Antiparallel Β-sheet Conformation Research Articles

Structure-Toxicity Relationship in Intermediate Fibrils from α-Synuclein Condensates.

The aberrant aggregation of α-synuclein (αS) into amyloid fibrils is associated with a range of highly debilitating neurodegenerative conditions, including Parkinson's disease. Although the structural properties of mature amyloids of αS are currently understood, the nature of transient protofilaments and fibrils that appear during αS aggregation remains elusive. Using solid-state nuclear magnetic resonance (ssNMR), cryogenic electron microscopy (cryo-EM), and biophysical methods, we here characterized intermediate amyloid fibrils of αS forming during the aggregation from liquid-like spherical condensates to mature amyloids adopting the structure of pathologically observed aggregates. These transient amyloid intermediates, which induce significant levels of cytotoxicity when incubated with neuronal cells, were found to be stabilized by a small core in an antiparallel β-sheet conformation, with a disordered N-terminal region of the protein remaining available to mediate membrane binding. In contrast, mature amyloids that subsequently appear during the aggregation showed different structural and biological properties, including low levels of cytotoxicity, a rearranged structured core embedding also the N-terminal region, and a reduced propensity to interact with the membrane. The characterization of these two fibrillar forms of αS, and the use of antibodies and designed mutants, enabled us to clarify the role of critical structural elements endowing intermediate amyloid species with the ability to interact with membranes and induce cytotoxicity.

Helical Superstructures from the Hierarchical Self-Assembly of Coil-Coil Block Copolymer Guided by Side Chain Amyloid-β(17-19) LVF Peptide.

The rational design of precisely controlled hierarchical chiral nanostructures from synthetic polymers garnered inspiration from sophisticated biological materials. Since chiral peptide motifs induce helix formation in macromolecules, herein we report the synthesis of a novel type of hybrid polymer consisting of a β-sheet forming a LVF [L = leucine, V = valine, and F = phenylalanine] tripeptide pendant polymethacrylate block and a poly[poly(ethylene glycol) methyl ether methacrylate] (PPEGMA) block. The designed block copolymer self-organized into helical superstructures with a left-handed twisting sense, as visualized by field emission scanning electron microscopy, transmission electron microscopy, and atomic force microscopy. This intriguing hierarchical self-assembly is driven by the minimalistic peptide motif that itself has a high propensity to adopt an antiparallel β-sheet conformation. We also report the generation of a diverse array of nanostructures, including spherical micelles, spindle micelles, rod-like micelles, vesicles, helical supramolecular fibers, and helical toroids via self-assembly of the designed block copolymer in tetrahydrofuran/water mixed solvents. To realize the observable helical superstructure, a twisted two-dimensional core-shell tape is proposed as a structure model in which the peptide segments form an antiparallel β-sheet with a polymer shell. The findings contribute to the advancement of a helical polymer or the superhelical self-assembly of polymers, paving the way for diverse applications in materials science and related fields.

Comparative stability study and aggregate analysis of Bevacizumab marketed formulations using advanced analytical techniques

Bevacizumab (Bvz) is the most preferred recombinant humanized monoclonal antibody in biosimilar development due to its prominence as a standard treatment in the oncology space. Therapeutic monoclonal antibodies are typically more complex and unlikely to produce a replica. As a result, regulatory agencies allow approval of biosimilars that differ structurally and functionally from their reference product, but these differences should not have any clinical significance. To identify these significant discrepancies, it is essential to perform a thorough characterization of critical product attributes both in real-time and after storage until the product's expiration. In the present study, two Bvz biosimilar brands (Bio-1 and Bio-2) marketed in India were evaluated and compared with the reference product Avastin® to assess their degree of similarity. A comprehensive physicochemical characterization of biosimilars and reference product was performed using orthogonal techniques including LC-ESI-QTOF, MALDI-TOF, FTIR-ATR, iCIEF, rCE, nrCE, UV280, and RP-HPLC. Furthermore, Bvz formulations under study were subjected to various stress conditions of thermal (elevated temperature 50 ± 2 °C), chemical (acidic pH 3.0 ± 0.2, neutral pH 7.0 ± 0.2, and basic pH 10.0 ± 0.2), and mechanical (agitation 200 rpm) for comparative stability evaluation. Any alteration in the secondary structure of the native protein was detected and quantified using far-UV circular dichroism (CD), indicating an average of 15% and 11% loss in native antiparallel β-sheet conformation respectively in Bio-1 and Bio-2 upon exposure to elevated temperature and high pH. Additionally, covalent or non-covalent aggregates formed as a function of elevated temperature and agitation were quantified using SEC-MALS.

A new synthetic protegrin as a promising peptide with antibacterial activity against MDR Gram-negative pathogens.

Protegrins are a family of natural peptides from the innate immune system of vertebrates, with broad-spectrum antimicrobial activity. However, the toxicity and haemolysis of protegrin-1 (PG-1) at low concentrations renders it useless for therapeutic application. We rationally designed PLP-3, a novel synthetic PG-1-like peptide, comprising key activity features of protegrins in a constrained bicyclic structure. Our main objective was to investigate PLP-3's activity against MDR strains of Acinetobacter baumannii, Pseudomonas aeruginosa and Klebsiella pneumoniae and to analyse its haemolysis and cytotoxicity. Peptide synthesis was performed via solid phase and intramolecular ligation in solution, and the correct folding of the peptide was verified by circular dichroism. Antimicrobial activity was performed through broth microdilution. The test panel contained 45 bacterial strains belonging to A. baumannii, P. aeruginosa and K. pneumoniae (15 strains per species) comprising colistin-resistant and MDR strains. Cytotoxicity was assessed by XTT cell viability assays using HeLa and A549 cells and haemolysis of human erythrocytes. PLP-3 was successfully synthesized, and its antiparallel β-sheet conformation was confirmed. Antimicrobial activity screening showed MIC90 values of 2 mg/L for A. baumannii, 16 mg/L for K. pneumoniae and 8 mg/L for P. aeruginosa. The haemolysis IC50 value was 48.53 mg/L. Cytotoxicity against human HeLa and A549 cells showed values of ca. 200 mg/L in both cell lines resulting in a 100-fold selectivity window for bacterial over human cells. PLP-3 has potent antimicrobial activity, especially against A. baumannii, while maintaining low haemolysis and toxicity against human cell lines at antimicrobial concentrations. These characteristics make PLP-3 a promising peptide with an interesting therapeutic window.

Structural Dissection of the First Events Following Membrane Binding of the Islet Amyloid Polypeptide

The islet amyloid polypeptide (IAPP) is the main constituent of the amyloid fibrils found in the pancreas of type 2 diabetes patients. The aggregation of IAPP is known to cause cell death, where the cell membrane plays a dual role: being a catalyst of IAPP aggregation and being the target of IAPP toxicity. Using ATR-FTIR spectroscopy, transmission electron microscopy, and molecular dynamics simulations we investigate the very first molecular steps following IAPP binding to a lipid membrane. In particular, we assess the combined effects of the charge state of amino-acid residue 18 and the IAPP-membrane interactions on the structures of monomeric and aggregated IAPP. Distinct IAPP-membrane interaction modes for the various IAPP variants are revealed. Membrane binding causes IAPP to fold into an amphipathic α-helix, which in the case of H18K-, and H18R-IAPP readily moves beyond the headgroup region. For all IAPP variants but H18E-IAPP, the membrane-bound helix is an intermediate on the way to amyloid aggregation, while H18E-IAPP remains in a stable helical conformation. The fibrillar aggregates of wild-type IAPP and H18K-IAPP are dominated by an antiparallel β-sheet conformation, while H18R- and H18A-IAPP exhibit both antiparallel and parallel β-sheets as well as amorphous aggregates. Our results emphasize the decisive role of residue 18 for the structure and membrane interaction of IAPP. This residue is thus a good therapeutic target for destabilizing membrane-bound IAPP fibrils to inhibit their toxic actions.

Heterochirality-Mediated Cross-Strand Nested Hydrophobic Interaction Effects Manifested in Surface-Bound Peptide Assembly Structures.

Amino acid chirality has been envisioned as an important strategy to regulate structure and function of peptide self-assembled architectures. However, the molecular mechanism of chirality effects in peptide assemblies remains largely elusive. Here, the assembly structures of l-peptide polyphenylalanine F10 (FFFFFFFFFF) and block heterochiral peptide F5f5 (FFFFFfffff) composed of two FFFFF repeat blocks with opposite chirality were characterized at the single-molecule level by using scanning tunneling microscopy. Each peptide formed two distinctively different assembly structures on the HOPG surface, in which peptide chains took parallel and antiparallel β-sheet conformations, respectively. The molecular-level observations revealed that the staggered arrangement of cross-strand side chains achieved in the antiparallel β-sheet structure of the block heterochiral peptide facilitated intimate packing of side chains and maximized inter-residue van der Waals interactions, which led to more residues participating in assembly and greatly stabilized the β-sheet structure of the surface-bound peptide assembly, but such cross-strand nested interactions were not accessible in the heterochiral parallel β-sheet structure and the enantiomerically pure assembly structures. This work could contribute to the molecular insights of stereochemical interactions in peptide assemblies and feasibility of extending this block heterochirality pattern to other peptides with various lengths and amino acid compositions for structural regulations.

Structure analysis of the proteins associated with polyA repeat expansion disorders

Repeat regions are low-complexity regions in the human genome that largely code for intrinsic disorder in proteins. Expansions outside the normal thresholds in repeat regions are likely to be pathogenic, leading to the so-called repeat expansion diseases. There have been numerous studies on the most common group of repeat expansion diseases, which are the polyglutamine (polyQ) repeat expansion diseases, but there has been much less work done on the second-largest group of expansion repeats disorders, which involves the expansion of polyalanine (polyA) repeat tracts. In this article, we present a comprehensive study of the structural changes predicted using I-TASSER when comparing the wild type and enlarged structures of all known polyA expansion disorders. The results show that there is a reduction in α helices, an increase in extended strands in parallel and/or anti-parallel β-sheet conformation, an increase in random coils/loops and irregular elements, and a large increase in solvent-accessible surface area. When compared to the findings in polyQ expansions disorders we see similar trends, suggesting that the polyQ and polyA repeat expansion causes similar effects on the respective proteins, which lead to higher misfolding and aggregation propensities. Communicated by Ramaswamy H. Sarma

Hemopressin-Based pH-Sensitive Hydrogel: A Potential Bioactive Platform for Drug Delivery.

Peptides with proper sequences are capable of self-assembling into well-defined nanostructures, which can subsequently grow and entangle into three-dimensional nanomatrices. In this study, hemopressin, a cannabinoid receptor-modulating peptide derived from the α-chain of hemoglobin known to self-assemble into nanofibrils, was examined for its potential applicability as a gelator. The results indicated that hemopressin's gel formation was dependent on pH and salt concentration. Although hemopressin's macroscopic states showed differences, its microscopic structure remained largely unchanged in which it consisted mainly of the antiparallel β-sheet conformation as confirmed by FTIR (C=O stretch peaks at 1630 and 1695 cm-1) and CD (β-sheet peak at 195 nm). The major difference between the gel and sol states was displayed in the fibril length in which the gelation at pH 7.4 resulted in 4 μm fibrils, whereas the solution at pH 5.0 showed 800 nm fibrils. The pH-dependent sol-gel phase transition property was then utilized for the investigation of the pH-responsive release of FITC-dextran (4-40 kDa) from hemopressin fibrillary gel. Finally, the biocompatibility of the peptide was demonstrated by proliferation assay of cultured bone marrow mesenchymal stem cells. Altogether, the results suggested that hemopressin is a potentially promising candidate as a therapeutically active platform for drug delivery.

Biocompatible Materials Based on Self-Assembling Peptides on Ti25Nb10Zr Alloy: Molecular Structure and Organization Investigated by Synchrotron Radiation Induced Techniques.

In this work, we applied advanced Synchrotron Radiation (SR) induced techniques to the study of the chemisorption of the Self Assembling Peptide EAbuK16, i.e., H-Abu-Glu-Abu-Glu-Abu-Lys-Abu-Lys-Abu-Glu-Abu-Glu-Abu-Lys-Abu-Lys-NH2 that is able to spontaneously aggregate in anti-parallel β-sheet conformation, onto annealed Ti25Nb10Zr alloy surfaces. This synthetic amphiphilic oligopeptide is a good candidate to mimic extracellular matrix for bone prosthesis, since its β-sheets stack onto each other in a multilayer oriented nanostructure with internal pores of 5–200 nm size. To prepare the biomimetic material, Ti25Nb10Zr discs were treated with aqueous solutions of EAbuK16 at different pH values. Here we present the results achieved by performing SR-induced X-ray Photoelectron Spectroscopy (SR-XPS), angle-dependent Near Edge X-ray Absorption Fine Structure (NEXAFS) spectroscopy, FESEM and AFM imaging on Ti25Nb10Zr discs after incubation with self-assembling peptide solution at five different pH values, selected deliberately to investigate the best conditions for peptide immobilization.

Polyglutamine Fibrils: New Insights into Antiparallel β-Sheet Conformational Preference and Side Chain Structure.

Understanding the structure of polyglutamine (polyQ) amyloid-like fibril aggregates is crucial to gaining insights into the etiology of at least ten neurodegenerative disorders, including Huntington's disease. Here, we determine the structure of D2Q10K2 (Q10) fibrils using ultraviolet resonance Raman (UVRR) spectroscopy and molecular dynamics (MD). Using UVRR, we determine the fibril peptide backbone Ψ and glutamine (Gln) side chain χ3 dihedral angles. We find that most of the fibril peptide bonds adopt antiparallel β-sheet conformations; however, a small population of peptide bonds exist in parallel β-sheet structures. Using MD, we simulate three different potential fibril structural models that consist of either β-strands or β-hairpins. Comparing the experimentally measured Ψ and χ3 angle distributions to those obtained from the MD simulated models, we conclude that the basic structural motif of Q10 fibrils is an extended β-strand structure. Importantly, we determine from our MD simulations that Q10 fibril antiparallel β-sheets are thermodynamically more stable than parallel β-sheets. This accounts for why polyQ fibrils preferentially adopt antiparallel β-sheet conformations instead of in-register parallel β-sheets like most amyloidogenic peptides. In addition, we directly determine, for the first time, the structures of Gln side chains. Our structural data give new insights into the role that the Gln side chains play in the stabilization of polyQ fibrils. Finally, our work demonstrates the synergistic power and utility of combining UVRR measurements and MD modeling to determine the structure of amyloid-like fibrils.

Steric Crowding of the Turn Region Alters the Tertiary Fold of Amyloid-β18–35 and Makes It Soluble

Aβ self-assembles into parallel cross-β fibrillar aggregates, which is associated with Alzheimer's disease pathology. A central hairpin turn around residues 23-29 is a defining characteristic of Aβ in its aggregated state. Major biophysical properties of Aβ, including this turn, remain unaltered in the central fragment Aβ18-35. Here, we synthesize a single deletion mutant, ΔG25, with the aim of sterically hindering the hairpin turn in Aβ18-35. We find that the solubility of the peptide goes up by more than 20-fold. Although some oligomeric structures do form, solution state NMR spectroscopy shows that they have mostly random coil conformations. Fibrils ultimately form at a much higher concentration but have widths approximately twice that of Aβ18-35, suggesting an opening of the hairpin bend. Surprisingly, two-dimensional solid state NMR shows that the contact between Phe(19) and Leu(34) residues, observed in full-length Aβ and Aβ18-35, is still intact in these fibrils. This is possible if the monomers in the fibril are arranged in an antiparallel β-sheet conformation. Indeed, IR measurements, supported by tyrosine cross-linking experiments, provide a characteristic signature of the antiparallel β-sheet. We conclude that the self-assembly of Aβ is critically dependent on the hairpin turn and on the contact between the Phe(19) and Leu(34) regions, making them potentially sensitive targets for Alzheimer's therapeutics. Our results show the importance of specific conformations in an aggregation process thought to be primarily driven by nonspecific hydrophobic interactions.

Effect of pH on the Aggregation of α-syn12 Dimer in Explicit Water by Replica-Exchange Molecular Dynamics Simulation.

The dimeric structure of the N-terminal 12 residues drives the interaction of α-synuclein protein with membranes. Moreover, experimental studies indicated that the aggregation of α-synuclein is faster at low pH than neutral pH. Nevertheless, the effects of different pH on the structural characteristics of the α-syn12 dimer remain poorly understood. We performed 500 ns temperature replica exchange molecular dynamics (T-REMD) simulations of two α-syn12 peptides in explicit solvent. The free energy surfaces contain ten highly populated regions at physiological pH, while there are only three highly populated regions contained at acidic pH. The anti-parallel β-sheet conformations were found as the lowest free energy state. Additionally, these states are nearly flat with a very small barrier which indicates that these states can easily transit between themselves. The dimer undergoes a disorder to order transition from physiological pH to acidic pH and the α-syn12 dimer at acidic pH involves a faster dimerization process. Further, the Lys6–Asp2 contact may prevent the dimerization.

Heterodyne-Detected Achiral and Chiral Vibrational Sum Frequency Generation of Proteins at Air/Water Interface

We present complex achiral and chiral vibrational sum frequency generation (VSFG) spectra at the air/water interface of protein solutions by using heterodyne-detected VSFG. Bovine serum albumin, pepsin, concanavalin A, and α-chymotrypsin were measured as model proteins. The obtained achiral Im[χ(2)] spectra gave us insights into the molecular orientation of protein molecules and water at the interface. From the chiral Im[χ(2)] spectra in the NH stretching and amide I regions, the secondary structures of the interfacial proteins were deduced. We attributed the chiral signals in the amide I and NH stretching regions to the interface on the basis of the phase of the signals. All the achiral and chiral spectra in each region showed the same sign despite different secondary-structure contents of the examined proteins. Real-time observation of the spectral change of α-chymotrypsin was also performed by heterodyne-detected chiral VSFG. The signal intensity of the chiral Im[χ(2)] spectra in the NH stretching and amide I regions decreased on the scale of 10 min, originating from the decrease of the portion of antiparallel β-sheet conformation in the molecule. The conformational change occurred not in the bulk but at the interface. Heterodyne-detected achiral and chiral VSFG are capable of addressing the molecular orientation and conformation of proteins at air/water interfaces.

Alkynyl β-sheet peptidomimetics retain their anti-parallel sheet conformation when coordinated to tungsten

Peptide derivatives (8 and 9) of 2-amino-2′-carboxydiphenylacetylene (1) were prepared using the Sonogashira reaction and standard peptide chemistry. As reported earlier by Kemp and Li (Ref. [1]), 8 and 9 adopt anti-parallel β-sheet conformations in solution. Reaction of 8 and 9 with W(CO)3(dmtc)2 produced the tungsten mono-alkyne complexes 10 and 11 as a mixture of two inseparable diastereomers (10a and 10b, and 11a and 11b) resulting from two different spatial arrangements of the dmtc ligands. The respective mixtures of the two diastereomers were characterized by ES-MS and 1H NMR spectroscopy. The solution conformations of the diastereomers were probed using NOESY and DMSO titration experiments. The data from these experiments show that the peptide portions of 10 and 11 maintain their anti-parallel β-sheet conformations following coordination to tungsten.

Transformation between α-helix and β-sheet structures of one and two polyglutamine peptides in explicit water molecules by replica-exchange molecular dynamics simulations.

Aggregation of polyglutamine peptides with β-sheet structures is related to some important neurodegenerative diseases such as Huntington's disease. However, it is not clear how polyglutamine peptides form the β-sheets and aggregate. To understand this problem, we performed all-atom replica-exchange molecular dynamics simulations of one and two polyglutamine peptides with 10 glutamine residues in explicit water molecules. Our results show that two polyglutamine peptides mainly formed helix or coil structures when they are separated, as in the system with one-polyglutamine peptide. As the interpeptide distance decreases, the intrapeptide β-sheet structure sometimes appear as an intermediate state, and finally the interpeptide β-sheets are formed. We also find that the polyglutamine dimer tends to form the antiparallel β-sheet conformations rather than the parallel β-sheet, which is consistent with previous experiments and a coarse-grained molecular dynamics simulation.

Interaction between Cell-Penetrating Peptides and Acid-Sensitive Anionic Oligopeptides as a Model for the Design of Targeted Drug Carriers

Overcoming the nonspecific cellular uptake of cell-penetrating peptides (CPPs) is a major hurdle in their clinical application. Using pH as the activation switch, histidine-glutamic acid (HE) dipeptide repeats were fused to CPPs to trigger the membrane-penetrating activity at mildly acidic pH environments (i.e., pH 6.5 or below) while masking the internalization at neutral pH (i.e., pH 7.0 or above). In this study, a series of recombinant GST-fusion proteins containing an HE oligopeptide sequence (i.e., (HE)n with n = 8, 10, or 12) and a cationic CPP (i.e., YG(RG)6, YGR6G6, or Tat) were engineered for a pH-sensitive study comparing their cellular uptake and surface binding in cultured HeLa cells. Circular dichroism (CD) spectroscopy was performed to correlate differences between CPPs in secondary structure with the pH sensitivity. YGR6G6 with clustered arginine residues exhibited greater pH sensitivity in cellular uptake than YG(RG)6 with separated arginine residues. Increasing the stretch of HE repeats decreased cellular uptake and surface binding for both YG(RG)6 and YGR6G6. The ratio of cellular internalization at pH 7.5 vs 6.0 was not changed by the presence of serum. CD spectral data revealed that both (HE)10-Tat and (HE)10-YGR6G6 exhibited an unordered secondary structure, whereas (HE)10-YG(RG)6 adopted an antiparallel β-sheet conformation. This β-sheet conformation presumably stabilized the association of (HE)10 with YG(RG)6, leading to weakened pH sensitivity of (HE)10-YG(RG)6. On the other hand, the random-coiled structures, that is, (HE)10-YGR6G6 and (HE)10-Tat, both showed higher pH sensitivity as determined in cell experiments. The data presented in this study provide a basis for the future design of pH-sensitive HE-CPP carrier for targeted drug delivery.

Native and recombinant Pg-AMP1 show different antibacterial activity spectrum but similar folding behavior

Glycine-rich proteins (GRPs) derived from plants compose a family of proteins and peptides that share a glycine repeat domain and they can perform diverse functions. Two structural conformations have been proposed for GRPs: glycine loops arranged as a Velcro and an anti-parallel β-sheet with several β-strands. The antimicrobial peptide Pg-AMP1 is the only plant GRP with antibacterial activity reported so far and its structure remains unclear. Recently, its recombinant expression was reported, where the recombinant peptide had an additional methionine residue at the N-terminal and a histidine tag at the C-terminal (His6-tag). These changes seem to change the peptide's activity, generating a broader spectrum of antibacterial activity. In this report, through ab initio molecular modelling and molecular dynamics, it was observed that both native and recombinant peptide structures were composed of an N-terminal α-helix and a dynamic loop that represents two-thirds of the protein. In contrast to previous reports, it was observed that there is a tendency to adopt a globular fold instead of an extended one, which could be in both, glycine loops or anti-parallel β-sheet conformation. The recombinant peptide showed a slightly higher solvated potential energy compared to the native form, which could be related to the His6-tag exposition. In fact, the His6-tag could be mainly responsible for the broader spectrum of activity, but it does not seem to cause great structural changes. However, novel studies are needed for a better characterization of its pharmacological properties so that in the future novel drugs may be produced based on this peptide.

Assessing the Utility of Infrared Spectroscopy as a Structural Diagnostic Tool for β-Sheets in Self-Assembling Aromatic Peptide Amphiphiles

β-Sheets are a commonly found structural motif in self-assembling aromatic peptide amphiphiles, and their characteristic "amide I" infrared (IR) absorption bands are routinely used to support the formation of supramolecular structure. In this paper, we assess the utility of IR spectroscopy as a structural diagnostic tool for this class of self-assembling systems. Using 9-fluorene-methyloxycarbonyl dialanine (Fmoc-AA) and the analogous 9-fluorene-methylcarbonyl dialanine (Fmc-AA) as examples, we show that the origin of the band around 1680-1695 cm(-1) in Fourier transform infrared (FTIR) spectra, which was previously assigned to an antiparallel β-sheet conformation, is in fact absorption of the stacked carbamate group in Fmoc-peptides. IR spectra from (13)C-labeled samples support our conclusions. In addition, DFT frequency calculations on small stacks of aromatic peptides help to rationalize these results in terms of the individual vibrational modes.

Self-Assembly of Arg–Phe Nanostructures via the Solid–Vapor Phase Method

We report for the first time on the self-assembly of nanostructures composed exclusively of alternating positively charged and hydrophobic amino acids. A novel arginine/phenylalanine octapeptide, RF8, was synthesized. Because the low hydrophobicity of this sequence makes its spontaneous ordering through solution-based methods difficult, a recently proposed solid-vapor approach was used to obtain nanometric architectures on ITO/PET substrates. The formation of the nanostructures was investigated under different preparation conditions, specifically, under different gas-phase solvents (aniline, water, and dichloromethane), different peptide concentrations in the precursor solution, and different incubation times. The stability of the assemblies was experimentally studied by electron microscopy and thermogravimetric analysis coupled with mass spectrometry. The secondary structure was assessed by infrared and Raman spectroscopy, and the arrays were found to assume an antiparallel β-sheet conformation. FEG-SEM images clearly reveal the appearance of fibrillar structures that form extensive homogeneously distributed networks. A close relationship between the morphology and preparation parameters was found, and a concentration-triggered mechanism was suggested. Molecular dynamics simulations were performed to address the thermal stability and nature of intermolecular interactions of the putative assembly structure. Results obtained when water is considered as solvent shows that a stable lamellar structure is formed containing a thin layer of water in between the RF8 peptides that is stabilized by H-bonding.

S100A6 Amyloid Fibril Formation Is Calcium-modulated and Enhances Superoxide Dismutase-1 (SOD1) Aggregation

S100A6 is a small EF-hand calcium- and zinc-binding protein involved in the regulation of cell proliferation and cytoskeletal dynamics. It is overexpressed in neurodegenerative disorders and a proposed marker for Amyotrophic Lateral Sclerosis (ALS). Following recent reports of amyloid formation by S100 proteins, we investigated the aggregation properties of S100A6. Computational analysis using aggregation predictors Waltz and Zyggregator revealed increased propensity within S100A6 helices H(I) and H(IV). Subsequent analysis of Thioflavin-T binding kinetics under acidic conditions elicited a very fast process with no lag phase and extensive formation of aggregates and stacked fibrils as observed by electron microscopy. Ca(2+) exerted an inhibitory effect on the aggregation kinetics, which could be reverted upon chelation. An FT-IR investigation of the early conformational changes occurring under these conditions showed that Ca(2+) promotes anti-parallel β-sheet conformations that repress fibrillation. At pH 7, Ca(2+) rendered the fibril formation kinetics slower: time-resolved imaging showed that fibril formation is highly suppressed, with aggregates forming instead. In the absence of metals an extensive network of fibrils is formed. S100A6 oligomers, but not fibrils, were found to be cytotoxic, decreasing cell viability by up to 40%. This effect was not observed when the aggregates were formed in the presence of Ca(2+). Interestingly, native S1006 seeds SOD1 aggregation, shortening its nucleation process. This suggests a cross-talk between these two proteins involved in ALS. Overall, these results put forward novel roles for S100 proteins, whose metal-modulated aggregation propensity may be a key aspect in their physiology and function.