Thermodynamics of oligomerization and Helix-to-sheet structural transition of amyloid ß-protein on anionic phospholipid vesicles.

Understanding oligomerization and aggregation of the amyloid-ß protein is important to elucidate the pathological mechanisms of Alzheimer's disease, and lipid membranes play critical roles in this process. In addition to studies reported by other groups, our group has also reported that the negatively-charged lipid bilayers with a high positive curvature induced α-helix-to-ß-sheet conformational transitions of amyloid-ß-(1-40) upon increase in protein density on the membrane surface and promoted amyloid fibril formation of the protein. Herein, we investigated detailed mechanisms of the conformational transition and oligomer formation of the amyloid-ß protein on the membrane surface. Changes in the fractions of the three protein conformers (free monomer, membrane-bound α-helix-rich conformation, and ß-sheet-rich conformation) were determined from the fluorescent spectral changes of the tryptophan probe in the protein. The helix-to-sheet structural transition on the surface was described by a thermodynamic model of octamer formation driven by entropic forces including hydrophobic interactions. These findings provide useful information for understanding the self-assembly of amyloidogenic proteins on lipid membrane surfaces.

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 ß-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.

Antimicrobial Activities of α-Helix and ß-Sheet Peptides against the Major Bovine Respiratory Disease Agent, Mannheimia haemolytica.

Bovine respiratory disease (BRD) is the leading cause of morbidity and mortality in cattle raised in North America. At the feedlot, cattle are subject to metaphylactic treatment with macrolides to prevent BRD, a practice that may promote antimicrobial resistance and has resulted in an urgent need for novel strategies. Mannheimia haemolytica is one of the major bacterial agents of BRD. The inhibitory effects of two amphipathic, α-helical (PRW4, WRL3) and one ß-sheet (WK2) antimicrobial peptides were evaluated against multidrug-resistant (MDR) M. haemolytica isolated from Alberta feedlots. WK2 was not cytotoxic against bovine turbinate (BT) cells by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. All three peptides inhibited M. haemolytica, with WK2 being the most efficacious against multiple isolates. At 8-16 µg/mL, WK2 was bactericidal against Mh 330 in broth, and at 32 µg/mL in the presence of BT cells, it reduced the population by 3 logs CFU/mL without causing cytotoxic effects. The membrane integrity of Mh 330 was examined using NPN (1-N-phenylnaphthylamine) and ONPG (o-Nitrophenyl ß-D-galactopyranoside), with both the inner and outer membranes being compromised. Thus, WK2 may be a viable alternative to the use of macrolides as part of BRD prevention and treatment strategies.

Resolving the Nanoscale Structure of ß-Sheet Peptide Self-Assemblies Using Single-Molecule Orientation-Localization Microscopy.

Synthetic peptides that self-assemble into cross-ß fibrils are versatile building blocks for engineered biomaterials due to their modularity and biocompatibility, but their structural and morphological similarities to amyloid species have been a long-standing concern for their translation. Further, their polymorphs are difficult to characterize by using spectroscopic and imaging techniques that rely on ensemble averaging to achieve high resolution. Here, we utilize Nile red (NR), an amyloidophilic fluorogenic probe, and single-molecule orientation-localization microscopy (SMOLM) to characterize fibrils formed by the designed amphipathic enantiomers KFE8L and KFE8D and the pathological amyloid-beta peptide Aß42. Importantly, NR SMOLM reveals the helical (bilayer) ribbon structure of both KFE8 and Aß42 and quantifies the precise tilt of the fibrils' inner and outer backbones in relevant buffer conditions without the need for covalent labeling or sequence mutations. SMOLM also distinguishes polymorphic branched and curved morphologies of KFE8, whose backbones exhibit much more heterogeneity than those of typical straight fibrils. Thus, SMOLM is a powerful tool to interrogate the structural differences and polymorphism between engineered and pathological cross-ß-rich fibrils.

ß-sheets mediate the conformational change and allosteric signal transmission between the AsLOV2 termini.

Avena sativa phototropin 1 light-oxygen-voltage 2 domain (AsLOV2) is a model protein of Per-Arnt-Sim (PAS) superfamily, characterized by conformational changes in response to external environmental stimuli. This conformational change begins with the unfolding of the N-terminal A'α helix in the dark state followed by the unfolding of the C-terminal Jα helix. The light state is characterized by the unfolded termini and the subsequent modifications in hydrogen bond patterns. In this photoreceptor, ß-sheets are identified as crucial components for mediating allosteric signal transmission between the two termini. Through combined experimental and computational investigations, the Hß and Iß strands are recognized as the most critical and influential ß-sheets in AsLOV2's allosteric mechanism. To elucidate the role of these ß-sheets, we introduced 13 distinct mutations (F490L, N492A, L493A, F494L, H495L, L496F, Q497A, R500A, F509L, Q513A, L514A, D515V, and T517V) and conducted comprehensive molecular dynamics simulations. In-depth hydrogen bond analyses emphasized the role of two hydrogen bonds, Asn482-Leu453 and Gln479-Val520, in the observed distinct behaviors of L493A, L496F, Q497A, and D515V mutants. This illustrates the role of ß-sheets in the transmission of the allosteric signal upon the photoactivation of the light state.

Fusion of amyloid beta with ferritin yields an isolated oligomeric beta-sheet-rich aggregate inside the ferritin cage.

Alzheimer's disease is a severe brain condition caused by the formation of amyloid plaques composed of amyloid beta (Aß) peptides. These peptides form oligomers, protofibrils, and fibrils before deposition into amyloid plaques. Among these intermediates, Aß oligomers (AßOs) were found to be the most toxic and therefore an appealing target for drug development and understanding their role in the disease. However, precise isolation and characterization of AßOs have proven challenging because AßOs tend to aggregate and form heterogeneous mixtures in solution. As a solution, we genetically fused the Aß peptide with a ferritin monomer. Such fusion allowed the encapsulation of precisely 24 Aß peptides inside the 24-mer ferritin cage. Using high-speed atomic force microscopy (HS-AFM), we disassembled ferritin and directly visualized the Aß core enclosed within the cage. The thioflavin-T assay (ThT) and attenuated total reflection infrared spectroscopy (ATR-IR) revealed the presence of a ß-sheet structure in the encapsulated oligomeric aggregate. Gallic acid, an amyloid inhibitor, can inhibit the fluorescence of ThT bound AßOs. Our approach represents a significant advancement in the isolation and characterization of ß-sheet rich AßOs and is expected to be useful for future studies of other disordered peptides such as α-synuclein and tau.

The structural landscape of the immunoglobulin fold by large-scale de novo design.

De novo designing immunoglobulin-like frameworks that allow for functional loop diversification shows great potential for crafting antibody-like scaffolds with fully customizable structures and functions. In this work, we combined de novo parametric design with deep-learning methods for protein structure prediction and design to explore the structural landscape of 7-stranded immunoglobulin domains. After screening folding of nearly 4 million designs, we have assembled a structurally diverse library of ~50,000 immunoglobulin domains with high-confidence AlphaFold2 predictions and structures diverging from naturally occurring ones. The designed dataset enabled us to identify structural requirements for the correct folding of immunoglobulin domains, shed light on ß-sheet-ß-sheet rotational preferences and how these are linked to functional properties. Our approach eliminates the need for preset loop conformations and opens the route to large-scale de novo design of immunoglobulin-like frameworks.

The Name Is Barrel, ß-Barrel.

ß-barrels are a class of membrane proteins made up of a cylindrical, anti-parallel ß-sheet with a hydrophobic exterior and a hydrophilic interior. The majority of proteins found in the outer membranes (OMs) of Gram-negative bacteria, mitochondria, and chloroplasts are ß-barrel outer membrane proteins (OMPs). ß-barrel OMPs have a diverse repertoire of functions, including nutrient transport, secretion, bacterial virulence, and enzymatic activity. Here, we discuss the broad functional classes of ß-barrel OMPs, how they are folded into the membrane, and the future of ß-barrel OMP research and its applications.

Spidroin-mimetic Engineered Protein Fibers with High Toughness and Minimized Batch-to-batch Variations through ß-sheets Co-assembly.

Synthetic spidroin fibers have not yet attained the same level of toughness and stability as natural spider silks due to the complexity of composition and hierarchical structure. Particularly, understanding the intricate interactions between spidroin components in spider fiber is still elusive. Herein, we report modular design and preparation of spidroin-mimetic fibers composed of a conservative C-terminus spidroin module, two different natural ß-sheets modules, and a non-spidroin random-coil module. The resulting fibers exhibit a toughness of ~200 MJ/m3, reaching the highest value among the reported artificial spider silks. The interactions between two components of recombinant spidroins facilitate the intermolecular co-assembly of ß-sheets, thereby enhancing the mechanical strength and reducing batch-to-batch variability in the dual-component spidroin fibers. Additionally, the dual-component spidroin fibers offer potential applications in implantable or even edible devices. Therefore, our work presents a generic strategy to develop high-performance protein fibers for diverse translations in different scenarios.

Maturation-dependent changes in the size, structure and seeding capacity of Aß42 amyloid fibrils.

Many proteins self-assemble to form amyloid fibrils, which are highly organized structures stabilized by a characteristic cross-ß network of hydrogen bonds. This process underlies a variety of human diseases and can be exploited to develop versatile functional biomaterials. Thus, protein self-assembly has been widely studied to shed light on the properties of fibrils and their intermediates. A still open question in the field concerns the microscopic processes that underlie the long-time behaviour and properties of amyloid fibrillar assemblies. Here, we use atomic force microscopy with angstrom-sensitivity to observe that amyloid fibrils undergo a maturation process, associated with an increase in both fibril length and thickness, leading to a decrease of their density, and to a change in their cross-ß sheet content. These changes affect the ability of the fibrils to catalyse the formation of new aggregates. The identification of these changes helps us understand the fibril maturation processes, facilitate the targeting of amyloid fibrils in drug discovery, and offer insight into the development of biocompatible and sustainable protein-based materials.

Elucidating the reversible and irreversible self-assembly mechanisms of low-complexity aromatic-rich kinked peptides and steric zipper peptides.

Many RNA-binding proteins such as fused-in sarcoma (FUS) can self-assemble into reversible liquid droplets and fibrils through the self-association of their low-complexity (LC) domains. Recent experiments have revealed that SYG-rich segments in the FUS LC domains play critical roles in the reversible self-assembly behaviors of FUS. These FUS LC segments alone can self-assemble into reversible kinked fibrils, which are markedly different from the canonical irreversible steric zipper ß-sheet fibrils. However, the molecular determinants underlying the reversible and irreversible self-assembly are poorly understood. Herein we conducted extensive all-atom and coarse-grained molecular dynamics simulations of four representative hexapeptides: two low-complexity aromatic-rich kinked peptides from the amyotrophic lateral sclerosis-related FUS protein, FUS37-42 (SYSGYS) and FUS54-59 (SYSSYG); and two steric zipper peptides from Alzheimer's-associated Aß and Tau proteins, Aß16-21 (KLVFFA) and Tau306-311 (VQIVYK). We dissected their reversible and irreversible self-assembly dynamics, predicted their phase separation behaviors, and elucidated the underpinning molecular interactions. Our simulations showed that alternating stickers (Tyr) and spacers (Gly and Ser) in FUS37-42 and FUS54-59 facilitate the formation of highly dynamic coil-rich oligomers and lead to reversible self-assembly, while consecutive hydrophobic residues of LVFF in Aß16-21 and IVY in Tau306-311 act as hydrophobic patches, favoring the formation of stable ß-sheet-rich oligomers and driving the irreversible self-assembly. Intriguingly, we found that FUS37-42 and FUS54-59 peptides, possessing the same amino acid composition and the same number of sticker and spacer residues, display differential self-assembly propensities. This finding suggests that the self-assembly behaviors of FUS peptides are fine-tuned by the site-specific patterning of spacer residues (Ser and Gly). This study provides significant mechanistic insights into reversible and irreversible peptide self-assembly, which would be helpful for understanding the molecular mechanisms underlying the formation of biological liquid condensates and pathological solid amyloid fibrils.

What happens when left meets right under equimolar and non-equimolar co-assembly of short peptide stereoisomers?

The co-assembly of different peptide chains usually leads to the formation of intricate architectures and sophisticated functions in biological systems. Although the co-assembly of stereoisomeric peptides represents a facile and flexible strategy for the synthesis of peptide-based nanomaterials with novel structures and potentially interesting properties, there is a lack of a general knowledge on how different isomers pack during assembly. Through the combined use of simulations and experimental observations, we report that heterochiral pairing is preferred to homochiral pairing at the molecular scale but self-sorting dictates beyond the molecular level for the mixtures of the short stereoisomeric ß-sheet peptides I3K (Ile-Ile-Ile-Lys). Furthermore, we demonstrate that flat ß-sheets and fibril morphology are always preferred to twisted ones during heterochiral pairing and subsequent assembly. However, the heterochiral pairing into flat morphology is not always at an equimolar ratio. Instead, a non-equimolar ratio (1:2) is observed for the mixing of homochiral LI3LK and heterochiral LI3DK, whose strand twisting degrees differ greatly. Such a study provides a paradigm for understanding the co-assembly of stereoisomeric peptides at the molecular scale and harnessing their blending for targeted nanostructures.

Structural Consequences of Introducing Bioactive Domains to Designer ß-Sheet Peptide Self-Assemblies.

We applied solid- and solution-state nuclear magnetic resonance spectroscopy to examine the structure of multidomain peptides composed of self-assembling ß-sheet domains linked to bioactive domains. Bioactive domains can be selected to stimulate specific biological responses (e.g., via receptor binding), while the ß-sheets provide the desirable nanoscale properties. Although previous work has established the efficacy of multidomain peptides, molecular-level characterization is lacking. The bioactive domains are intended to remain solvent-accessible without being incorporated into the ß-sheet structure. We tested for three possible anticipated molecular-level consequences of introducing bioactive domains to ß-sheet-forming peptides: (1) the bioactive domain has no effect on the self-assembling peptide structure; (2) the bioactive domain is incorporated into the ß-sheet nanofiber; and (3) the bioactive domain interferes with self-assembly such that nanofibers are not formed. The peptides involved in this study incorporated self-assembling domains based on the (SL)6 motif and bioactive domains including a VEGF-A mimic (QK), an IGF-mimic (IGF-1c), and a de novo SARS-CoV-2 binding peptide (SBP3). We observed all three of the anticipated outcomes from our examination of peptides, illustrating the unintended structural effects that could adversely affect the desired biofunctionality and biomaterial properties of the resulting peptide hydrogel. This work is the first attempt to evaluate the structural effects of incorporating bioactive domains into a set of peptides unified by a similar self-assembling peptide domain. These structural insights reveal unmet challenges in the design of highly tunable bioactive self-assembling peptide hydrogels.

Metal-driven folding and assembly of a minimal ß-sheet into a 3D-porous honeycomb framework.

In contrast to short helical peptides, constrained peptides, and foldamers, the design and fabrication of crystalline 3D frameworks from the ß-sheet peptides are rare because of their high self-aggregation propensity to form 1D architectures. Herein, we demonstrate the formation of a 3D porous honeycomb framework through the silver coordination of a minimal ß-sheet forming a peptide having terminal metal coordinated 4- and 3-pyridyl ligands.

Controlled Assembly and Disassembly of Higher-Order Peptide Nanotubes.

The controlled peptide self-assembly and disassembly are not only implicated in many cellular processes but also possess huge application potential in a wide range of biotechnology and biomedicine. ß-sheet peptide assemblies possess high kinetic stability, so it is usually hard to disassemble them rapidly. Here, we reported that both the self-assembly and disassembly of a designed short ß-sheet peptide IIIGGHK could be well harnessed through the variations of concentration, pH, and mechanical stirring. Microscopic imaging, neutron scattering, and infrared spectroscopy were used to track the assembly and disassembly processes upon these stimuli, especially the interconversion between thin, left-handed protofibrils and higher-order nanotubes with superstructural right-handedness. The underlying rationale for these controlled disassembly processes mainly lies in the fact that the specific His-His interactions between protofibrils were responsive to these stimuli. By taking advantage of the peptide self-assembly and disassembly, the encapsulation of the hydrophobic drug curcumin and its rapid release upon stimuli were achieved. Additionally, the peptide hydrogels facilitated the differentiation of neural cells while maintaining low cell cytotoxicity. We believe that such dynamic and reversible structural transformation in this work provides a distinctive paradigm for controlling the peptide self-assembly and disassembly, thus laying a foundation for practical applications of peptide assemblies.

Formation, structural characteristics and specific peptide identification of gluten amyloid fibrils.

This research investigates the formation of amyloid fibrils using enzymatically hydrolyzed peptides from gluten, including its components glutenin and gliadin. After completing the fibrillation incubation, the gluten group demonstrated the most significant average particle size (908.67 nm) and conversion ratio (57.64 %), with a 19.21 % increase in thioflavin T fluorescence intensity due to self-assembly. The results indicated increased levels of ß-sheet structures after fibrillation. The gliadin group exhibited the highest zeta potential (∼13 mV) and surface hydrophobicity (H0 = 809.70). Around 71.15 % of predicted amyloidogenic regions within gliadin peptides showed heightened hydrophobicity. These findings emphasize the collaborative influence of both glutenin and gliadin in the formation of gluten fibrils, influenced by hydrogen bonding, hydrophobic, and electrostatic interactions. They also highlight the crucial role played by gliadin with amyloidogenic fragments such as ILQQIL and SLVLQTL, aiming to provide a theoretical basis for understanding the utilization of gluten proteins.

Design of Functional Globular ß-Sheet Miniproteins.

The design of discrete ß-sheet peptides is far less advanced than e. g. the design of α-helical peptides. The reputation of ß-sheet peptides as being poorly soluble and aggregation-prone often hinders active design efforts. Here, we show that this reputation is unfounded. We demonstrate this by looking at the ß-hairpin and WW domain. Their structure and folding have been extensively studied and they have long served as model systems to investigate protein folding and folding kinetics. The resulting fundamental understanding has led to the development of hyperstable ß-sheet scaffolds that fold at temperatures of 100 °C or high concentrations of denaturants. These have been used to design functional miniproteins with protein or nucleic acid binding properties, in some cases with such success that medical applications are conceivable. The ß-sheet scaffolds are not always completely rigid, but can be specifically designed to respond to changes in pH, redox potential or presence of metal ions. Some engineered ß-sheet peptides also exhibit catalytic properties, although not comparable to those of natural proteins. Previous reviews have focused on the design of stably folded and non-aggregating ß-sheet sequences. In our review, we now also address design strategies to obtain functional miniproteins from ß-sheet folding motifs.

Molecular insights into atypical modes of ß-arrestin interaction with seven transmembrane receptors.

ß-arrestins (ßarrs) are multifunctional proteins involved in signaling and regulation of seven transmembrane receptors (7TMRs), and their interaction is driven primarily by agonist-induced receptor activation and phosphorylation. Here, we present seven cryo-electron microscopy structures of ßarrs either in the basal state, activated by the muscarinic receptor subtype 2 (M2R) through its third intracellular loop, or activated by the ßarr-biased decoy D6 receptor (D6R). Combined with biochemical, cellular, and biophysical experiments, these structural snapshots allow the visualization of atypical engagement of ßarrs with 7TMRs and also reveal a structural transition in the carboxyl terminus of ßarr2 from a ß strand to an α helix upon activation by D6R. Our study provides previously unanticipated molecular insights into the structural and functional diversity encoded in 7TMR-ßarr complexes with direct implications for exploring novel therapeutic avenues.

Zn2+ Binding Increases Parallel Structure in the Aß(16-22) Oligomer by Disrupting Salt Bridge in Antiparallel Structure.

The aggregation of monomeric amyloid ß protein (Aß) into oligomers and amyloid plaque in the brain is associated with Alzheimer's disease. The hydrophobic central core Aß16-22 has been widely studied due to its essential role in the fibrillization of full-length Aß peptides. Compared to the homogeneous antiparallel structure of Aß16-22 at the late stage, the early-stage prefibrillar aggregates contain varying proportions of different ß structures. In this work, we studied the appearance probabilities of various self-assembly structures of Aß16-22 and the effects of Zn2+ on these probabilities by replica exchange molecular dynamics simulations. It was found that at room temperature, Aß16-22 can readily form assembled ß-sheet structures in pure water, where a typical antiparallel arrangement dominates (24.8% of all sampled trimer structures). The addition of Zn2+ to the Aß16-22 solution will dramatically decrease the appearance probability of antiparallel trimer structures to 12.5% by disrupting the formation of the Lys16-Glu22 salt bridge. Meanwhile, the probabilities of hybrid antiparallel/parallel structures increase. Our simulation results not only reveal the competition between antiparallel and parallel structures in the Aß16-22 oligomers but also show that Zn2+ can affect the oligomer structures. The results also provide insights into the role of metal ions in the self-assembly of short peptides.

Structural Analyses of Designed α-Helix and ß-Sheet Peptide Nanofibers Using Solid-State Nuclear Magnetic Resonance and Cryo-Electron Microscopy and Introduction of Structure-Based Metal-Responsive Properties.

The 21-residue peptide α3, which is artificially designed and consists of three repeats of 7 residues, is known to rapidly assemble into the α-helix nanofiber. However, its molecular structure within the fiber has not yet been fully elucidated. Thus, we conducted a thorough investigation of the fiber's molecular structure using solid-state NMR and other techniques. The molecules were found to be primarily composed of the α-helix structure, with some regions near the C- and N-terminal adopting a 310-helix structure. Furthermore, it was discovered that ß-sheet hydrogen bonds were formed between the molecules at both ends. These intermolecular interactions caused the molecules to assemble parallelly in the same direction, forming helical fibers. In contrast, we designed two molecules, CaRP2 and ßKE, that can form ß-sheet intermolecular hydrogen bonds using the entire molecule instead of just the ends. Cryo-EM and other measurements confirmed that the nanofibers formed in a cross ß structure, albeit at a slow rate, with the formation times ranging from 1 to 42 days. To create peptide nanofibers that instantaneously respond to changes in the external environment, we designed several molecules (HDM1-3) based on α3 by introducing metal-binding sites. One of these molecules was found to be highly responsive to the addition of metal ions, inducing α-helix formation and simultaneously assembling into nanofibers. The nanofibers lost their structure upon removal of the metal ion. The change occurred promptly and was reversible, demonstrating that the intended level of responsiveness was attained.