. In vitro SOD1, in its immature reduced and apo state, forms fibrillar aggregates. Previous literature data have suggested that a monomeric SOD1 construct, lacking loops IV and VII, (apoSODΔIV-VII), shares the same fibrillization properties of apoSOD1, both proteins having the common structural feature of the central ß-barrel. In this work, we show that structural information can be obtained at a site-specific level from solid-state NMR. The residues that are sequentially assignable are found to be located at the putative nucleation site for fibrillar species formation in apoSOD, as detected by other experimental techniques.