β-Folded amyloid fibrils are fibrous insoluble oligomers formed by the aggregation of amyloid proteins through β-folded secondary structures; they are the core component of amyloid plaques in Alzheimer's disease and were first discovered by Glenner and Wong in 1984^[18].

Under normal physiological conditions, Aβ primarily exists as amorphous soluble monomers. However, under specific pathological conditions, Aβ can self-assemble into β-sheet secondary structures and spontaneously aggregate into cross-β amyloid fibrils. Early researchers believed that the deposition of these amyloid fibrils was the main toxic product causing neuronal cell damage,Figure 1shows the structure of Aβ_1-40determined by solid-state NMR, where adjacent β strands form parallel β-sheet secondary structures through inter-side-chain interactions^[19]. Furthermore,Tauprotein abnormal phosphorylation also leads to the protein transitioning from its normal α-helix or random coil conformation into neurofibrillary tangles with a β-sheet conformation, which is another hallmark pathological feature used to identify Alzheimer's disease.

Building on this, Hardy and Higgins^[20]proposed the amyloid cascade hypothesis in 1992, which posits that Aβ deposition in the brain is the initiating event leading toTauprotein fibrillary tangle formation, loss of neuronal function, cognitive impairment, and a series of other pathological changes. The amyloid cascade hypothesis suggests that Alzheimer's disease begins with neuronal injury and subsequent neurodegeneration triggered by excessive production or impaired clearance of Aβ in the brain. Therefore, the key factor leading to Alzheimer's disease lies not in the final pathological product of β-amyloid fibril deposition (senile plaques), but in the alterations in synaptic plasticity induced by the appearance of soluble Aβ oligomers and earlier aggregates. Zhang et al.^[21]summarized research literature, clinical trials, and therapeutic strategies based on the "β-amyloid cascade hypothesis" over the past 30 years, pointing out that soluble Aβ oligomers play a central role in inducing Alzheimer's disease, further confirming that intervention strategies targeting early soluble Aβ oligomers are key to blocking the onset of the disease.

In recent years, more genetic and clinical evidence regarding Aβ has further provided strong support for the amyloid cascade hypothesis. DeMattos et al.^[22]used a transgenic mouse model carrying mutant human amyloid precursor protein to compare the effects of single versus double gene knockout of ApoE or clusterin on brain amyloid production. The study indicated that these two genes have an additive effect in inhibiting Aβ deposition; knocking out either gene significantly increased Aβ levels in the cerebrospinal fluid. Furthermore, both genes participate in the clearance pathway of soluble Aβ in the brain, playing a role in regulating Aβ metabolism, transport, and clearance. Liu et al.^[23]further confirmed through clinical trials that clusterin gene expression is significantly upregulated in neurons of patients with aging, stroke, and type II diabetes, making them more prone to Aβ aggregation.

The aforementioned genetic and clinical evidence has greatly promoted drug development targeting toxic Aβ oligomers, such as Verubecestat, an inhibitor drug targeting Beta-secretase 1 (BACE-1)^[24], and inhibitors targetingγ-secretase, such as Atabecestat and Semagacestat^[25-26]; however, these drugs failed to meet expectations in clinical trials, even triggering skepticism within the clinical medical community regarding the amyloid-beta hypothesis. It was not until the new generation of monoclonal antibody drugs targeting Aβ oligomers (Aducanumab^[27], Lecanemab^[28], etc.) demonstrated the potential to clear Aβ and slow cognitive decline in clinical trials involving patients in the early stages of the disease, thereby reconfirming the key role of Aβ in the pathogenesis of Alzheimer's disease. However, to date, the causes of Aβ production under pathological conditions, its assembly mechanisms, and conformational transition processes remain unclear, urging further research.

α-Since the concept of folding was proposed, it has long lacked crystallographic evidence and has therefore been regarded as an unstable conformation that appears transiently during protein folding. Until research confirmed that α-folding secondary structures are closely related to the occurrence of various neurodegenerative diseases: α-folding soluble oligomers can disrupt cell membrane integrity, induce calcium overload and mitochondrial dysfunction, ultimately leading to neuronal apoptosis^[29-32].

α-The concept of α-sheets as key intermediates in neurodegenerative diseases was first proposed by Armen et al. in 2004^[33]. Building on this, Daggett^[34]discovered during further molecular dynamics simulations of key toxic oligomeric proteins (prion protein, lysozyme variants, transthyretin) in various amyloid diseases that, under low pH conditions, partial denaturation of amyloid proteins can manifest as rare α-sheet conformations, and pointed out that α-sheet soluble oligomers are the core structures responsible for neurotoxicity. Asshown in Figure 2a, traditional β-sheet chains exhibit a zigzag cross-arrangement of hydrogen bonds, with single amino acids adopting a β-sheet conformation; α-helical chains present a coiled-coil structure with consistent hydrogen bond directionality, with single amino acids adopting a right-handed α-helical conformation; whereas α-sheets possess partial characteristics of both, with their —NH and —C$\stackrel{\mathrm{ }\mathrm{ }\mathrm{ }\mathrm{ }}{̿}$O groups oriented on opposite sides of the backbone, exhibiting consistent hydrogen bond directionality and polar arrangement, with single amino acids alternating between right-handed α-helix (α_R) and left-handed α-helix (α_L) conformations, and the entire peptide chain presenting a sheet-like state similar to β-sheets, where the sheets formed by inter-chain hydrogen bonds can be either parallel or antiparallel. The conformational distribution of single amino acids in α-sheets can be further corroborated from the Ramachandran plot obtained via molecular dynamics simulations (Figure 2b).

Meanwhile, circular dichroism (CD) spectroscopy, as a spectral analysis method based on the difference in absorption of left- and right-circularly polarized light by chiral molecules, can sensitively reflect peptide secondary structures in the far-ultraviolet region (190~260 nm). Different peptide secondary structures exhibit typical circular dichroism spectral characteristics, such asFigure 2cshown, where α-helices display negative peaks at approximately 222 and 208 nm, and a strong positive peak at approximately 193 nm; β-sheets (β-sheets) exhibit a weak negative peak at approximately 218 nm and a positive peak between 195~200 nm; random coils show only a single negative peak between 195~200 nm. In contrast, the circular dichroism spectral characteristics of α-sheets are entirely different from the classical peptide secondary structures mentioned above. Their typical features are: a weak positive peak appearing at approximately 218 nm, and a negative peak appearing around 200 nm, with the spectral signal exhibiting an almost mirror-symmetric relationship to that of β-sheets (Figure 2c, α-sheets and β-sheets). The main reasons for this uniqueness are: (1) Different backbone conformations: in β-sheets, all backbone amino acids adopt the β conformation, whereas α-sheet backbones are formed by alternating α_Rand α_Lconformations; (2) Different dipole arrangements: in β-sheets, the backbone C$\stackrel{\mathrm{ }\mathrm{ }\mathrm{ }\mathrm{ }}{̿}$O and N—H dipoles are arranged alternately, whereas in α-sheet backbones, all groups are aligned in the same direction, presenting highly ordered dipoles. This change in dipole orientation causes a reversal in the optical rotation sign of electronic transitions, ultimately leading to the mirrored differences observed in the circular dichroism spectra of α-sheets. Therefore, research on the circular dichroism spectra of α-sheets not only provides direct optical evidence for their existence and conformational identification in solution but also establishes an experimental foundation for the early screening and diagnosis of amyloid diseases.

To further confirm the role of α-helices in neurodegenerative diseases, Hopping et al.^[35]designed five peptides with different secondary structures based on Daggett's molecular dynamics simulation results: a β-sheet control peptide (β), a random coil control peptide (rc), and three differently modified α-helical peptides (α_1-3), among which the α-helical peptides employed an alternating D-/L-chiral amino acid strategy to stabilize the conformation. Studies confirmed that α-helical peptides can target and bind toxic oligomers, thereby inhibiting amyloid aggregation, asshown in Figures 3a and c, in transthyretin (TTR) and amyloid-β (Aβ) systems, α-helical peptides (α₁ and α₃) significantly inhibited the aggregation of both TTR and Aβ, with maximum inhibition rates of 72% and 87%, respectively, whereas the control β-sheet peptide (β) and random coil peptide (rc) showed no significant effect. Further cytotoxicity experiments (Figures 3b and d) also confirmed that α-helical peptides almost completely suppressed cytotoxicity. These results collectively demonstrate that α-helical peptides can specifically recognize and neutralize soluble toxic oligomers, effectively blocking the amyloid fibrillation process, preventing toxic oligomer-induced damage to cell membranes and mitochondria, and indirectly confirming that the soluble oligomers truly responsible for neurotoxicity in various neurodegenerative diseases possess an α-helical secondary structure, rather than a β-sheet structure.

Based on α-helix secondary structure's special hydrogen bond arrangement, soluble α-helical oligomers in amyloid proteins have positively charged amide hydrogens and negatively charged carbonyl oxygen groups on both sides of the peptide chain arranged neatly and exhibiting polar orientation. Asshown in Figure 4, adjacent peptide chains easily form hydrogen bonds under the attraction of interchain polar charges, subsequently stacking into early soluble amyloid oligomers. The dipole moment formed by this polar orientation greatly enhances the hydrophilic-lipophilic distribution difference of oligomer molecules, facilitating their crossing of cell membranes and binding to membrane protein receptors, thereby leading to neuronal damage. This conformation not only serves as a key mediator in the Aβ oligomer aggregation process but is also consistent with specific binding to antibodies for various neurodegenerative disease drugs^[34,36-37], which further provides clinical experimental support for the α-helix secondary structure serving as a toxic conformation of amyloid proteins.

Since the proposal of the β-amyloid cascade hypothesis^[38]targeting the elimination of β-amyloid fibrils and inhibiting pathways of β-amyloid fibril formation has remained a primary strategy for treating neurodegenerative diseases, led by Alzheimer's disease; however, clinical efficacy has been minimal over the past few decades^[39-40], leading to skepticism within the medical community regarding the β-amyloid cascade hypothesis. If the aforementioned α-sheet soluble oligomers are the primary conformations inducing neurotoxicity, this would explain why targeted antibody drugs in multiple clinical trials can specifically bind to soluble oligomers but fail to recognize the final mature amyloid fibrils^[36,41].

In molecular dynamics simulations of various proteins associated with neurodegenerative diseases, researchers found that under specific conditions such as pathogenic mutations or mechanical stress, the original β-sheet conformation in amyloid proteins is weakened, and the β-sheet backbone can undergo peptide-plane flipping^[42,44]to open a conversion channel between the ββ conformation and α_Rα_Lconformations, thereby triggering hydrogen bond rearrangement and side chain reorganization to achieve the conformational transition from β-sheet to α-helix (Fig. 5a, b). Peptide-plane flipping is very common in biological processes, such as the structural transition during the catalytic process of the Fig. 5cshown UvrB-DNA helicase^[43]. Therefore, in the early stages of neurodegenerative diseases, β-sheets form soluble α-helices through ββ to α_Rα_L-type peptide-plane flipping; this process can be regarded as a core event constituting early toxic oligomers. If the α-helical conformation serves as the true toxic intermediate, it will eventually undergo an inverse conversion from α_Rα_Lback to ββ to restore the β-sheet conformation, completing the assembly of the final β-amyloid fibrils.

To further analyze the reverse process of the conformational transition from α-helix to β-sheet, Hayward et al.^[45]proposed two distinct peptide plane flipping pathways via torsion-angle driving simulations: concerted flipping and sequential flipping. In concerted flipping, the entire peptide chain rotates synchronously, whereas in sequential flipping, individual amino acid residues flip one by one along the peptide chain from the N-terminus to the C-terminus. Simulation results indicate that during the sequential peptide plane flipping process in antiparallel β-sheets, geometric mismatches occur on the peptide chain when the peptide plane flips to 90°, preventing the maintenance of interchain hydrogen bonds. This ultimately leads to the collapse of the hydrogen bond network and causes peptide chain slippage and lateral separation (Figure 6). In contrast, for parallel β-sheets, both concerted and sequential flipping can maintain the hydrogen bond network without peptide chain dissociation, thereby achieving mutual conversion with the α-helix conformation (Figure 7a~d). Therefore, the transition from antiparallel β-sheet to α-helix cannot be completed without chain breakage, unlike parallel β-sheets; it must undergo brief chain separation followed by re-pairing. This explains why parallel β-sheets are more prevalent in mature amyloid fibers (with parallel β-sheet content exceeding 80%)^[46-47].

In recent years, multidisciplinary evidence has further consolidated the critical role of α-sheet soluble oligomers in neurodegenerative diseases. Shea et al.^[48]combined multiple characterization methods, including circular dichroism (CD), microfluidic modulation-infrared spectroscopy (MMS-IR), and nuclear magnetic resonance (NMR), to directly identify and determine the structural information of α-sheets during the Aβ lag phase. Furthermore, they confirmed in C. elegans and transgenic mouse model experiments that the key structure exerting cytotoxicity is α-sheet soluble oligomers rather than β-sheet fibrils (Figure 8a, d), and simultaneously designed α-sheet peptides capable of specifically neutralizing toxic oligomers to inhibit cytotoxicity. Prosswimmer et al.^[49]used transthyretin (TTR) as a model to capture, for the first time at the atomic scale, the entire process of β-sheets converting into α-sheets via peptide plane flipping,Figure 8edetailedly demonstrating the key transition steps during the conformational conversion: early changes in external conditions disrupt the native hydrogen bond network of β-sheets, causing rapid flipping of peptide planes and exposure of polar interfaces; subsequently, mediated by temporary hydrogen bonds formed between water molecules and the polar interfaces, the backbone ultimately forms an α-sheet conformation. Meanwhile, this study also confirmed that the "peptide plane flipping-water mediation-dipole driving" conformational transition pathway is universal across various neurodegenerative diseases, further clarifying the generation mechanism of α-sheet toxic oligomers.

Not only that, α-sheet conformation toxic oligomers have also been further confirmed in recent clinical studies; Serwer et al.^[50]utilized thin-section electron microscopy to discover, for the first time, a large number of light-dark biphasic lipofuscin inclusions in cerebral cortical cell sections from Alzheimer's patients. Among these, the light-colored inclusions have relatively smooth surfaces and lower contrast, whereas the dark-colored inclusions are filled with α-sheet filamentous oligomers (Fig. 8b, c). Further research revealed that dark inclusions represent an intermediate state in the conformational transition from α-sheet to β-sheet; as the α-to-β conformational transition proceeds and lipids are redistributed, they will eventually bleach into light-colored inclusions. Therefore, α-sheet toxic oligomers are a key transitional state in the maturation process of β-sheet amyloid fibrils. If the α-to-β conformational transition is incomplete, residual α-sheet toxic oligomers will disrupt lysosomal membranes and be released into neighboring cells to exert toxicity, thereby driving pathological propagation within the brain and ultimately leading to further damage to cerebral cortical cells.

As a rare polypeptide secondary structure, the α-sheet is closely related to the early pathogenesis of various neurodegenerative diseases, such as Alzheimer's disease. In the soluble oligomer stage prior to amyloid fibril deposition into amyloid plaques, polypeptides temporarily adopt an α-sheet conformation and exhibit strong cytotoxicity, thereby severely damaging neuronal cells. As the earliest and most pathogenic detectable structures, if α-sheet toxic oligomers can be specifically recognized and captured at the initial stage of neurodegenerative disease onset, the α-to-β conformational transition process can be intervened, significantly advancing the intervention window and providing possibilities for early screening and blockade of the disease. This is sufficient to subvert current conventional targeted therapies aimed at amyloid fibrils.^[51-52].

Shea and Daggett^[31]Systematically summarized the changes in dominant secondary structures during amyloid fibril deposition: from initial monomers with random coil conformations to low-molecular-weight soluble oligomers with α-helical conformations, then to high-molecular-weight fibril precursors with β-sheet conformations, and finally depositing as amyloid fibril plaques. Cytotoxicity analysis of different conformations revealed that cytotoxicity continuously decreases during the process of amyloid plaque deposition, with α-helical intermediates and the α-to-β conformational transition playing a key toxic role. Furthermore, based on an analysis of existing monitoring methods for the various conformations involved and the target sites of inhibitory drugs for different disease stages (Figure 9b), it is concluded that most antibodies and targeted inhibitors currently under development or already on the market^[27,53]target the β-sheet conformation or amyloid plaque stage occurring in the late phase of the disease; therefore, they cannot effectively clear the early α-helical oligomers that possess genuine neurotoxicity, leading to limited clinical efficacy over the long term.

Building on the aforementioned research, Shea, Chen, et al.^[32,54]further proposed an early diagnostic method for Alzheimer's disease called SOBA (Soluble Oligomer Binding Assay). This method utilizes an artificially synthesized α-sheet peptide (AP193) with alternating D-/L-chiral amino acids to specifically bind to the toxic α-sheet conformations within soluble oligomers, thereby reducing the damage caused by these toxic conformations to nerve cells. In a study of 379 plasma samples, SOBA monitoring achieved a specificity of up to 99% for identifying Alzheimer's disease and was capable of detecting high-risk patients 1 to 13 years (average 6 years) before symptom onset.Figure 10confirms that SOBA can specifically recognize and effectively capture toxic α-sheet oligomers, providing direct evidence for the application of SOBA in clinical early screening of body fluids. Meanwhile, since this method only requires complementary pairing of the α-sheet backbone, it can effectively avoid limitations imposed by side-chain sequences. Furthermore, by changing the monitoring sequence, the scope of detection can be expanded to include the diagnosis of other related neurodegenerative diseases such as Parkinson's disease. SOBA monitoring targeting toxic α-sheet conformations pioneers a diagnostic approach that integrates detection and therapy: as an early screening tool capable of monitoring both blood and cerebrospinal fluid, SOBA can identify high-risk populations as early as possible, providing them with a sufficient "window of intervention." As a targeted peptide drug against toxic α-sheet oligomers, its therapeutic scope can also be expanded through rational sequence design, holding promise for playing a role in the diagnosis of a wider range of neurodegenerative diseases.

Therefore, the rational design of α-sheet peptides that can specifically bind to toxic α-sheet oligomers shows great potential for early disease screening and targeted therapy. This is highly likely to replace existing amyloid fibril-targeting drugs and become the primary strategy for treating neurodegenerative diseases, with Alzheimer's disease being the foremost. However, there are currently few reports on research into α-sheet secondary structures, and issues regarding how to design and synthesize α-sheet peptides, as well as their assembly mechanisms and formation principles, urgently need further clarification.

β-sheets are formed by two or more fully extended β-strands cross-linked via hydrogen bonds between the backbone carbonyl oxygen and the amide hydrogen of adjacent strands, oriented perpendicular to the backbone direction; the inter-strand orientation can be parallel or antiparallel. Due to strong lateral stacking, assemblies rich in β-sheets exhibit high morphological polymorphism, similar to amyloid fibrils; the most typical β-sheet in nature is Aβ, which induces neurodegenerative diseases._1-42amyloid protein^[81].

In recent years, extensive experimental evidence and simulation work have indicated that the β-sheet secondary structure is the most common self-assembly scaffold for amphiphilic peptides^[80], as shown in Figure 15a, Han et al.^[82]used the amphiphilic tetrapeptide I₃K as a prototype and designed and synthesized L₃K by replacing isoleucine with leucine, which has weaker hydrophobicity. This confirmed that after the hydrophobic strength was reduced, the assembly morphology of L₃K degraded into random coil spherical micelles, whereas further extending the hydrophobic chain length to synthesize L₅K restored the β-sheet nanofibers, revealing the synergistic driving force of hydrogen bonding and hydrophobic interactions during peptide assembly. Furthermore, Zhou et al.^[83]conducted systematic simulations using REMD on the aforementioned I₃K, L₃K, and L₅K series peptides from monomers to trimers, confirming the competitive relationship between amino acid residue conformational preferences and interchain hydrogen bonding and hydrophobic interactions during the peptide assembly process (Figure 15b). Zhao et al.^[84]then designed and synthesized the homopeptide sequences I₄K₂, KI₄K, and I₂K₂I₂ based on this foundation. Experiments revealed that peptides composed of the same amino acids could achieve precise regulation of β-sheet side-chain packing merely by altering the arrangement order of hydrophilic/hydrophobic residues, ultimately realizing reversible transitions among three morphologies: nanofibers, nanotubes, and disordered aggregates (Figure 15c). The above studies provide the following fundamental strategies for hierarchical regulation of peptide self-assembly: (1) precisely exchanging the positions of hydrophilic/hydrophobic amino acid residues; (2) adjusting the hydrophobic chain length and side-chain geometric arrangement; (3) introducing amino acids with different chiralities.

The α-helix is the most common and abundant secondary structure in natural proteins. Its backbone carbonyl oxygen forms an intrachain hydrogen bond with the amide hydrogen of the fourth residue downstream (n,n+4), constituting the helical skeleton. The hydrogen bonds are parallel to the helix axis, exhibiting a periodic repetition of 3.6 residues per turn. With an average of 13 atoms per helix, the α-helix is also known as the 3.6₁₃-helix. Through complementary electrostatic and hydrophobic interactions between side chains, α-helical backbones can further wind into structurally stable coiled-coil higher-order assemblies. Due to their compact and regular hydrogen bond networks, they effectively shield polar groups in the backbone; thus, α-helices are widely distributed in the transmembrane regions of membrane proteins.

Therefore, self-assembling peptides designed and synthesized based on α-helical secondary structures typically possess relatively long and orderly arranged repeating sequences (Figure 16). Limited by sequence length, the design of α-helical peptide sequences necessitates the insertion of special structural components such as coiled-coil sticky ends at specific positions (Figure 16b).^[85-86]to achieve their higher-order self-assembly.

Unlike β-sheets, which form extensive cross-β interchain hydrogen bonds early during assembly, the hydrogen bonds maintaining α-helices are intrachain hydrogen bonds, all pointing in the same direction from the N-terminus to the C-terminus of the peptide chain. As early as the early 21st century, Woolfson et al.^[85]proposed a method for constructing α-helical peptide nanofibers using "sticky end" structural components: designing α-helices according to a heptad repeat sequence (abcdefg)_x, where positions a/d in the heptad repeat employ isoleucine/leucine to construct a hydrophobic core, positions e/g employ glutamic acid/lysine to form charge complementarity, and positions b/c/f are filled with alanine to maintain the basic propensity for α-helix formation. AsFigure 16ashows, two 28-residue self-assembling peptides (SAF-p1 and SAF-p2), each composed of four groups of heptad sequences, form parallel staggered sticky ends under the action of salt bridges at the e/g positions, inducing the peptides to achieve higher-order self-assembly in a coiled-coil form. Recently, this method of designing α-helical self-assembling peptides based on the heptad repeat sequence template (abcdefg)_xhas been confirmed to be universal^[87]. Based on this, Li et al.^[86]designed three groups of α-helical peptides, namely NN, NK, and HH, by mutating the 'a' position of the 2nd and 4th groups of heptad sequences to asparagine, lysine, and histidine, respectively. Among them, the NN sequence locks parallel coiled-coil dimers via buried intrachain Asn-Asn hydrogen bonds while exposing sticky ends; although the NK variant with introduced lysine can still form coiled-coil dimers, electrostatic repulsion weakens the connection of the sticky ends, preventing axial extension and significantly shortening the assembly length at high concentrations; whereas the HH variant, using histidine instead of asparagine, possesses metal ion responsiveness and can achieve axial assembly induced by copper ions.

The aforementioned study meticulously elucidates the division of labor and collaboration among four distinct types of non-covalent interactions during the hierarchical self-assembly of peptides: Throughout the hierarchical assembly process of α-helical peptides, peptide molecules first form an α-helical backbone driven by backbone hydrogen bonds; subsequently, under the synergy of side-chain hydrophobic and electrostatic interactions, they form parallel coiled-coil dimers; finally, driven by sticky ends formed through complementary sequences and charge interactions at the peptide termini, these dimers undergo axial association to assemble into nanoscale long fibers. Meanwhile, metal ion-histidine coordination can achieve precise regulation of axial relationships under specific pH conditions.

The templated design rules for mature α-helical peptides not only elucidate the mechanisms of different non-covalent interactions during peptide assembly, but also their specific hydrogen bonding orientation and amino acid conformational preferences are highly similar to α-folded peptides, which provides a theoretical foundation for our subsequent further design and synthesis of α-folded self-assembling peptides.

As early as the 1950s, Pauling and Corey^[10], while proposing classic secondary structures such as the α-helix and β-sheet, predicted an unusual sheet structure known as the "α-pleated sheet." Although the α-pleated sheet exhibits an extended polypeptide chain arrangement similar to that of the β-sheet overall, the carbonyl and amino groups on a single polypeptide chain are directionally arranged on opposite sides of the chain. This results in one side of the entire sheet being rich in negatively charged carbonyl groups, while the other side is rich in positively charged amino groups, thereby presenting a significant dipole moment (Figure 4,Figure 17). However, since its proposal, the α-pleated sheet secondary structure has been regarded by researchers as a transient intermediate during protein folding due to its instability under conventional conditions, and thus has been largely overlooked.

With advances in molecular dynamics simulations and crystallographic analysis techniques, the α-sheet secondary structure has received unprecedented attention in the past five years. Daggett et al.^[34,54]confirmed through molecular dynamics simulations that the α-sheet is the primary toxic conformation inducing neurodegenerative diseases, and based on this, constructed relevant early screening, intervention, and treatment strategies. Furthermore, α-sheet structures have also been discovered in natural proteins,Figure 17shows that the transmembrane region of the potassium ion channel protein is composed of four α-sheet strands, each containing five residues. This design fully utilizes the neatly arranged carbonyl negative charge array on one side of the α-sheet strands, enabling specific capture of K⁺while avoiding aggregation and toxicity of the α-sheet conformation itself^[42,88]. This special channel protein based on α-sheet strands also provides a paradigm for subsequent molecular design of artificial ion pore proteins^[89].

To date, preliminary progress has been made in research on α-sheet secondary structures; however, evidence supporting the existence of α-sheets still relies heavily on molecular dynamics simulations. To further confirm their structural authenticity, researchers are currently focusing on the construction of α-sheet self-assembling peptides and the search for relevant crystallographic evidence.

Unlike the classic β-sheet molecular arrangement, in α-sheets, the amide hydrogens and carbonyl oxygens are located on opposite sides of the α-sheet chains (Figure 18). To construct this special arrangement, researchers introduced amino acids with different chiralities into the peptide chains, employing strategies such as block heterochirality design or heterochiral enantiomer co-assembly to build a series of self-assembling peptides with α-sheet secondary structures. Kuhn et al.^[90]utilized equimolar co-assembly of all-L-type FFF and all-D-type^DF^DF^DF to obtain crystals, and confirmed the actual existence of the α-sheet structure through X-ray crystallographic analysis (Figure 18b). This study confirmed Pauling and Corey's 1951 prediction at the atomic level and simultaneously provided a theoretical foundation for subsequently designing true α-sheet polypeptides through amino acid chirality engineering.

However, FFF and ^DF^DF^DF co-assembled crystal analysis only captured the dimer structure and did not obtain periodically extended continuous sheets; therefore, it was impossible to determine whether the α-pleated sheet structure could be expanded to the entire sheet. Based on this, Hazari et al.^[91]through three sets of chiral mirror-complementary tripeptide sequences (FYF and ^DF^DY^DF, FWF and ^DF^DW^DF, FWF and ^DF^DY^DF) co-assembly, successfully overcame the stacking limitations of FFF aromatic side chains, expanding the α-pleated sheet dimer to the entire sheet (Figure 18a), providing true experimental evidence for the α-pleated sheet structure that previously existed only in theory, and thoroughly validating Pauling and Corey's prediction of the α-pleated sheet structure. Meanwhile, this study also demonstrated two peptide assembly regulation methods: central aromatic amino acid substitution and co-assembly with corresponding chiral racemates, offering insights for the design of subsequent supramolecular peptide structures.

Although the aforementioned work successfully resolved atomic-level α-sheet molecular structures in polypeptide crystals, as mentioned above, soluble α-sheet oligomers are the key toxic conformations leading to neurodegenerative diseases such as Alzheimer's disease. Therefore, the α-sheets that truly function in biological activities should be the soluble state formed by polypeptide self-assembly, which further highlights the importance of constructing self-assembling peptides based on α-sheets. In previous molecular dynamics simulations of α-sheet structures, researchers have found that under specific conditions, β-sheets can spontaneously transform into α-sheets through peptide plane flipping, with amino acids on the peptide chain flipping from a single ββ conformation to α_Rα_Lalternating arrangement conformation^[45].Figure 19illustrates the process of peptide plane flipping forming α-sheet peptide nanotubes. The α-sheet peptide nanotubes are formed by the coiling and closure of multiple continuous α-sheet chains, creating a continuous double-layer hydrogen bond network on the outer wall of the tube. Their carbonyl groups are uniformly distributed along the axis pointing inward, while the inner and outer side chains alternately point inside and outside the tube wall. The above research provides the following ideas for the subsequent design of α-sheet self-assembling peptides: (1) block heterochirality design of polypeptide sequences (alternating arrangement of D-/L-chiral amino acids); (2) co-assembly of polypeptides with different chiral enantiomers; (3) introduction of α_R or α_Lconformation amino acids to regulate the helical propensity of the peptide chain; (4) precise control of nanotube diameter by regulating the length of hydrophilic and hydrophobic chains.

Based on this, Zhou et al.^[13]utilized a peptide block heterochirality strategy to design and synthesize a series of amphiphilic self-assembling short peptides with alternating D-/L-chiral amino acids. The peptide sequences are Ac-L^DLL^DLK-NH₂(2D-L₄K) and Ac-^DLL^DLL^DK-NH₂(3D-L₄K). The peptide chain consists of four hydrophobic leucines linked to one hydrophilic lysine. To match the α-sheet conformational characteristics, the entire peptide chain was designed following an alternating D-/L-chiral amino acid strategy. Asshown in Figure 20a, cryo-EM characterization reveals that, unlike the β-sheet nanofibers formed by the assembly of all-L-type L₄K, the block heterochirality-designed 2D-L₄K and 3D-L₄K both assemble into nanotubes with morphologies distinctly different from β-sheet peptides. In previous predictions regarding the assembly morphology of α-sheet peptides, researchers speculated that parallel α-sheet chains would maintain hydrogen bonds throughout the assembly process and curl into a nanotube structure (Figure 19)^[45]. This result preliminarily confirms that 2D-L₄K and 3D-L₄K, designed using the alternating D-/L-chiral amino acid strategy, form stable soluble α-sheet structures. To further verify the authenticity of the α-sheet conformation, the REMD simulation results inFigure 20bshow that the four leucines on the entire peptide chain exhibit an α_Lα_Rα_Lα_R(α-helix) alternating arrangement conformation, which is completely consistent with the previously predicted α-sheet conformational distribution (Figure 2b). On this basis, the authors constructed a hybrid arrangement model featuring alternating sliding of parallel and antiparallel α-sheet layers. Under this arrangement, the α-sheet chains possess bending stress during axial extension to counteract the positive charge effect of lysine, ultimately curling and closing asshown in Figure 20cinto nanotubes with a wall thickness of approximately 2.1 nm, consistent with cryo-EM and neutron scattering experimental characterization data. The study also found that hydrogen bonds between α-sheet layers exist in a dynamically stable equilibrium state; these hydrogen bonds rapidly recover and reorganize after disruption. This research represents the first report to date successfully constructing an α-sheet secondary structure based on a self-assembling short peptide system, systematically confirming that α-sheets can be obtained through sequence-level chiral programming and can maintain stable structures in aqueous phases. This breakthrough not only fills the gap in non-canonical secondary structures within the field of short peptide self-assembly but also provides a design blueprint for subsequently simulating and designing multifunctional peptide nanotubes with polarized cavities, tunable diameters, and other features.

Considering the special hydrogen bonding arrangement of α-sheets, the strong dipole moment between their molecular chains makes them more prone to linear assembly under electrostatic interactions, generating morphological features distinctly different from typical secondary structures, thus holding greater potential as biomaterials. To date, based on the 2D-L₄K and 3D-L₄K α-sheet self-assembling short peptide sequences, we have designed more α-sheet self-assembling short peptides capable of stable existence. Meanwhile, we have successfully achieved preliminary progress in multiple application areas such as protein protection, biocatalysis, and piezoelectric response using α-sheet peptide nanotube materials. Currently, we are continuing to explore expanding their industrial applications.