The 75-99 C-Terminal Peptide of URG7 Protein Promotes α-Synuclein Disaggregation.

Up Regulation Gene seven (URG7) is the pseudogene 2 of the transporter ABCC6. The translated URG7 protein is localized with its single transmembrane α-helix in the endoplasmic reticulum (ER) membrane, orienting the N- and C-terminal regions in the lumen and cytoplasm, respectively, and it plays a crucial role in the folding of ER proteins. Previously, the C-terminal region of URG7 (PU, residues 75-99) has been shown to modify the aggregation state of α-synuclein in the lysate of HepG2 cells. PU analogs were synthesized, and their anti-aggregation potential was tested in vitro on α-synuclein obtained using recombinant DNA technology. Circular dichroism (CD), differential scanning calorimetry (DSC), Fourier-transform infrared (FTIR) spectroscopy, and microscopic techniques were used to assess the sample's behavior. The results show that the peptides studied by themselves are prone to clathrate-like structure formation of variable stability. Aggregation of α-synuclein is accompanied by desolvation of its peptide chain and an increase in intermolecular ß-sheets. The PU analogs all interact with α-synuclein aggregates and those possessing the most stable clathrate-like structures have the highest disaggregating effect. These findings suggest that the C-terminal region of URG7 may have a role in interacting and modulating α-synuclein structures and could be used to generate interesting therapeutic candidates as disaggregators of α-synuclein.

Peptide Inhibitors of Insulin Fibrillation: Current and Future Challenges.

Amyloidoses include a large variety of local and systemic diseases that share the common feature of protein unfolding or refolding into amyloid fibrils. The most studied amyloids are those directly involved in neurodegenerative diseases, while others, such as those formed by insulin, are surprisingly far less studied. Insulin is a very important polypeptide that plays a variety of biological roles and, first and foremost, is at the basis of the therapy of diabetic patients. It is well-known that it can form fibrils at the site of injection, leading to inflammation and immune response, in addition to other side effects. In this concise review, we analyze the current knowledge on insulin fibrillation, with a focus on the development of peptide-based inhibitors, which are promising candidates for their biocompatibility but still pose challenges to their effective use in therapy.

Macrocyclic ß-Sheets Stabilized by Hydrogen Bond Surrogates.

Mimics of protein secondary and tertiary structure offer rationally-designed inhibitors of biomolecular interactions. ß-Sheet mimics have a storied history in bioorganic chemistry and are typically designed with synthetic or natural turn segments. We hypothesized that replacement of terminal inter-ß-strand hydrogen bonds with hydrogen bond surrogates (HBS) may lead to conformationally-defined macrocyclic ß-sheets without the requirement for natural or synthetic ß-turns, thereby providing a minimal mimic of a protein ß-sheet. To access turn-less antiparallel ß-sheet mimics, we developed a facile solid phase synthesis protocol. We surveyed a dataset of protein ß-sheets for naturally observed interstrand side chain interactions. This bioinformatics survey highlighted an over-abundance of aromatic-aromatic, cation-π and ionic interactions in ß-sheets. In correspondence with natural ß-sheets, we find that minimal HBS mimics show robust ß-sheet formation when specific amino acid residue pairings are incorporated. In isolated ß-sheets, aromatic interactions endow superior conformational stability over ionic or cation-π interactions. Circular dichroism and NMR spectroscopies, along with high-resolution X-ray crystallography, support our design principles.

Tailored Cross-ß Assemblies Establish Peptide "Dominos" Structures for Anchoring Undruggable Pharmacophores.

ß-sheets have the ability to hierarchically stack into assemblies, and much effort has been spent on designing different peptides to regulate their assembly behaviors. Although the progress is remarkable, it remains challenging to manipulate them in a controllable way for achieving both tailored structures and specific functions. In this study, we obtained bola-like peptides using de novo design and combinatorial chemical screening. By regulating the solvent-accessible surface area of the peptide chain, a series of assemblies with different tilt angles and active sites of the ß-sheet were obtained, resembling collapsed dominos. The structure-activity relationship of the optimized peptide NQ40 system was established and its ability to target the PD-L1 was demonstrated. This study successfully established the structure-function relationship of ß-sheets assemblies and has positive implications on the rational design of peptide assemblies that possess recognition abilities.

Recombinant Silk Proteins with Additional Polyalanine Have Excellent Mechanical Properties.

This paper explores the structures of exogenous protein molecules that can effectively improve the mechanical properties of silkworm silk. Several transgenic vectors fused with the silkworm fibroin light chain and type 3 repeats in different multiples of the ampullate dragline silk protein 1 (MaSp1) from black widow spider with different lengths of the polyalanine motifs were constructed for this study. Transgenic silkworms were successfully obtained by piggyBac-mediated microinjection. Molecular detection showed that foreign proteins were successfully secreted and contained within the cocoon shells. According to the prediction of PONDR® VSL2 and PONDR® VL-XT, the type 3 repeats and the polyalanine motif of the MaSp1 protein were amorphous. The results of FTIR analysis showed that the content of ß-sheets in the silk of transgenic silkworms engineered with transgenic vectors with additional polyalanine was significantly higher than that of wild-type silkworm silk. Additionally, silk with a higher ß-sheet content had better fracture strength and Young's modulus. The mechanical properties of silk with longer chains of exogenous proteins were improved. In general, our results provide theoretical guidance and technical support for the large-scale production of excellent bionic silk.

ß-Strand-mediated interactions of protein domains.

Protein domains exist by themselves or in combination with other domains to form complex multidomain proteins. Defining domain boundaries in proteins is essential for understanding their evolution and function but is not trivial. More specifically, partitioning domains that interact by forming a single ß-sheet is known to be particularly troublesome for automatic structure-based domain decomposition pipelines. Here, we study edge-to-edge ß-strand interactions between domains in a protein chain, to help define the boundaries for some more difficult cases where a single ß-sheet spanning over two domains gives an appearance of one. We give a number of examples where ß-strands belonging to a single ß-sheet do not belong to a single domain and highlight the difficulties of automatic domain parsers on these examples. This work can be used as a baseline for defining domain boundaries in homologous proteins or proteins with similar domain interactions in the future.

The Impact of Composition and Morphology on Ionic Conductivity of Silk/Cellulose Bio-Composites Fabricated from Ionic Liquid and Varying Percentages of Coagulation Agents.

Blended biocomposites created from the electrostatic and hydrophobic interactions between polysaccharides and structural proteins exhibit useful and unique properties. However, engineering these biopolymers into applicable forms is challenging due to the coupling of the material's physicochemical properties to its morphology, and the undertaking that comes with controlling this. In this particular study, numerous properties of the Bombyx mori silk and microcrystalline cellulose biocomposites blended using ionic liquid and regenerated with various coagulation agents were investigated. Specifically, the relationship between the composition of polysaccharide-protein bio-electrolyte membranes and the resulting morphology and ionic conductivity is explored using numerous characterization techniques, including scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR), thermal gravimetric analysis (TGA), differential scanning calorimetry (DSC), X-ray scattering, atomic force microscopy (AFM) based nanoindentation, and dielectric relaxation spectroscopy (DRS). The results revealed that when silk is the dominating component in the biocomposite, the ionic conductivity is higher, which also correlates with higher ß-sheet content. However, when cellulose becomes the dominating component in the biocomposite, this relationship is not observed; instead, cellulose semicrystallinity and mechanical properties dominate the ionic conduction.

Structural Characterization of the CD44 Stem Region for Standard and Cancer-Associated Isoforms.

CD44 is widely expressed in most vertebrate cells, whereas the expression of CD44v6 is restricted to only a few tissues and has been considered to be associated with tumor progression and metastasis. Thus, CD44v6 has been recognized as a promising prognostic biomarker and therapeutic target for various cancers for more than a decade. However, despite many experimental studies, the structural dynamics and differences between CD44s and CD44v6, particularly in their stem region, still remain elusive. Here, a computational study was conducted to address these problems. We found that the stem of CD44s adopted predominantly two conformations, one featuring antiparallel ß-sheets and the other featuring parallel ß-sheets, whereas the stem of CD44v6 adopted mainly one conformation with relatively highly suppressed ß-sheet contents. Moreover, Phe215 was found to be essential in the ß-sheets of both CD44s and CD44v6. We finally found intramolecular Phe215-Trp224 hydrogen-bonding interactions and hydrophobic interactions with Phe215 that cooperatively drove conformational differences upon the addition of the v6 region to CD44. Our study elucidated the structural differences between the stem regions of CD44s and CD44v6 and thus can offer useful structural information for drug design to specifically target CD44v6 in promising clinical applications.

Improved Stability and Tunable Functionalization of Parallel ß-Sheets via Multicomponent N-Alkylation of the Turn Moiety.

In contrast to the myriad of methods available to produce α-helices and antiparallel ß-sheets in synthetic peptides, just a few are known for the construction of stable, non-cyclic parallel ß-sheets. Herein, we report an efficient on-resin approach for the assembly of parallel ß-sheet peptides in which the N-alkylated turn moiety enhances the stability and gives access to a variety of functionalizations without modifying the parallel strands. The key synthetic step of this strategy is the multicomponent construction of an N-alkylated turn using the Ugi reaction on varied isocyano-resins. This four-component process assembles the orthogonally protected turn fragment and incorporates handles serving for labeling/conjugation purposes or for reducing peptide aggregation. NMR and circular dichroism analyses confirm the better-structured and more stable parallel ß-sheets in the N-alkylated peptides compared to the non-functionalized variants.

Structural insights into the aggregation mechanism of huntingtin exon 1 protein fragment with different polyQ-lengths.

Huntington disease is a neurodegenerative disorder caused by the expansion of polyglutamine (polyQ) at the N-terminal of the huntingtin exon 1 protein. The detailed structure and the mechanism behind this aggregation remain unclear and it is assumed that the polyQ undergoes a conformational transition to the ß-sheet structure when it aggregates. Investigating the misfolding of polyQ facilitates the determination of the molecular mechanism of aggregation and can potentially help in developing a novel approach to inhibit polyQ aggregation. Moreover, the flanking sequences of the polyQ region play a vital role in structural changes and the aggregation mechanism. We performed all-atom molecular dynamics simulations to gain structural insights into the aggregation mechanism using eight different models with glutamine repeat lengths Q27 , Q27 P11 , Q34 , Q35 , Q36 , Q40 , Q50 , and Q50 P11 . In the models without flanking polyPs, we noticed that the transformation of a random coil to ß-sheet occurs when the number of Q increases. We also found that the flanking polyPs prevent aggregation by decreasing the probability of forming a ß-sheet structure. When polyQ length increases, the 17 N-terminal flanking residues are more likely to adopt a ß-sheet conformation from α-helix and coil. From our simulations, we suggest that at least 34 glutamines are required for initiating aggregation and 40 residues length is critical for the aggregation of huntingtin exon 1 protein for disease onset. This study provides structural insights into misfolding and the role of flanking sequences in huntingtin aggregation which will further help in developing therapeutic strategies for Huntington's disease.

Preparation and characterization of curcumin-loaded polymeric nanomicelles to interference with amyloidogenesis through glycation method.

Amyloid fibrils, including ß-amyloid (Aß) fibrils, are protein aggregates that form under certain conditions, associated with neurodegeneration that interfere with neural synaptic transmission resulting in some neural disorders, such as Alzheimer's disease. The aim of this study is to inhibit amyloidogenesis by using preparatory polymeric nanomicelles as therapeutic agents and also as nanocarriers for curcumin to target Aß fibrils through the glycation method of bovine serum albumin (BSA) in the presence of phosphate-buffered saline. Polymeric nanomicelles were prepared from phosphatidylethanolamine-distearoyl methoxypolyethylene glycol conjugates in the presence and absence of curcumin and then the morphological and structural characteristics of the nanomicelles were characterized in detail. Following the preparation of unloaded and curcumin-loaded nanomicelles with the desired size and properties, their effects on BSA glycation/fibrillation process were investigated. The samples were analyzed by thioflavin T (ThT) fluorescence and advanced glycation end (AGE) products autofluorescence measurements. The results showed that ThT fluorescence related to the formation of ß-sheets and AGE autofluorescence (associated with AGE production) decreased in the presence of curcumin-loaded nanomicelles more than other samples. In conclusion, the promising effect of curcumin-loaded nanomicelles on inhibition of amyloidogenesis through glycation process due to curcumin release and thus their ability to prevent the formation and accumulation of amyloid fibrils and so to suppress the Alzheimer's disease progression has been proven and can go for further investigations.

Rippled ß-Sheet Formation by an Amyloid-ß Fragment Indicates Expanded Scope of Sequence Space for Enantiomeric ß-Sheet Peptide Coassembly.

In 1953, Pauling and Corey predicted that enantiomeric ß-sheet peptides would coassemble into so-called "rippled" ß-sheets, in which the ß-sheets would consist of alternating l- and d-peptides. To date, this phenomenon has been investigated primarily with amphipathic peptide sequences composed of alternating hydrophilic and hydrophobic amino acid residues. Here, we show that enantiomers of a fragment of the amyloid-ß (Aß) peptide that does not follow this sequence pattern, amyloid-ß (16-22), readily coassembles into rippled ß-sheets. Equimolar mixtures of enantiomeric amyloid-ß (16-22) peptides assemble into supramolecular structures that exhibit distinct morphologies from those observed by self-assembly of the single enantiomer pleated ß-sheet fibrils. Formation of rippled ß-sheets composed of alternating l- and d-amyloid-ß (16-22) is confirmed by isotope-edited infrared spectroscopy and solid-state NMR spectroscopy. Sedimentation analysis reveals that rippled ß-sheet formation by l- and d-amyloid-ß (16-22) is energetically favorable relative to self-assembly into corresponding pleated ß-sheets. This work illustrates that coassembly of enantiomeric ß-sheet peptides into rippled ß-sheets is not limited to peptides with alternating hydrophobic/hydrophilic sequence patterns, but that a broader range of sequence space is available for the design and preparation of rippled ß-sheet materials.

3D 14 N/1 H Double Quantum/1 H Single Quantum Correlation Solid-State NMR for Probing the Parallel and Anti-Parallel Beta-Sheet Arrangement of Oligo-Peptides at Natural Abundance.

The beta (ß)-sheet structures of oligopeptides and polypeptides can be formed in anti-parallel (AP) and parallel (P) forms, which is an important feature to understand such structures. In principle, P- and AP-ß-sheet structures can be identified by the presence (AP) or absence (P) of inter-strand 1 HNH /1 HNH correlations on a diagonal in the corresponding 2D 1 H double quantum (DQ)/1 H single quantum (SQ) spectrum due to the different inter-strand 1 HNH /1 HNH distances between the two arrangements. However, the 1 HNH /1 HNH peaks overlap with the 1 HNH3+ /1 HNH3+ peaks, which always give cross-peaks regardless of the ß-sheet arrangement. The 1 HNH3+ /1 HNH3+ peaks disturb the observation of the presence/absence of 1 HNH /1 HNH correlations and the assignment of 1 HNH and 1 HNH3+ is not always available. Here, 3D 14 N/1 H DQ/1 H SQ correlation solid-state NMR experiments at fast magic angle spinning (70 kHz) are introduced to distinguish AP- and P-ß-sheet structures. The 14 N dimension allows the distinction of 1 HNH /1 HNH peaks from 1 HNH3+ /1 HNH3+ peaks with clear assignments of 1 HNH and 1 HNH3+ . In addition, the high natural abundance of 1 H and 14 N enables 3D 14 N/1 H DQ/1 H SQ experiments of oligo-alanines (Ala3-6 ) in four hours without isotope labelling.

Secondary Structure-Driven Hydrogelation Using Foldable Telechelic Polymer-Peptide Conjugates.

The synthesis of ABA and ABA' triblock polyethylene glycol-and polysarcosine-peptide conjugates is reported. The A/A' peptides are based on phenylalanine(F)-histidine(H) pentapeptide sequences FHFHF, which promote pH-switchable ß-sheet self-assembly into nanorods in water. Only parallel ß-sheet-driven folding and intermolecular assembly using ABA triblock polymer-peptide conjugates leads to interstrand cross-linking and hydrogelation, highlighting the impact of supramolecular interactions-directed structure formation at the nano- and mesoscopic level.

Segmental 13 C-Labeling and Raman Microspectroscopy of α-Synuclein Amyloid Formation.

Mapping conformational changes of α-synuclein (α-syn) from soluble, unstructured monomers to ß-sheet- rich aggregates is crucial towards understanding amyloid formation. Raman microspectroscopy is now used to spatially resolve conformational heterogeneity of amyloid aggregates and monitor amyloid formation of segmentally 13 C-labeled α-syn in real-time. As the 13 C-isotope shifts the amide-I stretching frequency to lower energy, the ligated construct, 13 C1-8612 CS87C-140 -α-syn, exhibits two distinct bands allowing for simultaneous detection of secondary structural changes in N-terminal 1-86 and C-terminal 87-140 residues. The disordered-to-ß-sheet conformational change is first observed for the N-terminal followed by the C-terminal region. Finally, Raman spectroscopic changes occurred prior to Thioflavin T fluorescence enhancement, indicating that the amide-I band is a superior probe of amyloid formation.

Structural motif of polyglutamine amyloid fibrils discerned with mixed-isotope infrared spectroscopy.

Polyglutamine (polyQ) sequences are found in a variety of proteins, and mutational expansion of the polyQ tract is associated with many neurodegenerative diseases. We study the amyloid fibril structure and aggregation kinetics of K2Q24K2W, a model polyQ sequence. Two structures have been proposed for amyloid fibrils formed by polyQ peptides. By forming fibrils composed of both (12)C and (13)C monomers, made possible by protein expression in Escherichia coli, we can restrict vibrational delocalization to measure 2D IR spectra of individual monomers within the fibrils. The spectra are consistent with a ß-turn structure in which each monomer forms an antiparallel hairpin and donates two strands to a single ß-sheet. Calculated spectra from atomistic molecular-dynamics simulations of the two proposed structures confirm the assignment. No spectroscopically distinct intermediates are observed in rapid-scan 2D IR kinetics measurements, suggesting that aggregation is highly cooperative. Although 2D IR spectroscopy has advantages over linear techniques, the isotope-mixing strategy will also be useful with standard Fourier transform IR spectroscopy.

A pH Switch for ß-Sheet Protein Folding.

Protein design advancements have led to biotechnological strategies based on more stable and more specific structures. Herein we present a 6-residue sequence (HPATGK) that acts as a stable structure-nucleating turn at physiological and higher pH but is notably unfavorable for chain direction reversal at low pH. When placed into the turn of a ß-sheet, this leads to a pH switch of folding. Using a standard 3-stranded ß-sheet model, the WW domain, it was found that the pH switch sequence insertion caused minimal change at pH 8 but a ca. 50 °C drop in the melting temperature (Tm ) was observed at pH 2.5: ΔΔGF ≥11.3 kJ mol-1 . Using the strategies demonstrated in this article, the redesign of ß-sheets to contain a global, or local, pH-dependent conformational switch should be possible.

Tuning the Inter-nanofibril Interaction To Regulate the Morphology and Function of Peptide/DNA Co-assembled Viral Mimics.

The ability to tune the inter-subunit interaction within the virus capsid may be critical to assembly and biological function. This process was extended here with peptide/DNA co-assembled viral mimics. The resulting co-assemblies, formed and stabilized by both peptide nanofibril-DNA and peptide nanofibril-nanofibril interactions, were tuned through hydrophobic packing interactions of the peptide sequences. By strengthening peptide side-chain complementarity and/or elongating the peptide chain (from 4 to 8 residues), we report strengthening the inter-nanofibril interaction to create stable nanococoons that give high gene-transfection efficacy.

pH-Controlled Formation of a Stable ß-Sheet and Amyloid-like Fibers from an Amphiphilic Peptide: The Importance of a Tailor-Made Binding Motif for Secondary Structure Formation.

The new amphiphilic peptide 1 is composed of alternating cyclohexyl side chains and guanidiniocarbonyl pyrrole (GCP) groups. In contrast to analogue 2, which contains lysine instead of the GCP groups and only exists as a random coil owing to charge repulsion, peptide 1 forms a stable ß-sheet at neutral pH in aqueous medium. The weakly basic GCP groups (pKa ≈7) are key for secondary structure formation as they stabilize the ß-sheet through mutual interactions (formation of a "GCP zipper"). The ß-sheets further aggregate into left-handed helically twisted fibers. However, ß-sheet formation is completely reversible as a function of pH. At low pH (ca. 4), peptide 1 is unstructured (random coil) as all GCP units are protonated. Only round colloidal particles are observed. The amyloid nature of the fibers formed at neutral pH was confirmed by staining experiments with Congo Red and thioflavin T. Furthermore, at millimolar concentrations, peptide 1 forms a stable hydrogel.

Racemic Amino Acid Assembly Enables Supramolecular ß-Sheet Transition with Property Modulations.

Amino acids are the most simplistic bio-building blocks and perform a variety of functions in metabolic activities. Increasing publications report that amino acid-based superstructures present amyloid-like characteristics, arising from their supramolecular ß-sheet secondary structures driven by hydrogen-bonding-connected supramolecular ß-strands, which are formed by head-to-tail hydrogen bonds between terminal amino and carboxyl groups of the adjacent residues. Therefore, the establishment of the structure-function relationships is critical for exploring the properties and applications of amino acid assemblies. Among the naturally encoded self-assembling amino acids, tyrosine (Y)-based superstructures have been found to show diverse properties and functions including high rigidity, promoting melanin formations, mood regulations, and preventing anxiety, thus showing promising potential as next-generation functional biomaterials for biomedical and bio-machine interface applications. However, the development of Y-based organizations of functional features is severely limited due to the intrinsic difficulty of modulating the energetically stable supramolecular ß-sheet structures. Herein, we report that by the racemic assembly of l-Y and d-Y, the supramolecular secondary structures are modulated from the antiparallel ß-sheets in the enantiomeric assemblies to the parallel ones in the racemate counterparts, thus leading to higher degrees of freedom, which finally induce distinct organization kinetics and modulation of the physicochemical properties including the optical shifts, elastic softening, and the piezoelectric outputs of the superstructures.