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Disrupted protein folding or decreased protein stability can lead to the accumulation of (partially) un- or misfolded proteins, which ultimately cause the formation of protein aggregates. Much of the interest in protein aggregation is associated with its involvement in a wide range of human diseases and the challenges it poses for large-scale biopharmaceutical manufacturing and formulation of therapeutic proteins and peptides. On the other hand, protein aggregates can also be functional, as observed in nature, which triggered its use in the development of biomaterials or therapeutics as well as for the improvement of food characteristics. Thus, unmasking the various steps involved in protein aggregation is critical to obtain a better understanding of the underlying mechanism of amyloid formation. This knowledge will allow a more tailored development of diagnostic methods and treatments for amyloid-associated diseases, as well as applications in the fields of new (bio)materials, food technology and therapeutics. However, the complex and dynamic nature of the aggregation process makes the study of protein aggregation challenging. To provide guidance on how to analyse protein aggregation, in this review we summarize the most commonly investigated aspects of protein aggregation with some popular corresponding methods. ispartof: FEBS JOURNAL vol:290 issue:3 pages:554-583 ispartof: location:England status: published

Aggregation of the tau protein plays a central role in several neurodegenerative diseases collectively known as tauopathies, including Alzheimer’s and Parkinson’s disease. Tau misfolds into fibrillar beta sheet structures that constitute the paired helical filaments found in Neurofibrillary tangles. It is known that there can be significant structural heterogeneities in tau aggregates associated with different diseases. However, while structures of mature fibrils have been studied, the structural distributions in early stage tau aggregates is not well understood. In the present study, we use AFM-IR to investigate nanoscale spectra of individual tau fibrils at different stages of aggregation and demonstrate the presence of multiple fibrillar polymorphs that exhibit different secondary structures. We further show that mature fibrils contain significant amounts of antiparallel beta sheets. Our results are the very first application of nanoscale infrared spectroscopy to tau aggregates and underscore the promise of spatially resolved infrared spectroscopy for investigating protein aggregation.

Peptide amphiphiles (PAs) are a class of molecules comprised of short amino acid sequences conjugated to hydrophobic moieties that may exhibit self-assembly in water into supramolecular structures. We investigate here how mechanical properties of hydrogels formed by PA supramolecular nanofibers are affected by hydrogen bond densities within their internal structure by substituting glycine for aza-glycine (azaG) residues. We found that increasing the number of PA molecules that contain azaG up to 5 mol% in PA supramolecular nanofibers increases their persistence length fivefold and decreases their diffusion coefficients as measured by fluorescence recovery after photobleaching. When these PAs are used to create hydrogels, their bulk storage modulus (G') was found to increase as azaG PA content in the supramolecular assemblies increases up to a value of 10 mol% and beyond this value a decrease was observed, likely due to diminished levels of nanofiber entanglement in the hydrogels as a direct result of increased supramolecular rigidity. Interestingly, we found that the bioactivity of the scaffolds toward dopaminergic neurons derived from induced pluripotent stem cells can be enhanced directly by persistence length independently of storage modulus. We hypothesize that this is due to interactions between the cells and the extracellular environment across different size scales: from filopodia adhering to individual nanofiber bundles to cell adhesion sites that interact with the hydrogel as a bulk substrate. Fine tuning of hydrogen bond density in self-assembling peptide biomaterials such as PAs provides an approach to control nanoscale stiffness as part of an overall strategy to optimize bioactivity in these supramolecular systems. supramolecular biomaterials. STATEMENT OF SIGNIFICANCE: Hydrogen bonding is an important driving force for the self-assembly of peptides in both biological and artificial systems. Here, we increase the amount of hydrogen bonding within self-assembled peptide amphiphile (PA) nanofibers by substituting glycine for an aza-glycine (azaG). We show that increasing the molar concentration of azaG increases the internal order of individual nanofibers and increases their persistence length. We also show that these changes are sufficient to increase survival and tyrosine hydroxylase expression in induced pluripotent stem cell-derived dopaminergic neurons cultured in 3D gels made of these materials. Our strategy of tuning the number of hydrogen bonds in a supramolecular assembly provides mechanical customization for 3D cell culture and tissue engineering.

Peptide-based supramolecular hydrogels have attracted great attention due to their good biocompatibility and biodegradability and have become promising candidates for biomedical applications. The bottom-up self-assembly endows the peptides with a highly ordered secondary structure, which has proven to be an effective strategy to improve the mechanical properties of hydrogels through strong physical interactions and energy dissipation. Inspired by the excellent mechanical properties of spider-silk, which can be attributed to the rich β-sheet crystal formation by the hydrophobic peptide fragment, a hydrophobic peptide (HP) that can form a β-sheet assembly was designed and introduced into a poly(vinyl alcohol) (PVA) scaffold to improve mechanical properties of hydrogels by the cooperative intermolecular physical interactions. Compared with hydrogels without peptide grafting (P-HP0), the strong β-sheet self-assembly domain endows the hybrid hydrogels (P-HP20, P-HP29, and P-HP37) with high strength and toughness. The fracture tensile strength increased from 0.3 to 2.1 MPa (7 times), the toughness increased from 0.4 to 21.6 MJ m-3 (54 times), and the compressive strength increased from 0.33 to 10.43 MPa (31 times) at 75% strain. Moreover, the hybrid hydrogels are enzymatically degradable due to the dominant contribution of the β-sheet assembly for network cross-linking. Combining the good biocompatibility and sustained drug release of the constructed hydrogels, this hydrophobic β-sheet peptide represents a promising candidate for the rational design of hydrogels for biomedical applications.

This study uses differential scanning calorimetry, X-ray crystallography and molecular dynamics simulations to investigate the structural basis for the high thermal stability (melting temperature 97.5 °C) of a FN3-like protein domain from thermophilic bacteria Thermoanaerobacter tengcongensis (FN3tt). FN3tt adopts a typical FN3 fold with a three-stranded beta sheet packing against a four-stranded beta sheet. We identified three solvent exposed arginine residues (R23, R25 and R72), which stabilize the protein through salt bridge interactions with glutamic acid residues on adjacent strands. Alanine mutation of the three arginine residues reduced melting temperature by up to 22 °C. Crystal structures of the wild type and a thermally destabilized (∆Tm -19.7 °C) triple mutant (R23L/R25T/R72I) were found to be nearly identical, suggesting that the destabilization is due to interactions of the arginine residues. Molecular dynamics simulations showed that the salt bridge interactions in the wild type were stable and provided a dynamical explanation for the cooperativity observed between R23 and R25 based on calorimetry measurements. In addition, folding free energy changes computed using free energy perturbation molecular dynamics simulations showed high correlation with melting temperature changes. This work is another example of surface salt bridges contributing to the enhanced thermal stability of thermophilic proteins. The molecular dynamics simulation methods employed in this study may be broadly useful for in silico surface charge engineering of proteins. This article is protected by copyright. All rights reserved.

The aggregation of the amyloid beta (Aβ) protein into plaques is a pathological feature of Alzheimer's disease (AD). While amyloid aggregates have been extensively studied in vitro, their structural aspects and associated chemistry in the brain are not fully understood. In this report, we demonstrate, using infrared spectroscopic imaging, that Aβ plaques exhibit significant heterogeneities in terms of their secondary structure and phospholipid content. We show that the capabilities of discrete frequency infrared imaging (DFIR) are ideally suited for characterization of amyloid deposits in brain tissues and employ DFIR to identify nonplaque β-sheet aggregates distributed throughout brain tissues. We further demonstrate that phospholipid-rich β-sheet deposits exist outside of plaques in all diseased tissues, indicating their potential clinical significance. This is the very first application of DFIR toward a characterization of protein aggregates in an AD brain and provides a rapid, label-free approach that allows us to uncover β-sheet heterogeneities in the AD, which may be significant for targeted therapeutic strategies in the future.

An early step in cellular infection by a membrane-enveloped virus like HIV or influenza is joining (fusion) of the viral and cell membranes. Fusion is catalyzed by a viral protein that typically includes an apolar “fusion peptide” (fp) segment that binds the target membrane prior to fusion. In this study, the effects of non-homologous HIV and influenza fp’s on lipid acyl chain motion are probed with (2)H NMR transverse relaxation rates (R(2)’s) of perdeuterated DMPC membrane. Measurements were made between 35 and 0 °C which brackets the membrane liquid-crystalline-to-gel phase transitions. Samples were made with either HIV “GPfp” at pH 7 or influenza “HAfp” at pH 5 or 7. GPfp induces vesicle fusion at pH 7 and HAfp induces more fusion at pH 5 vs 7. GPfp bound to DMPC adopts intermolecular antiparallel β sheet structure, whereas HAfp is a monomer helical hairpin. The R(2)’s of the no peptide and HAfp, pH 7 samples increase gradually as temperature is lowered. The R(2)’s of GPfp and HAfp, pH 5 samples have very different temperature dependence, with ~10× increase in R(2)(CD2) when temperature is reduced from 25 to 20 °C, and smaller but still substantial R(2)’s at 10 and 0 °C. The large R(2)’s with GPfp and HAfp, pH 5 are consistent with large-amplitude motions of lipid acyl chains that can aid fusion catalysis by increasing the population of chains near the aqueous phase, which is the chain location for transition states between membrane fusion intermediates.

Circular dichroism (CD) spectroscopy is a widely‐used method for characterizing the secondary structures of proteins. The well‐established and highly used analysis website, DichroWeb (located at: http://dichroweb.cryst.bbk.ac.uk/html/home.shtml) enables the facile quantitative determination of helix, sheet, and other secondary structure contents of proteins based on their CD spectra. DichroWeb includes a range of reference datasets and algorithms, plus graphical and quantitative methods for determining the quality of the analyses produced. This article describes the current website content, usage and accessibility, as well as the many upgraded features now present in this highly popular tool that was originally created nearly two decades ago.

The rheological properties and the secondary structures of date palm pollen concentrates were investigated in the present study. The sonication pretreatment and the succinylation procedure were applied to the native protein, as physical and chemical modifications, respectively. Each modification affected differently the native concentrate. In fact, in the flow measurement, results, fitted with the power low model, showed that sonication increased the consistency coefficient and decreased the flow index implying a more shear-thinning character. However, for succinylation modification, an increase of the flow index and a decrease of the consistency coefficient were noticed which convert the protein concentrate to a less viscous and more Newtonian-like state. Sonication also enhanced the thixotropic behavior which was expressed by the increase of the hysteresis loop, whereas the succinylation suppressed it, which was deduced from the disappearance of the loop area. The frequency sweep analysis proved that the sonicated concentrate was characterized by a strong gel structure while the succinylated protein had a viscoelastic liquid character. Only the native and the succinylated concentrates gelified, in the temperature sweep test, at 53.1 and 54.4 °C, respectively. All discerned differences were attributed to the secondary structure content. The findings revealed that the sonicated protein had a high β sheet content, leading to greater thermal stability which proved that a higher temperature is required to ensure the gelation of the protein. However, native and succinylated concentrates were found to have an equilibrium between α helix and β sheet which made it possible to form a stable gel at a lower temperature. The present study concluded that the modifications had a great impact on the rheological properties which was strongly attributed to the submitted structural changes.

Protein aggregation, such as amyloid fibril formation, is molecular hallmark of many neurodegenerative disorders including Alzheimer's, Parkinson's, and Prion disease. Indole alkaloids are well-known as the compounds having the ability to inhibit protein fibrillation. In this study, we experimentally and computationally have investigated the anti-amyloid property of a derivative of a synthesized tetracyclic indole alkaloid (TCIA), possessing capable functional groups. The fibrillation reaction of Hen White Egg Lysozyme (HEWL) was performed in absence and presence of the indole alkaloid. For quantitative analysis, we used Thioflovin T binding assay which showed ~50% reduction in fibril formation in the presence of 20 μM TCIA. Using TEM imaging, we observed a significant morphological change in our model protein in the presence of TCIA. In addition, we exploited FT-IR assay by which Amide I peak's shifting toward lower wavenumber was clearly observed. Using Molecular Docking, the interaction of the inhibitor (TCIA) with the protein's amyloidogenic region was modeled. Also, different biophysical parameters were calculated by Molecular Dynamics (MD) simulation. Various biochemical assays, conformational change, and hydrophobicity exposure of the protein during amyloid formation indicated that the compound assists HEWL to keep its native structure via destabilizing β-sheet structure.

Plant cryptochromes are central blue light receptors in land plants and algae. Photoreduction of the flavin bound to the photolyase homology region (PHR) causes a dissociation of the C-terminal extension (CCT) as effector via an unclear pathway. We applied the recently developed in-cell infrared difference (ICIRD) spectroscopy to study the response of the full-length pCRY from Chlamydomonas reinhardtii in living bacterial cells, because the receptor degraded upon isolation. We demonstrate a stabilization of the flavin neutral radical as photoproduct and of the resulting β-sheet reorganization by binding of cellular ATP. Comparison between light-induced structural responses of full-length pCRY and PHR reveals a downshift in frequency of the β-sheet signal, implying an association of the CCT close to the only β-sheet of the PHR in the dark. We provide a missing link in activation of plant cryptochromes after flavin photoreduction by indicating that β-sheet reorganization causes the CCT release and restructuring.

Creating stimulus-responsive materials solely by controlling polypeptide secondary nanostructures is challenging. We synthesized a methyl poly(ethylene glycol)-b-poly(O-benzyl-L-threonine) (mPEG-PBnLT) diblock copolymer that exhibited gel-to-sol UCST (Upper Critical Solution Temperature) transition behavior in an aqueous solution. The transition temperature window was easily adjusted by changing the copolymer concentration or length of the PBnLT block. Disassembly of the initial β-sheet nanoassemblies caused nanofibril transformation to spherical aggregates with increasing temperature, resulting in a gel-to-sol UCST transition. This result inspires a brand-new strategy for the structural design and functional control of materials. A novel thermoresponsive diblock copolymer of methyl poly (ethylene glycol)-b-poly (O-benzyl-L-threonine) was synthesized. The copolymer solutions exhibited gel-to-sol UCST transition behavior with temperature. The gel-to-sol transition was due to the disassembly of the initial β-sheet layered nanoassemblies that induced the transformation of self-organized morphology from nanosized fibrils to spherical aggregates.

Many neurodegenerative diseases such as Alzheimer's disease are characterized by pathological s-sheet filaments of the tau protein, which spread in a prion-like manner in patient brains. To date, high-resolution structures of tau filaments obtained from patient brains show that the s-sheet core only includes portions of the microtubule-binding repeat domains and excludes the C-terminal residues, indicating that the C-terminus is dynamically disordered. Here, we use solid-state NMR spectroscopy to identify the s-sheet core of full-length 0N3R tau fibrillized using heparin. Assignment of 13C and 15N chemical shifts of the rigid core of the protein revealed a single predominant s-sheet conformation, which spans not only the R3, R4, R' repeats but also the entire C-terminal domain (CT) of the protein. This massive s-sheet core qualitatively differs from all other tau fibril structures known to date. Using long-range correlation NMR experiments, we found that the R3 and R4 repeats form a s-arch, similar to that seen in some of the brain-derived tau fibrils, but the R1 and R3 domains additionally stack against the CT, reminiscent of previously reported transient interactions of the CT with the microtubule-binding repeats. This expanded s-sheet core structure suggests that the CT may have a protective effect against the formation of pathological tau fibrils by shielding the amyloidogenic R3 and R4 domains, preventing side-on nucleation. Truncation and post-translational modification of the CT in vivo may thus play an important role in the progression of tauopathies.

Protein complexes are defined by the three‐dimensional structure of participating binding partners. Knowledge about these structures can facilitate the design of peptidomimetics which have been applied for example, as inhibitors of protein–protein interactions (PPIs). Even though β‐sheets participate widely in PPIs, they have only rarely served as the basis for peptidomimetic PPI inhibitors, in particular when addressing intracellular targets. Here, we present the structure‐based design of β‐sheet mimetics targeting the intracellular protein β‐catenin, a central component of the Wnt signaling pathway. Based on a protein binding partner of β‐catenin, a macrocyclic peptide was designed and its crystal structure in complex with β‐catenin obtained. Using this structure, we designed a library of bicyclic β‐sheet mimetics employing a late‐stage diversification strategy. Several mimetics were identified that compete with transcription factor binding to β‐catenin and inhibit Wnt signaling in cells. The presented design strategy can support the development of inhibitors for other β‐sheet‐mediated PPIs., Starting from a 52 amino acid protein binding epitope, a bicyclic β‐hairpin structure was developed to bind the transcriptional coactivator β‐catenin. Our structure‐based design approach was supported by screening a focused library of bicyclic mimetics which was generated via late‐stage diversification. The most active bicyclic β‐hairpin shows cell‐penetration and inhibits Wnt signaling in a cell‐based assay.

Coassembling peptides offer an additional degree of freedom in the design of nanostructured biomaterials when compared to analogous self-assembling peptides. Yet, our understanding of how amino acid sequences encodes coassembled nanofiber structure is limited. Prior work on a charge-complementary pair, CATCH+ and CATCH- peptides, detected like-peptide nearest neighbors (CATCH+:CATCH+ and CATCH-:CATCH-) within coassembled β-sheet nanofibers; these self-associated peptide pairs marked a departure from an "ideal" coassembled structure. In this work, we employ solid-state NMR, isotope-edited FTIR, and coarse-grained molecular dynamics simulations to evaluate the alignment of β-strands within CATCH peptide nanofibers. Both experimental and computational results suggest that CATCH molecules coassemble into structurally heterogeneous nanofibers, which is consistent with our observations in another coassembling system, the King-Webb peptides. Within β-sheet nanofibers, β-strands were found to have nearest neighbors aligned in-register parallel, in-register antiparallel, and out-of-register. In comparison to the King-Webb peptides, CATCH nanofibers exhibit a greater degree of structural heterogeneity. By comparing the amino acid sequences of CATCH and King-Webb peptides, we can begin to unravel sequence-to-structure relationships, which may encode more precise coassembled β-sheet nanostructures.

Mechanical training is an operation where a sample is cyclically stretched in a solvent. It is accepted as an effective strategy to strengthen and stiffen the highly hydrated silk materials (HHSMs). However, the detailed reinforcement mechanism of the process still remains to be understood. Herein, this process is studied by the integration of experimental characterization and theoretical analysis. The results from time-resolved Fourier transform infrared spectroscopy and real-time birefringent characterization reveal that the silk proteins rapidly formed a molecular cross-linking network (MCN) during the mechanical training. The cross-links were the β-sheet nanocrystals generated from the conformation transition of silk proteins. With the progress in mechanical training, these MCNs gradually remodeled to a highly oriented molecular network structure. The final structure of the silk proteins in HHSMs is highly similar to the structural organization of silk proteins in the natural animal silk. The training process significantly improved the mechanical strength and modulus of the material. With regards to the dynamic behavior of conformation transition and MCN orientation, the structural evaluation of silk proteins during mechanical training was divided into three distinct stages, namely, the MCN-forming stage, MCN-orienting stage, and oriented-MCN stage. Such division is in complete agreement with the three-stage viscoelastic behavior observed in the cyclic loading and unloading tests. Hence, a five-parameter viscoelastic model has been established to elucidate the structure-property relationship of these three stages. This work improves in-depth understanding of the fundamental issues related to structure-property relationships of HHSMs and thus provides inspiration and guidance in the design of soft silk functional materials.

The structure-function relationships of plant-based proteins that give rise to desirable texture attributes in order to mimic meat products are generally unknown. In particular, it is not clear how to engineer viscoelasticity to impart cohesiveness and proper mouthfeel; however, it is known that intermolecular β-sheet structures have the potential to enhance the viscoelastic property. Here, we investigated the propensity of selected peptide segments within common corn α-zein variants to maintain stable aggregates and β-sheet structures. Simulations on dimer systems showed that stability was influenced by the initial orientation and the presence of contiguous small hydrophobic residues. Simulations using eight-peptide β-sheet oligomers revealed that peptide sequences without proline had higher levels of β-sheet structuring. Additionally, we identified that sequences with a dimer hydrogen-bonding density of >22% tended to have a larger percent β-sheet conformation. These results contribute to understanding how the viscoelasticity of zein can be increased for use in plant-based meat analogues.

The VQIVYK fragment from the Tau protein, also known as PHF6, is essential for aggregation of Tau into neurofibrillary lesions associated with neurodegenerative diseases. VQIVYK itself forms amyloid fibrils composed of paired β-sheets. Therefore, the full Tau protein and VQIVYK fibrils have been intensively investigated. A central issue in these studies is polymorphism, the ability of a protein to fold into more than one structure. Using all-atom molecular simulations, we generate five stable polymorphs of VQIVYK fibrils, establish their relative free energy with umbrella sampling methods, and identify the side chain interactions that provide stability. The two most stable polymorphs, which have nearly equal free energy, are formed by interdigitation of the mostly hydrophobic VIY “face” sides of the β-sheets. Another stable polymorph is formed by interdigitation of the QVK “back” sides. When we turn to examine structures from cryo-electron microscopy experiments on Tau filaments taken from diseased patients or generated in vitro, we find that the pattern of side chain interactions found in the two most stable face-to-face as well as the back-to-back polymorphs are recapitulated in amyloid structures of the full protein. Thus, our studies suggest that the interactions stabilizing PHF6 fibrils explain the amyloidogenicity of the VQIVYK motif within the full Tau protein and provide justification for the use of VQIVYK fibrils as a test bed for the design of molecules that identify or inhibit amyloid structures.

In humans, cystatin C mediated amyloid aggregation is quite detrimental. It is deemed to actuate intra-cerebral haemorrhaging, stroke, paralysis and further, turns out to be fatal under exigent circumstances. Especially, the domain swapped dimerization of cystatin C is the pathogenic trigger that propels its amyloidosis. Targeting the protein at this initial pathogenic stage could be effective in arresting cystatin C amyloid formation and its eventual aggregation. In our current analysis, using a logically assertive coherent computational pipeline, we have proffered an efficacious peptide based therapeutic (due to its benefits over small molecules) against the domain swapped cystatin C amyloid like dimer (CCAD) pre-fibril. Wherein, the tripeptide, ‘LVN’ has effectually decreased the average beta sheet content (a distinct pathogenic aspect of amyloids) of CCAD from 49 to 43%. Findings from discrete molecular dynamics simulation through intramolecular hydrogen bond analysis, covariance matrix, free energy landscape and solvent accessible surface area have further substantiated that, upon interaction, LVN has steered CCAD from its distinct pathogenic behaviour. Besides, the thermal unfolding simulation indicated that due to LVN’s confluence with CCAD, the phase transition of the complex to a flexible state from a rigid phase, occurs with a relatively lower temperature and energy, compared to peptide unbound pathogenic CCAD. Hence, the consistent findings from various computational tools anticipate that LVN tripeptide could be a potent promising candidate in mitigating cystatin C amyloid aggregation in humans.

Poly(l-cysteine)s with methylthio pendants (PMTLCs) were synthesized by ring-opening polymerization of a new l-cysteine-based N-carboxyanhydride. The thioether bonds of PMTLC can be readily oxidize...

The field of de novo protein design has met with considerable success over the past few decades. Heme, a cofactor has often been introduced to impart a diverse array of functions to a protein, ranging from electron transport to respiration. In nature, heme is found to occur predominantly in α-helical structures over β-sheets, which has resulted in significant designs of heme-proteins utilizing coiled coil helices. By contrast, there are only a few known β-sheet proteins that bind heme and designs of β-sheets frequently result in amyloid-like aggregates. This review reflects on our success with designing a series of multi-stranded β-sheet heme binding peptides that are well folded both in aqueous and membrane-like environments. Initially, we designed a β-hairpin peptide that self-assembles to bind heme and performs peroxidase activity in membrane. The β-hairpin was optimized further to accommodate a heme binding pocket within multi-stranded β-sheets for catalysis and electron transfer in membranes. Furthermore, we de novo designed and characterized β-sheet peptides and mini-proteins soluble in aqueous environment capable of binding single and multiple hemes with high affinity and stability. Collectively, these studies highlight substantial progress made towards the design of functional β-sheets. Ministry of Education (MOE) Accepted version AS would like to thank Ministry of Education, Singapore and Nanyang Technological University for Graduate Research Scholarship.

Alzheimer's disease is characterized by amyloid-β aggregation. Currently, all the approved medications are to treat the symptoms but there is no clinically approved treatment for the cure or to prevent the progression of Alzheimer's disease (AD). Earlier reports suggest the use of small molecules and peptides to target and destabilize the amyloid fibril. The use of Beta Sheet Breaker (BSB) peptides seems to be a promising and attractive therapeutic approach as it can strongly bind and destabilize the preformed amyloid fibril. There are experimental studies describing the destabilization role of various BSB peptides, but the exact mechanism remains elusive. In the current work, an attempt is made to study the destabilization mechanism of different BSB peptides on preformed amyloid protofibril using molecular docking and simulations. Molecular docking of eight different BSB peptides of varying length (5-mer to 10-mer) on the Abeta protofibril was done. Docking was followed by multiple sets of molecular simulations for the Abeta protofibril-BSB peptide complex for each of the top ranked poses of the eight BSB peptides. As a control, multiple sets of simulations for the Abeta protofibril (APO) were also carried out. An increase in the RMSD, decrease in the number of interchain hydrogen bonds, destabilization of important salt bridge interactions (D23-K28), and destabilization of interchain hydrophobic interactions suggested the destabilization of Abeta protofibril by BSB peptides. The MM-GBSA free energy of binding for each of the BSB peptides was calculated to measure the binding affinity of BSB peptides to Abeta protofibril. Further residue wise contribution of free energy of binding was also calculated. The study showed that 7-mer peptides tend to bind strongly to Abeta protofibril as compared to other BSB peptides. The KKLVFFA peptide showed better destabilization potential as compared to the other BSB peptides. The details about the destabilization mechanism of BSB peptides will help in the design of other peptides for the therapeutic intervention for AD.

Short peptides can self-assemble into supramolecular materials and be used for promising biomedical applications. This area of research has seen tremendous development in recent years. However, the preparation of nanofibers with a predefined secondary structure through rational design remains a challenge. Herein, a peptide-based building block, Fmoc-KKYpYp-COOH (P1), was rationally designed for dual-triggered self-assembly by pH and enzyme for the first time. By delicately modulating the surface charges of the peptides using different triggers, i.e. pH or enzyme, we have designed peptides that are capable of self-assembling into nanoscale structures with distinct secondary structures. Upon ALP treatment, precursor P1 was efficiently transformed into a hydrogelator, Fmoc-KKYY-COOH (P2), and self-assembled to nanofibers with a partial α-helix structure. In contrast, P1 displays a typical fibrous network structure with a predominant β-sheet arrangement upon pH stimulus. Furthermore, P1 was successfully used to co-assemble with a prodrug under ALP catalysis without compromising the anti-cancer activity of the drug. The combination index (CI) values indicated a synergistic effect between P1 and Ep. We envision that this study will provide a powerful approach for rationally designing peptide nanofibers with a predefined secondary structure in the future.

Helicobacter pylori (H. pylori) uses several outer membrane proteins for adhering to its host's gastric mucosa, an important step in establishing and preserving colonization. Several adhesins (SabA, BabA, HopQ) have been characterized in terms of their three-dimensional structure. A recent addition to the growing list of outer membrane porins is LabA ( La cdiNAc- b inding a dhesin), which is thought to bind specifically to GalNAcβ1-4GlcNAc, occurring in the gastric mucosa. LabA47-496 protein expressed as His-tagged protein in the periplasm of E. coli and purified via subtractive IMAC after TEV cleavage and subsequent size exclusion chromatography, resulted in bipyramidal crystals with good diffraction properties. Here, we describe the 2.06 ​A resolution structure of the exodomain of LabA from H. pylori strain J99 (PDB ID: 6GMM ). Strikingly, despite the relatively low levels of sequence identity with the other three structurally characterized adhesins (20–49%), LabA shares an L-shaped fold with SabA and BabA. The ‘head’ region contains a 4 ​+ ​3 α-helix bundle, with a small insertion domain consisting of a short antiparallel beta sheet and an unstructured region, not resolved in the crystal structure. Sequence alignment of LabA from different strains shows a high level of conservation in the N- and C-termini, and identifies two main types based on the length of the insertion domain (‘crown’ region), the ‘J99-type’ (insertion ~31 ​amino acids), and the H. pylori ‘26695 type’ (insertion ~46 ​amino acids). Analysis of ligand binding using Native Electrospray Ionization Mass Spectrometry (ESI-MS) together with solid phase-bound, ELISA-type assays could not confirm the originally described binding of GalNAcβ1-4GlcNAc-containing oligosaccharides, in line with other recent reports, which also failed to confirm LacdiNAc binding.

Mitochondrial ATP synthase plays a key role in inducing membrane curvature to establish cristae. In Apicomplexa causing diseases such as malaria and toxoplasmosis, an unusual cristae morphology has been observed, but its structural basis is unknown. Here, we report that the apicomplexan ATP synthase assembles into cyclic hexamers, essential to shape their distinct cristae. Cryo-EM was used to determine the structure of the hexamer, which is held together by interactions between parasite-specific subunits in the lumenal region. Overall, we identified 17 apicomplexan-specific subunits, and a minimal and nuclear-encoded subunit-a. The hexamer consists of three dimers with an extensive dimer interface that includes bound cardiolipins and the inhibitor IF1. Cryo-ET and subtomogram averaging revealed that hexamers arrange into ~20-megadalton pentagonal pyramids in the curved apical membrane regions. Knockout of the linker protein ATPTG11 resulted in the loss of pentagonal pyramids with concomitant aberrantly shaped cristae. Together, this demonstrates that the unique macromolecular arrangement is critical for the maintenance of cristae morphology in Apicomplexa., Structural and functional analysis of mitochondria from the human parasite Toxoplasma gondii reveals that its ATP synthase assembles into cyclic hexamers, arranged together in a form of pentagonal pyramids required for maintenance of cristae morphology in Apicomplexa.

HU, a DNA-binding protein, has a helical N-terminal region (NTR) of ∼44 residues and a beta strand- and IDR-rich C-terminal region (CTR) of ∼46 residues. CTR binds to DNA through (i) a clasp (two arginine/lysine-rich, IDR-rich beta hairpins that bind to phosphate groups in the minor groove), (ii) a flat surface (comprising four antiparallel beta strands that abut the major groove), and (iii) a charge cluster (two lysine residues upon a short C-terminal helix). HU forms a dimer displaying extensive inter-subunit CTR-CTR contacts. A single-chain simulacrum of these contacts (HU-Simul) incorporating all DNA-binding elements was created by fusing together the CTRs of Escherichia coli HU-A and Thermus thermophilus HU. HU-Simul is monomeric, binds to dsDNA and cruciform DNA, but not to ssDNA.