Extracellular G-quadruplexes and Z-DNA protect biofilms from DNase I, and G-quadruplexes form a DNAzyme with peroxidase activity.

Many bacteria form biofilms to protect themselves from predators or stressful environmental conditions. In the biofilm, bacteria are embedded in a protective extracellular matrix composed of polysaccharides, proteins and extracellular DNA (eDNA). eDNA most often is released from lysed bacteria or host mammalian cells, and it is the only matrix component most biofilms appear to have in common. However, little is known about the form DNA takes in the extracellular space, and how different non-canonical DNA structures such as Z-DNA or G-quadruplexes might contribute to its function in the biofilm. The aim of this study was to determine if non-canonical DNA structures form in eDNA-rich staphylococcal biofilms, and if these structures protect the biofilm from degradation by nucleases. We grew Staphylococcus epidermidis biofilms in laboratory media supplemented with hemin and NaCl to stabilize secondary DNA structures and visualized their location by immunolabelling and fluorescence microscopy. We furthermore visualized the macroscopic biofilm structure by optical coherence tomography. We developed assays to quantify degradation of Z-DNA and G-quadruplex DNA oligos by different nucleases, and subsequently investigated how these enzymes affected eDNA in the biofilms. Z-DNA and G-quadruplex DNA were abundant in the biofilm matrix, and were often present in a web-like structures. In vitro, the structures did not form in the absence of NaCl or mechanical shaking during biofilm growth, or in bacterial strains deficient in eDNA or exopolysaccharide production. We thus infer that eDNA and polysaccharides interact, leading to non-canonical DNA structures under mechanical stress when stabilized by salt. We also confirmed that G-quadruplex DNA and Z-DNA was present in biofilms from infected implants in a murine implant-associated osteomyelitis model. Mammalian DNase I lacked activity against Z-DNA and G-quadruplex DNA, while Micrococcal nuclease could degrade G-quadruplex DNA and S1 Aspergillus nuclease could degrade Z-DNA. Micrococcal nuclease, which originates from Staphylococcus aureus, may thus be key for dispersal of biofilm in staphylococci. In addition to its structural role, we show for the first time that the eDNA in biofilms forms a DNAzyme with peroxidase-like activity in the presence of hemin. While peroxidases are part of host defenses against pathogens, we now show that biofilms can possess intrinsic peroxidase activity in the extracellular matrix.

The optimal docking strength for reversibly tethered kinases.

Many kinases use reversible docking interactions to augment the specificity of their catalytic domains. Such docking interactions are often structurally independent of the catalytic domain, which allow for a flexible combination of modules in evolution and in bioengineering. The affinity of docking interactions spans several orders of magnitude. This led us to ask how the affinity of the docking interaction affects enzymatic activity and how to pick the optimal interaction module to complement a given substrate. Here, we develop equations that predict the optimal binding strength of a kinase docking interaction and validate it using numerical simulations and steady-state phosphorylation kinetics for tethered protein kinase A. We show that a kinase-substrate pair has an optimum docking strength that depends on their enzymatic constants, the tether architecture, the substrate concentration, and the kinetics of the docking interactions. We show that a reversible tether enhances phosphorylation rates most when 1) the docking strength is intermediate, 2) the substrate is nonoptimal, 3) the substrate concentration is low, 4) the docking interaction has rapid exchange kinetics, and 5) the tether optimizes the effective concentration of the intramolecular reaction. This work serves as a framework for interpreting mutations in kinase docking interactions and as a design guide for engineering enzyme scaffolds.

A Targetable N-Terminal Motif Orchestrates α-Synuclein Oligomer-to-Fibril Conversion.

Oligomeric species populated during α-synuclein aggregation are considered key drivers of neurodegeneration in Parkinson's disease. However, the development of oligomer-targeting therapeutics is constrained by our limited knowledge of their structure and the molecular determinants driving their conversion to fibrils. Phenol-soluble modulin α3 (PSMα3) is a nanomolar peptide binder of α-synuclein oligomers that inhibits aggregation by blocking oligomer-to-fibril conversion. Here, we investigate the binding of PSMα3 to α-synuclein oligomers to discover the mechanistic basis of this protective activity. We find that PSMα3 selectively targets an α-synuclein N-terminal motif (residues 36-61) that populates a distinct conformation in the mono- and oligomeric states. This α-synuclein region plays a pivotal role in oligomer-to-fibril conversion as its absence renders the central NAC domain insufficient to prompt this structural transition. The hereditary mutation G51D, associated with early onset Parkinson's disease, causes a conformational fluctuation in this region, leading to delayed oligomer-to-fibril conversion and an accumulation of oligomers that are resistant to remodeling by molecular chaperones. Overall, our findings unveil a new targetable region in α-synuclein oligomers, advance our comprehension of oligomer-to-amyloid fibril conversion, and reveal a new facet of α-synuclein pathogenic mutations.

Cell-Penetrating Peptides: Promising Therapeutics and Drug-Delivery Systems for Neurodegenerative Diseases.

Currently, one of the most significant and rapidly growing unmet medical challenges is the treatment of neurodegenerative diseases such as Alzheimer's disease (AD) and Parkinson's disease (PD). This challenge encompasses the imperative development of efficacious therapeutic agents and overcoming the intricacies of the blood-brain barrier for successful drug delivery. Here we focus on the delivery aspect with particular emphasis on cell-penetrating peptides (CPPs), widely used in basic and translational research as they enhance drug delivery to challenging targets such as tissue and cellular compartments and thus increase therapeutic efficacy. The combination of CPPs with nanomaterials such as nanoparticles (NPs) improves the performance, accuracy, and stability of drug delivery and enables higher drug loads. Our review presents and discusses research that utilizes CPPs, either alone or in conjugation with NPs, to mitigate the pathogenic effects of neurodegenerative diseases with particular reference to AD and PD.

4-Oxo-2-nonenal-Induced α-Synuclein Oligomers Interact with Membranes in the Cell, Leading to Mitochondrial Fragmentation.

Oxidative stress and formation of cytotoxic oligomers by the natively unfolded protein α-synuclein (α-syn) are both connected to the development of Parkinson's disease. This effect has been linked to lipid peroxidation and membrane disruption, but the specific mechanisms behind these phenomena remain unclear. To address this, we have prepared α-syn oligomers (αSOs) in vitro in the presence of the lipid peroxidation product 4-oxo-2-nonenal and investigated their interaction with live cells using in-cell NMR as well as stimulated emission depletion (STED) super-resolution and confocal microscopy. We find that the αSOs interact strongly with organellar components, leading to strong immobilization of the protein's otherwise flexible C-terminus. STED microscopy reveals that the oligomers localize to small circular structures inside the cell, while confocal microscopy shows mitochondrial fragmentation and association with both late endosome and retromer complex before the SOs interact with mitochondria. Our study provides direct evidence for close contact between cytotoxic α-syn aggregates and membraneous compartments in the cell.

Periostin C-Terminal Is Intrinsically Disordered and Interacts with 143 Proteins in an In Vitro Epidermal Model of Atopic Dermatitis.

Human periostin is a 78-91 kDa matricellular protein implicated in extracellular matrix remodeling, tumor development, metastasis, and inflammatory diseases like atopic dermatitis, psoriasis, and asthma. The protein consists of six domains, including an N-terminal Cys-rich CROPT domain, four fasciclin-1 domains, and a C-terminal domain. The exons encoding the C-terminal domain may be alternatively spliced by shuffling four exons, generating ten variants of unknown function. Here, we investigate the structure and interactome of the full-length variant of the C-terminal domain with no exons spliced out. The structural analysis showed that the C-terminal domain lacked a tertiary structure and was intrinsically disordered. In addition, we show that the motif responsible for heparin-binding is in the conserved very C-terminal part of periostin. Pull-down confirmed three known interaction partners and identified an additional 140 proteins, among which nine previously have been implicated in atopic dermatitis. Based on our findings, we suggest that the C-terminal domain of periostin facilitates interactions between connective tissue components in concert with the four fasciclin domains.

Induction, inhibition, and incorporation: Different roles for anionic and zwitterionic lysolipids in the fibrillation of the functional amyloid FapC.

Amyloid proteins are widespread in nature both as pathological species involved in several diseases and as functional entities that can provide protection and storage for the organism. Lipids have been found in amyloid deposits from various amyloid diseases and have been shown to strongly affect the formation and structure of both pathological and functional amyloid proteins. Here, we investigate how fibrillation of the functional amyloid FapC from Pseudomonas is affected by two lysolipids, the zwitterionic lipid 1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine and the anionic lipid 1-myristoyl-2-hydroxy-sn-glycero-3-phospho-(1'-rac-glycerol) (LPG). Small-angle X-ray scattering, circular dichroism, dynamic light scattering, and thioflavin T fluorescence measurements were performed simultaneously on the same sample to ensure reproducibility and allow a multimethod integrated analysis. We found that LPG strongly induces fibrillation around its critical micelle concentration (cmc) by promoting formation of large structures, which mature via accumulation of intermediate fibril structures with a large cross section. At concentrations above its cmc, LPG strongly inhibits fibrillation by locking FapC in a core-shell complex. In contrast, lipid 1-myristoyl-2-hydroxy-sn-glycero-3-phosphocholine induces fibrillation at concentrations above its cmc, not via strong interactions with FapC but by being incorporated during fibrillation and likely stabilizing the fibrillation nucleus to reduce the lag phase. Finally, we show that LPG is not incorporated into the fibril during assembly but rather can coat the final fibril. We conclude that lipids affect both the mechanism and outcome of fibrillation of functional amyloid, highlighting a role for lipid concentration and composition in the onset and mechanism of fibrillation in vivo.

Structural variations between small alarmone hydrolase dimers support different modes of regulation of the stringent response.

The bacterial stringent response involves wide-ranging metabolic reprogramming aimed at increasing long-term survivability during stress conditions. One of the hallmarks of the stringent response is the production of a set of modified nucleotides, known as alarmones, which affect a multitude of cellular pathways in diverse ways. Production and degradation of these molecules depend on the activity of enzymes from the RelA/SpoT homologous family, which come in both bifunctional (containing domains to both synthesize and hydrolyze alarmones) and monofunctional (consisting of only synthetase or hydrolase domain) variants, of which the structure, activity, and regulation of the bifunctional RelA/SpoT homologs have been studied most intensely. Despite playing an important role in guanosine nucleotide homeostasis in particular, mechanisms of regulation of the small alarmone hydrolases (SAHs) are still rather unclear. Here, we present crystal structures of SAH enzymes from Corynebacterium glutamicum (RelHCg) and Leptospira levettii (RelHLl) and show that while being highly similar, structural differences in substrate access and dimer conformations might be important for regulating their activity. We propose that a varied dimer form is a general property of the SAH family, based on current structural information as well as prediction models for this class of enzymes. Finally, subtle structural variations between monofunctional and bifunctional enzymes point to how these different classes of enzymes are regulated.

Glycation modulates alpha-synuclein fibrillization kinetics: A sweet spot for inhibition.

Glycation is a nonenzymatic posttranslational modification (PTM) known to be increased in the brains of hyperglycemic patients. Alpha-synuclein (αSN), a central player in the etiology of Parkinson's disease, can be glycated at lysine residues, thereby reducing αSN fibril formation in vitro and modulating αSN aggregation in cells. However, the molecular basis for these effects is unclear. To elucidate this, we investigated the aggregation of αSN modified by eight glycating agents, namely the dicarbonyl compound methylglyoxal (MGO) and the sugars ribose, fructose, mannose, glucose, galactose, sucrose, and lactose. We found that MGO and ribose modify αSN to the greatest extent, and these glycation products are the most efficient inhibitors of fibril formation. We show glycation primarily inhibits elongation rather than nucleation of αSN and has only a modest effect on the level of oligomerization. Furthermore, glycated αSN is not significantly incorporated into fibrils. For both MGO and ribose, we discovered that a level of ∼5 modifications per αSN is optimal for inhibition of elongation. The remaining sugars showed a weak but optimal inhibition at ∼2 modifications per αSN. We propose that this optimal level balances the affinity for the growing ends of the fibril (which decreases with the extent of modification) with the ability to block incorporation of subsequent αSN subunits (which increases with modification). Our results are not only relevant for other αSN PTMs but also for understanding PTMs affecting other fibrillogenic proteins and may thus open novel avenues for therapeutic intervention in protein aggregation disorders.

A penetratin-derived peptide reduces the membrane permeabilization and cell toxicity of α-synuclein oligomers.

Parkinson's disease is a neurodegenerative movement disorder associated with the intracellular aggregation of α-synuclein (α-syn). Cytotoxicity is mainly associated with the oligomeric species (αSOs) formed at early stages in α-syn aggregation. Consequently, there is an intense focus on the discovery of novel inhibitors such as peptides to inhibit oligomer formation and toxicity. Here, using peptide arrays, we identified nine peptides with high specificity and affinity for αSOs. Of these, peptides p194, p235, and p249 diverted α-syn aggregation from fibrils to amorphous aggregates with reduced ß-structures and increased random coil content. However, they did not reduce αSO cytotoxicity and permeabilization of large anionic unilamellar vesicles. In parallel, we identified a non-self-aggregating peptide (p216), derived from the cell-penetrating peptide penetratin, which showed 12-fold higher binding affinity to αSOs than to α-syn monomers (Kdapp 2.7 and 31.2 µM, respectively). p216 reduced αSOs-induced large anionic unilamellar vesicle membrane permeability at 10-1 to 10-3 mg/ml by almost 100%, was not toxic to SH-SY5Y cells, and reduced αSOs cytotoxicity by about 20%. We conclude that p216 is a promising starting point from which to develop peptides targeting toxic αSOs in Parkinson's disease.

Molecular-level insights into the surface-induced assembly of functional bacterial amyloid.

Protein coating material is important in many technological fields. The interaction between carbon nanomaterial and protein is especially interesting since it makes the development of novel hybrid materials possible. Functional bacterial amyloid (FuBA) is promising as a coating material because of its desirable features, such as well-defined molecular structure, robustness against harsh conditions, and easily engineerable functionality. Here, we report the systematic assembly of the functional amyloid protein, CsgA, from Escherichia coli (E. coli) on graphite. We characterize the assemblies using scanning tunneling microscopy (STM) and show that CsgA forms assemblies according to systematic patterns, dictated by the graphite lattice. In addition, we show that graphite flakes induce the fibrillization of CsgA, in vitro, suggesting a surface-induced conformational change of CsgA facilitated by the graphite lattice. Using coarse-grained molecular dynamics simulations, we model the adhesion and lamellar formation of a CsgA-derived peptide and conclude that peptides are adsorbed both as monomers and smaller aggregates leading initially to unordered graphite-bound aggregates, which are followed by rearrangement into lamellar structures. Finally, we show that CsgA-derived peptides can be immobilized in very systematic assemblies and their molecular orientation can be tuned using a small chaperone-like molecule. Our findings have implications for the development of FuBA-based biosensors, catalysts, and other technologies requiring well-defined protein assemblies on graphite.

Driving forces in amyloidosis: How does a light chain make a heavy heart?

Light-chain amyloidosis (AL) is a fatal disorder wherein the immunoglobulin light chain misfolds and aggregates, leading to amyloid plaques in various organs. Patient-specific mutations in the light chain variable domain (VL) are tightly linked to amyloidosis, but how these mutations drive AL is unknown. In recent work, Rottenaicher et al. analyze five mutations found in the VL of a patient with cardiac AL. Their data suggest that decreased VL stability and increased flexibility in the core of the VL, caused by mutations outside of this core, could be key to aggregation and highlight the delicate balancing act required for antibody maturation to enable antigen recognition while not altering protein biophysics.

Heparin promotes fibrillation of most phenol-soluble modulin virulence peptides from Staphylococcus aureus.

Phenol-soluble modulins (PSMs), such as α-PSMs, ß-PSMs, and δ-toxin, are virulence peptides secreted by different Staphylococcus aureus strains. PSMs are able to form amyloid fibrils, which may strengthen the biofilm matrix that promotes bacterial colonization of and extended growth on surfaces (e.g., cell tissue) and increases antibiotic resistance. Many components contribute to biofilm formation, including the human-produced highly sulfated glycosaminoglycan heparin. Although heparin promotes S. aureus infection, the molecular basis for this is unclear. Given that heparin is known to induce fibrillation of a wide range of proteins, we hypothesized that heparin aids bacterial colonization by promoting PSM fibrillation. Here, we address this hypothesis using a combination of thioflavin T-fluorescence kinetic studies, CD, FTIR, electron microscopy, and peptide microarrays to investigate the mechanism of aggregation, the structure of the fibrils, and identify possible binding regions. We found that heparin accelerates fibrillation of all α-PSMs (except PSMα2) and δ-toxin but inhibits ß-PSM fibrillation by blocking nucleation or reducing fibrillation levels. Given that S. aureus secretes higher levels of α-PSM than ß-PSM peptides, heparin is therefore likely to promote fibrillation overall. Heparin binding is driven by multiple positively charged lysine residues in α-PSMs and δ-toxins, the removal of which strongly reduced binding affinity. Binding of heparin did not affect the structure of the resulting fibrils, that is, the outcome of the aggregation process. Rather, heparin provided a scaffold to catalyze or inhibit fibrillation. Based on our findings, we speculate that heparin may strengthen the bacterial biofilm and therefore enhance colonization via increased PSM fibrillation.

How epigallocatechin gallate binds and assembles oligomeric forms of human alpha-synuclein.

The intrinsically disordered human protein α-synuclein (αSN) can self-associate into oligomers and amyloid fibrils. Several lines of evidence suggest that oligomeric αSN is cytotoxic, making it important to devise strategies to either prevent oligomer formation and/or inhibit the ensuing toxicity. (-)-epigallocatechin gallate (EGCG) has emerged as a molecular modulator of αSN self-assembly, as it reduces the flexibility of the C-terminal region of αSN in the oligomer and inhibits the oligomer's ability to perturb phospholipid membranes and induce cell death. However, a detailed structural and kinetic characterization of this interaction is still lacking. Here, we use liquid-state NMR spectroscopy to investigate how EGCG interacts with monomeric and oligomeric forms of αSN. We find that EGCG can bind to all parts of monomeric αSN but exhibits highest affinity for the N-terminal region. Monomeric αSN binds ∼54 molecules of EGCG in total during oligomerization. Furthermore, kinetic data suggest that EGCG dimerization is coupled with the αSN association reaction. In contrast, preformed oligomers only bind ∼7 EGCG molecules per protomer, in agreement with the more compact nature of the oligomer compared with the natively unfolded monomer. In previously conducted cell assays, as little as 0.36 EGCG per αSN reduce oligomer toxicity by 50%. Our study thus demonstrates that αSN cytotoxicity can be inhibited by small molecules at concentrations at least an order of magnitude below full binding capacity. We speculate this is due to cooperative binding of protein-stabilized EGCG dimers, which in turn implies synergy between protein association and EGCG dimerization.

Structural Basis for Dityrosine-Mediated Inhibition of α-Synuclein Fibrillization.

α-Synuclein (α-Syn) is an intrinsically disordered protein which self-assembles into highly organized ß-sheet structures that accumulate in plaques in brains of Parkinson's disease patients. Oxidative stress influences α-Syn structure and self-assembly; however, the basis for this remains unclear. Here we characterize the chemical and physical effects of mild oxidation on monomeric α-Syn and its aggregation. Using a combination of biophysical methods, small-angle X-ray scattering, and native ion mobility mass spectrometry, we find that oxidation leads to formation of intramolecular dityrosine cross-linkages and a compaction of the α-Syn monomer by a factor of √2. Oxidation-induced compaction is shown to inhibit ordered self-assembly and amyloid formation by steric hindrance, suggesting an important role of mild oxidation in preventing amyloid formation.

Size-Selective Phagocytic Clearance of Fibrillar α-Synuclein through Conformational Activation of Complement Receptor 4.

Aggregation of α-synuclein (αSN) is an important histological feature of Parkinson disease. Recent studies showed that the release of misfolded αSN from human and rodent neurons is relevant to the progression and spread of αSN pathology. Little is known, however, about the mechanisms responsible for clearance of extracellular αSN. This study found that human complement receptor (CR) 4 selectively bound fibrillar αSN, but not monomeric species. αSN is an abundant protein in the CNS, which potentially could overwhelm clearance of cytotoxic αSN species. The selectivity of CR4 toward binding fibrillar αSN consequently adds an important αSN receptor function for maintenance of brain homeostasis. Based on the recently solved structures of αSN fibrils and the known ligand preference of CR4, we hypothesize that the parallel monomer stacking in fibrillar αSN creates a known danger-associated molecular pattern of stretches of anionic side chains strongly bound by CR4. Conformational change in the receptor regulated tightly clearance of fibrillar αSN by human monocytes. The induced change coupled concomitantly with phagolysosome formation. Data mining of the brain transcriptome in Parkinson disease patients supported CR4 as an active αSN clearance mechanism in this disease. Our results associate an important part of the innate immune system, namely complement receptors, with the central molecular mechanisms of CNS protein aggregation in neurodegenerative disorders.

Functional Bacterial Amyloids: Understanding Fibrillation, Regulating Biofilm Fibril Formation and Organizing Surface Assemblies.

Functional amyloid is produced by many organisms but is particularly well understood in bacteria, where proteins such as CsgA (E. coli) and FapC (Pseudomonas) are assembled as functional bacterial amyloid (FuBA) on the cell surface in a carefully optimized process. Besides a host of helper proteins, FuBA formation is aided by multiple imperfect repeats which stabilize amyloid and streamline the aggregation mechanism to a fast-track assembly dominated by primary nucleation. These repeats, which are found in variable numbers in Pseudomonas, are most likely the structural core of the fibrils, though we still lack experimental data to determine whether the repeats give rise to ß-helix structures via stacked ß-hairpins (highly likely for CsgA) or more complicated arrangements (possibly the case for FapC). The response of FuBA fibrillation to denaturants suggests that nucleation and elongation involve equal amounts of folding, but protein chaperones preferentially target nucleation for effective inhibition. Smart peptides can be designed based on these imperfect repeats and modified with various flanking sequences to divert aggregation to less stable structures, leading to a reduction in biofilm formation. Small molecules such as EGCG can also divert FuBA to less organized structures, such as partially-folded oligomeric species, with the same detrimental effect on biofilm. Finally, the strong tendency of FuBA to self-assemble can lead to the formation of very regular two-dimensional amyloid films on structured surfaces such as graphite, which strongly implies future use in biosensors or other nanobiomaterials. In summary, the properties of functional amyloid are a much-needed corrective to the unfortunate association of amyloid with neurodegenerative disease and a testimony to nature's ability to get the best out of a protein fold.

A multimethod approach for analyzing FapC fibrillation and determining mass per length.

Amyloid proteins are found in a wide range of organisms owing to the high stability of the ß-sheet core of the amyloid fibrils. There are both pathological amyloids involved in various diseases and functional amyloids that play a beneficial role for the organism. The aggregation process is complex and often involves many different species. Full understanding of this process requires parallel acquisition of data by complementary techniques monitoring the time course of aggregation. This is not an easy task, given the often-stochastic nature of aggregation, which can lead to significant variations in lag time. Here, we investigate the aggregation process of the functional amyloid FapC by simultaneous use of four different techniques, namely dynamic light scattering, small-angle x-ray scattering (SAXS), circular dichroism, and Thioflavin T fluorescence. All these approaches are applied to the same FapC sample just after desalting. Our data allow us to construct a master time-course graph showing the same time-course of aggregation by all techniques. This allows us to integrate insights from approaches that report on different structural and length scales. During the lag phase, loosely aggregated oligomers with random-coil structure are formed, which subsequently transform to fibrils without accumulation of additional significant species. Subsequently, the loosely associated protofilaments/subfilaments, which form side by side, mature to more compact fibrils. Furthermore, we determine the mass per length of the mature fibrils, obtaining very similar results by SAXS (33 kDa/nm) and tilted-beam transmission electron microscopy (31 kDa/nm). Transmission electron microscopy showed that the fibrils consist of primarily two protofilaments and similar dimensions of the cross section of the fibrils as revealed by SAXS modeling when the number of protofilaments per fibril was taken into account. Mass per length information underscores the general usefulness of SAXS in fibrillation analysis and provides an important constraint for further modeling the fibril structures.

Cys-labeling kinetics of membrane protein GlpG: a role for specific SDS binding and micelle changes?

Empirically, α-helical membrane protein folding stability in surfactant micelles can be tuned by varying the mole fraction MFSDS of anionic (sodium dodecyl sulfate (SDS)) relative to nonionic (e.g., dodecyl maltoside (DDM)) surfactant, but we lack a satisfying physical explanation of this phenomenon. Cysteine labeling (CL) has thus far only been used to study the topology of membrane proteins, not their stability or folding behavior. Here, we use CL to investigate membrane protein folding in mixed DDM-SDS micelles. Labeling kinetics of the intramembrane protease GlpG are consistent with simple two-state unfolding-and-exchange rates for seven single-Cys GlpG variants over most of the explored MFSDS range, along with exchange from the native state at low MFSDS (which inconveniently precludes measurement of unfolding kinetics under native conditions). However, for two mutants, labeling rates decline with MFSDS at 0-0.2 MFSDS (i.e., native conditions). Thus, an increase in MFSDS seems to be a protective factor for these two positions, but not for the five others. We propose different scenarios to explain this and find the most plausible ones to involve preferential binding of SDS monomers to the site of CL (based on computational simulations) along with changes in size and shape of the mixed micelle with changing MFSDS (based on SAXS studies). These nonlinear impacts on protein stability highlights a multifaceted role for SDS in membrane protein denaturation, involving both direct interactions of monomeric SDS and changes in micelle size and shape along with the general effects on protein stability of changes in micelle composition.

Lipid Peroxidation Products HNE and ONE Promote and Stabilize Alpha-Synuclein Oligomers by Chemical Modifications.

The aggregation of α-synuclein (αSN) and increased oxidative stress leading to lipid peroxidation are pathological characteristics of Parkinson's disease (PD). Here, we report that aggregation of αSN in the presence of lipid peroxidation products 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE) increases the stability and the yield of αSN oligomers (αSO). Further, we show that ONE is more efficient than HNE at inducing αSO. In addition, we demonstrate that the two αSO differ in both size and shape. ONE-αSO are smaller in size than HNE-αSO, except when they are formed at a high molar excess of aldehyde. In both monomeric and oligomeric αSN, His50 is the main target of HNE modification, and HNE-induced oligomerization is severely retarded in the mutant His50Ala αSN. In contrast, ONE-induced aggregation of His50Ala αSN occurs readily, demonstrating the different pathways for inducing αSN aggregation by HNE and ONE. Our results show different morphologies of the HNE-treated and ONE-treated αSO and different roles of His50 in their modification of αSN, but we also observe structural similarities between these αSO and the non-treated αSO, e.g., flexible C-terminus, a folded core composed of the N-terminal and NAC region. Furthermore, HNE-αSO show a similar deuterium uptake as a previously characterized oligomer formed by non-treated αSO, suggesting that the backbone conformational dynamics of their folded cores resemble one another.