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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.

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.

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.

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.

Viscoelasticity of corn zein is associated with the formation of β-sheet secondary structures; however, studies of the fundamentals of this conformational change are limited due to zein insolubility and poor analytical resolution. Here, changes in soluble zein conformation were evaluated as the protein self-assembles in increasingly hydrophilic solvents to the concentration just before aggregation and precipitation. Circular dichroism spectra of zein showed that α-helix structures decrease in favor of random coil and β-sheets with increases in water content in an ethanol-water system, similar to observations of zein when it becomes viscoelastic in dough systems. This was further supported by changes in Thioflavin T fluorescence emission spectra and intrinsic viscosity measurements. Two widely recognized molecular models for α-zein (hairpin and superhelical conformations) were tested at 75 and 45% ethanol concentration using molecular dynamics simulation for agreement with experimental results. Increase in solvent hydrophilicity increased β-sheets and reduced distance between backbone anomeric carbons only for hairpin model, suggesting it to be the more valid of the two. These findings emphasize the importance of transformation to β-sheets during zein self-assembly and provide further insight into the mechanisms by which the protein is functionalized into viscoelastic systems.

CPAP is a centriolar protein and its C-terminal domain, G-box or TCP, has a very unique structure that comprises a single-layer β-sheet without hydrophobic core packing. Here we characterized its biophysical properties, including its stability against chemical denaturation. Interestingly, upon urea-induced equilibrium unfolding, the CPAP G-box showed cooperative unfolding behavior that is the hallmark of globular proteins. We analyzed the m-value, a measure of the cooperative transition, from the urea-induced unfolding and found that the estimated m-value from surface burial upon folding is consistent with the experimental value, supporting the two-state unfolding. Next, we constructed deletion mutants of the terminal β-strands and found that the mutants showed reduced stability. The unique structure and characteristics of CPAP G-box provides an interesting opportunity to observe how the core-less flat β-sheet protein can be folded in solution.

For the first time, the effect of two novel designed pentapeptides on amyloid growth of human insulin using combined biophysical, microscopic, cell viability and computational approaches. Collective experimental data from ThT, ANS, and TEM demonstrate that in spite of having contrasting features, both peptides can effectively inhibit amyloid formation by prolonging lag phase, slowing down aggregation rate, and reducing final fibril formation (up to 84.26% and 85.24% by P1 and P7 respectively). Although pure amyloid caused profound cellular toxicity in SH-SY5Y neuronal cells, amyloid formed in the presence of peptides showed much reduced cellular toxicity. Such an inhibitory effect can be attributed to interference with the structural transition of insulin toward β-sheet structure by peptides. Furthermore, molecular dynamic simulations confirm that peptide preferentially binds to nearby region which is more prone to form aggregates that consequently disrupts self-assembly into amyloid fibrils (P1 and P7 possess inhibition constant value of 0.000183 and 0.000216 nm, respectively), supporting our experimental observations. This study underscores the information about the sequence based inhibition mechanism of peptides that might dictate their inhibition or modulation capacity, which might be helpful in designing anti-amyloid therapeutics.

Protein-protein interactions involving β-sheet secondary structures have been questioned in many fatal human diseases such as cancer, autoimmune and neurodegenerative diseases. Small selective peptide derivatives and analogues are promising drug candidates for inhibiting this poorly known class of PPIs. In this review, we will highlight the main strategies developed for designing linear and cyclic peptide and peptidomimetic inhibitors of PPIs involving β-sheet structures. These compounds either do not adopt preferred conformations or can mimic protein secondary structures such as β-strands, β-hairpins or α-helices.

Amyloid-beta protein (Aβ) accumulation in the brain, which is influenced by several factors, is a hallmark of Alzheimer's disease (AD). Despite the important role of histidine in stabilizing the fibrillar structure of the Aβ peptide at neutral pH, the effect of histidine tautomerism on Aβ peptide aggregation is still largely unknown. Histidine is in equilibrium between δ and ε tautomers and there are three histidine residues (H6, H13, and H14) in the Aβ(1-40) peptide. We performed molecular dynamics simulation on (δδδ) and (εεε) histidine tautomers with different initial homodimeric configurations to elucidate structural and aggregation features. Results indicate that (εεε) homodimers have very low propensity or almost no tendency to form β-sheets, whereas (δδδ) dimers predominantly form β-sheets due to interactions between central hydrophobic core (CHC) residues and C-terminal residues. β-sheet formation occurred in the same regions of each dimer chain at the CHC and C-/N- terminals for different configurations of (δδδ). These results suggest that (δδδ) has an important role in AD progression. Our study provides deeper insight into the effect of tautomerism of histidine residues in Aβ(1-40) on amyloid aggregation.

Biomimetic synthesis of silica/polymer hybrid nanomaterials inspired by silica-condensing microorganisms has gained significant advances in recent years and the as-prepared hybrid materials have been explored for a variety of applications. In this work, silica/polypeptide hybrid nanoparticles (NPs) and coating films can be facilely fabricated by silica mineralization of poly( l -lysine)-block-poly( l -threonine)/poly( l -glutamic acid) (PLL-b-PLT/PGA) fibril complexes assembled in solutions or on substrates at benign conditions. The experimental data revealed that PLL-b-PLT can self-assemble to form fibrils via intermolecular hydrogen bonding interactions between PLT chains and, upon complexing with PGA, silicas were efficiently mineralized in both the sheet-like PLL/PGA complexes and PLT domains, resulting in the fabrication of silica/polypeptide hybrid materials. After removing the polypeptides, mesoporous silicas exhibiting pore size between 2 and 10 nm and large pores (>10 nm) were fabricated by the replication of the sheet-like polypeptides and fibril complexes/aggregates, respectively. This study demonstrates that these polypeptide fibril complexes can serve both as nucleating agents and as templates for the fabrication of oxide/polypeptide hybrid NPs, mesoporous oxides and oxide/polypeptide coating films, which have potential applications in a variety of areas.

With the advent of molybdenum disulfide nanosheets (MoS2 NSs) for biological applications, their complex interactions with human serum albumin (HSA) need to be understood in great detail for the molecular mechanisms of protein structure and activity. It was observed that MoS2 NSs quench the intrinsic fluorescence of HSA as a consequence of ground-state complex formation by the electron transfer, van der Waals, and hydrophobic forces. The presence of MoS2 NSs partly altered the conformation of HSA and destroyed the binding domain of HSA with bilirubin. In addition, MoS2 NSs can decrease the rate of the formation of beta sheet structures of HSA, reduce the non-enzymatic glycosylation, and increase the esterase-like activity of HSA. We hope that the present study will be helpful to understand the fundamental interactions of the two-dimensional materials with various biomacromolecules in human blood.

This work aimed to establish the impact of total solids (TS) concentration (9, 17 and 25%) of raw skim milk on the conformational properties of proteins during heating up to 121 °C. Conformational changes were determined using Fourier transform infrared spectroscopy and correlated to physicochemical properties of the system. The results showed high total solids dependence in rearrangement of β-sheet of β-lactoglobulin. Heat denaturation of whey proteins was concentration and temperature dependant. The micelle appeared to undergo temperature dependant dissociation that induced redistribution of helical and loop structures. The conformational changes were further evaluated with principal component analysis and normalising for a concentration effect for all samples. The most affected structures were located in the region 1620–1655 cm−1 including intra- and inter-molecular β-sheets, α-helix/loop structures and randomly distributed structures. These findings may assist in predicting the heat stability of concentrates.

d-amino acid-containing proteins have been found in several human tissues, and the spontaneous accumulation of d-amino acids in proteins is thought to be involved in age-dependent diseases including dementia. Tau, a microtubule-associated protein, is a major component of neurofibrillary tangles in Alzheimer's disease. Site-specific amino acid D-isomerization in Tau has been observed in the brains of patients with Alzheimer's disease. Here, we conducted amino acid D-isomerization at specific sites in microtubule-binding repeat peptides of Tau (Tau R2 and R3) and examined the effects on Tau structure and fibril formation. Our results demonstrate that amino acid D-isomerization in Tau R2 peptides decreased the rates of β-sheet transition and fibril formation compared with those of the wild-type peptide composed of all l-amino acids. In contrast, Tau R3 peptides that had undergone amino acid D-isomerization at either Asp314, Ser316, or Ser324 showed increased rates of β-sheet transition and fibril formation compared with those of the wild-type Tau R3 peptide.

The effect on protein conformation and thermal stability was studied for β-Galactosidase (β-Gal) encapsulated in the nanopores of a silicate matrix (Eβ-Gal). Circular dichroism spectra showed that, compared with the enzyme in buffer (Sβ-Gal), Eβ-Gal exhibited a higher content of α-helix structure. Heating Eβ-Gal up to 75 °C caused a decrease in the content of β-sheet structure and additional augments on Eβ-Gal components attributed to helical content, instead of the generalized loss of the ellipticity signal observed with Sβ-Gal. Steady state fluorescence spectroscopy analysis evidenced an Eβ-Gal structure less compact and more accessible to solvent and also less stable against temperature increase. While for Sβ-Gal the denaturation midpoint (Tm) was 59 °C, for Eβ-Galit was 48 °C. The enzymatic activity assays at increasing temperatures showed that in both conditions, the enzyme lost most of its hydrolytic activity against ONPG at temperatures above 65 °C and Eβ-Gal did it even at lower T values. Concluding, confinement in silica nanopores induced conformational changes on the tertiary/cuaternary structure of Eβ-Gal leading to the loss of thermal stability and enzymatic activity. Fil: Burgos, Martha Ines. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Instituto de Investigaciones Biológicas y Tecnológicas; Argentina. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Departamento de Química; Argentina Fil: Ochoa, Aylen. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Departamento de Química; Argentina Fil: Perillo, Maria Angelica. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Departamento de Química. Cátedra de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Córdoba. Instituto de Investigaciones Biológicas y Tecnológicas. Universidad Nacional de Córdoba. Facultad de Ciencias Exactas, Físicas y Naturales. Instituto de Investigaciones Biológicas y Tecnológicas; Argentina

The Systematic Evolution Ligands by Exponential Enrichment (SELEX) is common used for selection of high affinity single-stranded DNA (ssDNA) aptamer with target protein. However, we do not know what the most stable configuration of the selected aptamer bound with target protein is. Therefore, a systematic search process using the stochastic tunneling-basin hopping (STUN-BH) method is proposed to find the most stable configuration of the ssDNA aptamer specific for vascular endothelial growth factor (VEGF) capture (AptVEGF; 5′-TGTGGGGGTGGACGGGCCGGGTAGA-3′). After the most stable configuration was obtained by the STUN-BH method, molecular dynamics (MD) simulation was carried out to investigate the thermal stability of AptVEGF/VEGF at 300 K in both vacuum and water. All molecular simulations were conducted with the large-scale atomic/molecular massively parallel simulator (LAMMPS), and the AMBER99SB force field was used to describe the atomic interactions for the current AptVEGF/VEGF system. The three most stable AptVEGF/VEGF configurations obtained by the STUN-BH method indicated that AptVEGF residues exhibit greater affinity for VEGF surface loop fragments as compared with surface alpha helix and beta sheet fragments. Results indicated that after the first AptVEGF (AptVEGF I) occupies most of the VEGF loop fragment, the second AptVEGF (AptVEGF II) is adsorbed by the rest of the VEGF loop fragment and the VEGF Chain B beta sheet fragment, resulting in a 24.8% reduction in binding strength as compared to that of AptVEGF I. Furthermore, when AptVEGF I and AptVEGF II chains were stably adsorbed by VEGF, the third AptVEGF (AptVEGF III) chain can only partially attach to VEGF, as confirmed by real AptVEGF-VEGF binding experiments. Lastly, we demonstrated that the aptasensor constructed according to MD simulation is highly sensitive for VEGF with a linear detection range of 10 pg/mL–10 ng/mL.

Surfactant-induced amyloid fibrillation has many implications in laboratory and industry. Previously, cetyltrimethylammonium bromide (CTAB; C19H42BrN) induced amyloid formation in concanavalin A (ConA) has been reported by authors (Khan, JM et al., 2016 A). In this work, cationic gemini surfactants pentanediyl‑1,5‑bis (dimethylcetylammonium bromide); 16-5-16 (G5), and hexanediyl‑1,6‑bis (dimethyl cetyl ammonium bromide); 16-6-16 (G6) were found to induce amyloid-like aggregation in ConA as studied with turbidity at 350 nm, Rayleigh scattering and Thioflavin T (ThT) binding assay and Far UV CD. The sizes of the aggregates were characterized with dynamic light scattering (DLS) and atomic force microscopy (AFM). Hydrodynamic radii (Rh) of the aggregates were found to be 74 nm and 122 nm with 50 μM of G5 and G6 respectively. Even at 7.5 μM, gemini surfactants seems disrupting hydrophobic pockets of ConA and result in aggregation. On the contrary, aggregation disappeared around 250 μM surfactant concentration with β-sheet → α-helix transition. The re-establishment of intra-molecular hydrogen bonding may have resulted in the formation of non-native α-helical structures. Alcohols, predominantly 2,2,2‑trifuloroethanol (TFE), have been reported to induce α-helices in β-sheet proteins by solvent engineering. There are very few reports of β-sheet to α-helix transition induced by non-alcoholic substances. Our lab is pioneer in reporting surfactant (CTAB) induced β-sheet to α-helix transition in ConA (Khan, JM et al., 2016 A). This is the first report of G5 and G6 gemini surfactant induced β-sheet to α-helix transition, as per our knowledge. ConA being apoptotic to cancerous cells and having potential therapeutic effect on hepatoma, the observations done in this work could be of immense interest in vesicular delivery of ConA.

Organophosphate compounds that are used as pesticides affect the nervous system by binding irreversibly to the active site of the enzyme acetylcholine esterase (AChE) and disrupting neuro-signaling nerve cells. In this study we characterized adsorption of paraoxon to a set of designed peptides that present different arrangements of the three amino acids of the AChE catalytic site: histidine, glutamic-acid and serine. The peptides set included two β-strands with no net charge and three β-hairpins that differ in their net charge. Circular dichroism, Thioflavin T assays and TEM images provided only qualitative insights on paraoxon binding to the different peptides. Paraoxon binding to the different peptides was measured with dialysis membrane tubes filled with the peptide solutions and suspended in a reservoir of paraoxon solution. Among all the tested peptides, the single strand peptide, denoted ssESH exhibited at 100 μM in random conformation prefibrillar state, the maximum paraoxon adsorption, with a binding mol ratio of one paraoxon per two peptides and an estimated equilibrium binding constant 5 ∗ 104 M−1. The three β-hairpin peptides demonstrated that a net negative charge is unfavorable for paraoxon adsorption. Surface enhanced Raman spectroscopy measurements with ssESH enabled the detection of nanomolar adsorbed concentrations of paraoxon.

Cancer is one of the main causes of death worldwide. The current cancer treatment strategies often lack selectivity for cancer cells resulting in dose-limiting adverse effects and reduced quality of life. Recently, anticancer peptides (ACPs) have emerged as an alternative treatment with higher selectivity, less adverse effects, and lower propensity for drug resistance. However, most of the current studies on the ACPs are focused on α-helical ACPs and there is lack of systematic studies on β-sheet forming ACPs. Herein we report the development of a new series of rationally designed short cationic amphiphilic β-sheet forming ACPs and their structure activity relationship. The peptides had the general formula (XY1XY2)3, with X representing hydrophobic amino acids (isoleucine (I) or leucine (L)), Y1 and Y2 representing cationic amino acids (arginine (R) or lysine (K)). The cytotoxicity of the designed ACPs in HCT 116 colorectal cancer, HeLa cervical cancer and human dermal fibroblast (HDF) cells was assessed by MTT test. The physicochemical properties of the peptides were characterized by various techniques including RP-HPLC, LC-MS, and Circular Dichroism (CD) spectroscopy. The surface activity of the peptides at the air-water interface and their interaction with the lipid monolayers as models for cell membranes were studied by Langmuir trough. The peptides consisting of I with R and K had selective anticancer activity while the combination of L and R diminished the anticancer activity of the peptides but rendered them more toxic to HDFs. The anticancer activity of the peptides was directed by their surface activity (amphiphilicity) and their secondary structure in hydrophobic surfaces including cancer cell membranes. The selectivity of the peptides for cancer cells was a result of their higher penetration into cancer cell membranes compared to normal cell membranes. The peptides exerted their anticancer activity by disrupting the mitochondrial membranes and eventually apoptosis. The results presented in this study provide an insight into the structure-activity relationship of this class of ACPs which can be employed as guidance to design new ACPs with improved anticancer activity and lower toxicity against normal cells.

Islet amyloid polypeptide (IAPP) is a 37 residue intrinsically disordered protein whose aggregation is associated with Type II diabetes. Like most amyloids, it appears that the intermediate aggregates (“oligomers”) of IAPP are more toxic than the mature fibrils, and interaction with the cell membrane is likely to be an integral component of the toxicity. Here we probe the membrane affinity and the conformation of the peptide as a function of its aggregation state. We find that the affinity of the peptide for artificial lipid bilayers is more than 15 times higher in the small oligomeric state (hydrodynamic radius ~ 1.6 nm) compared to the monomeric state (hydrodynamic radius ~ 0.7 nm). Binding with RIN-m5F cell membranes also shows qualitatively similar behavior. The monomeric state, as determined by Forster Resonance Energy Transfer, has a much larger end to end distance than the oligomeric state, suggesting conformational change between the monomers and the oligomers. Raman and Infrared spectroscopic measurements show the presence of considerable alpha helical content in the oligomers, whereas the larger aggregates have largely beta sheet character. Therefore, the conformation of the small oligomers is distinct from both the smaller monomers and the larger oligomers, and this is associated with an enhanced membrane affinity. This provides a possible structural basis for the enhanced toxicity of amyloid oligomers. Such change is also reminiscent of amyloid beta, another aggregation prone amyloidogenic peptide, though the nature of the conformational change is quite different in the two cases. We infer that conformational change underlying oligomer formation is a key factor in determining the enhanced membrane affinity of disease causing oligomers, but the toxic “oligomer fold” may not be universal.

To resolve the contribution of ceramide-containing lipids to the aggregation of the amyloid-β protein into β-sheet rich toxic oligomers, we employed molecular dynamics simulations to study the effect of cholesterol-containing bilayers comprised of POPC (70% POPC, and 30% cholesterol) and physiologically relevant concentrations of sphingomyelin (SM) (30% SM, 40% POPC, and 30% cholesterol), and the GM1 ganglioside (5% GM1, 70% POPC, and 25% cholesterol). The increased bilayer rigidity provided by SM (and to a lesser degree, GM1) reduced the interactions between the SM-enriched bilayer and the N-terminus of Aβ42 (and also residues Ser26, Asn27, and Lys28), which facilitated the formation of a β-sheet in the normally disordered N-terminal region. Aβ42 remained anchored to the SM-enriched bilayer through hydrogen bonds with the side chain of Arg5. With β-sheets in the at the N and C termini, the structure of Aβ42 in the sphingomyelin-enriched bilayer most resembles β-sheet-rich structures found in higher-ordered Aβ fibrils. Conversely, when bound to a bilayer comprised of 5% GM1, the conformation remained similar to that observed in the absence of GM1, with Aβ42 only making contact with one or two GM1 molecules. This article is part of a Special Issue entitled: Protein Aggregation and Misfolding at the Cell Membrane Interface edited by Ayyalusamy Ramamoorthy.

Antimicrobial peptides attracted increasing interest in last decades due to the rising concern of multi-drug resistant pathogens. Dendrimeric peptides are branched molecules with multiple copies of one peptide functional unit bound to the central core. Compared to linear analogues, they usually show improved activity and lower susceptibility to proteases. Knowledge of structure-function relationship is fundamental to tailor their properties. This work is focused on SB056, the smallest example of dendrimeric peptide, whose amino acid sequence is WKKIRVRLSA. Two copies are bound to the α- and e- nitrogen of one lysine core. An 8-aminooctanamide was added at the C-terminus to improve membrane affinity. Its propensity for β-type structures is also interesting, since helical peptides were already thoroughly studied. Moreover, SB056 maintains activity at physiological osmolarity, a typical limitation of natural peptides. An optimized analogue with improved performance was designed, β-SB056, which differs only in the relative position of the first two residues (KWKIRVRLSA). This produced remarkable differences. Structure order and aggregation behavior were characterized by using complementary techniques and membrane models with different negative charge. Infrared spectroscopy showed different propensity for ordered β-sheets. Lipid monolayers’ surface pressure was measured to estimate the area/peptide and the ability to perturb lipid packing. Fluorescence spectroscopy was applied to compare peptide insertion into the lipid bilayer. Such small change in primary structure produced fundamental differences in their aggregation behavior. A regular amphipathic peptide’s primary structure was responsible for ordered β-sheets in a charge independent fashion, in contrast to unordered aggregates formed by the former analogue.

The influence of flanking structures (alpha helices and beta strands in the primary sequence) on amino acid content of the elements of secondary structure has been analyzed in seven sets of nonhomologous proteins. Elevated usage of beta structural amino acid residues and pentapeptides in beta strands between two alpha helices can be explained by the stabilization of secondary structure of those beta strands by natural selection. High usage of alpha helical amino acids and pentapeptides in beta strands situated between two other beta strands is an evidence of the relaxation of natural selection: "passive" beta strands in these fragments of polypeptide chains are frequently formed due to the influence of flanking "active" beta strands. Alpha helices situated between alpha helix and beta strand are enriched by alpha helical pentapeptides and have lower usage of beta structural pentapeptides than those situated between beta strand and alpha helix, their N-termini are more frequently protected from helix to beta transitions by Glu residues. Alpha helices between two beta strands are stabilized by Ala residues and pentapeptides specific to alpha helices. Dipeptide content of the most stable alpha helices and beta strands was used for the creation of the PentaFOLD 2.0 algorithm (http://chemres.bsmu.by) that finds stable fragments of these elements of secondary structure in PDB files.

Although many naturally occurring proteins consist of multiple domains, most studies on protein folding to date deal with single-domain proteins or isolated domains of multi-domain proteins. Studies of multi-domain protein folding are required for further advancing our understanding of protein folding mechanisms. Borrelia outer surface protein A (OspA) is a β-rich two-domain protein, in which two globular domains are connected by a rigid and stable single-layer β-sheet. Thus, OspA is particularly suited as a model system for studying the interplays of domains in protein folding. Here, we studied the equilibria and kinetics of the urea-induced folding-unfolding reactions of OspA probed with tryptophan fluorescence and ultraviolet circular dichroism. Global analysis of the experimental data revealed compelling lines of evidence for accumulation of an on-pathway intermediate during kinetic refolding and for the identity between the kinetic intermediate and a previously described equilibrium unfolding intermediate. The results suggest that the intermediate has the fully native structure in the N-terminal domain and the single layer β-sheet, with the C-terminal domain still unfolded. The observation of the productive on-pathway folding intermediate clearly indicates substantial interactions between the two domains mediated by the single-layer β-sheet. We propose that a rigid and stable intervening region between two domains creates an overlap between two folding units and can energetically couple their folding reactions.

In phylogenomic profiling, the genomic context based methods are based on the observation that two or more proteins having the same pattern of presence or absence in many diverse genomes most likely have a functional link. In this research work, a tool (PATSIM) has been developed to predict the protein patterns based on the SOPM tool. In this tool, the secondary structure for CATH database protein sequences, predicted by the SOPM (Self Optimized Prediction Method) server is passed as input to fulfill objectives such as, (i) Predict the Amino Acid Pattern using the proposed Hybrid KMP and BM algorithm, (ii) Predict the physiochemical properties such as Hydrophobic Non-Polar ALKYL Amino Acid groups, Hydrophobic Non-Polar AROMATIC Amino Acid groups, Hydrophilic Polar Neutral Amino Acid groups, Hydrophilic Polar Acidic Amino Acid groups and Hydrophilic Polar Basic Amino Acid groups of protein sequence, (iii) Predict the secondary structure of protein where the structure of protein sequence is unknown, and (iv) Similarity analysis of protein sequence (structure unknown) with the CATH database. From the results, it is inferred that this tool effectively predicts the similarity between the sequences and also identifies the protein patterns for four secondary structural classes, namely Alpha Helix (h), Beta Sheet (e), Turn (t) and Coil (c). Based on the experimental results, it is inferred that this tool identifies the physiochemical properties of the protein sequence in an effective manner. The source code and its documentation for the PATSIM tool is freely available in the GitHub public repository ( https://github.com/manimkn89/Protein-Sequence-Analysis ).

Arsenic trioxide is one of the most common metallic pollutants entering the food chain both by human activities and nature. Its entry inside the living organism through food, air and water results into the accumulation of heavy metal in several tissues which manifest several metabolic or hormonal disorders. Till now the effect of arsenic trioxide on protein misfolding and aggregation culminating into several neurodegenerative disorders is poorly understood. In the present study, we reveal the aggregation process of Hen Egg White Lysozyme (HEWL) in presence of arsenic trioxide (As2O3) at physiological conditions. We show that As2O3 promote the in vitro aggregation of HEWL in concentration dependent manner. Early phase of aggregation is observed to be induced by exposure of hydrophobic surfaces which later reorganized to promote further self-association leading to β sheet structure. Presence of lower ordered oligomers after two days and higher ordered oligomers along with amorphous aggregates after week long incubation indicate that As2O3 drives the self-assembly of lysozyme towards oligomeric form.

Protein aggregation and amyloid fibrillation are responsible for several serious pathological conditions (like type II diabetes, Alzheimer's and Parkinson's diseases etc.) and protein drugs ineffectiveness. Therefore, a molecule that can inhibit the amyloid fibrillation and potentially clear amyloid fibrils is of great therapeutic value. In this manuscript, we investigated the antiamyloidogenic, fibril disaggregating, as well as cell protective effect of an anti-tuberculosis drug, Capreomycin (CN). Aggregation kinetics data, as monitored by ThT fluorescence, inferred that CN retards the insulin amyloid fibrillation by primarily targeting the fibril elongation step with little effect on lag time. Increasing the dose of CN boosted its inhibitory potency. Strikingly, CN arrested the growth of fibrils when added during the elongation phase, and disaggregated mature insulin fibrils. Our Circular Dichroism (CD) results showed that, although CN is not able to maintain the alpha helical structure of protein during fibrillation, reduces the formation of beta sheet rich structure. Furthermore, Dynamic Light Scattering (DLS) and Transmission Electronic Microscopy (TEM) analysis confirmed that CN treated samples exhibited different size distribution and morphology, respectively. In addition, molecular docking results revealed that CN interacts with insulin through hydrophobic interactions as well as hydrogen bonding, and the Hemolytic assay confirmed the non-hemolytic activity of CN on human RBCs. For future research, this study may assist in the rational designing of molecules against amyloid formation.

The formation of specific patterns during evaporation of biofluids e.g. blood, serum etc. can be analyzed for quick identification of several diseases. Human Serum Albumin (HSA) is frequently used as the model protein to study amyloid fibril formation that is responsible for numerous neurodegenerative diseases like Parkinson’s disease, Alzheimer’s disease etc. In the present study the fibril growth of HSA is characterized by analyzing the drying patterns of the protein solution. The fibril formation of HSA is confirmed by spectrofluorimetry and the β-sheet content is quantified by far-UV Circular Dichroism spectrometry. Examinations of the drying patterns of HSA, in presence of fluorescent nanoparticles, reveals distinctive pattern transformations with increase in the β-sheet content visualized through fluorescence microscopy and FESEM. The fluorescence images of the dried droplet are analyzed using image processing by the color thresholding method. A rapid quantification methodology of the β-sheet content of HSA and the fraction of unfolded protein has been developed by comparing the results of the image analysis with those of the far-UV CD data.

NMR spectroscopy is an established tool in drug discovery, but its strength is commonly regarded to be largely confined to the early stages of hit discovery and fragment based drug design, where NMR offers unique capabilities of characterizing the binding modes of ligand molecules that bind sufficiently weakly to be in rapid exchange between bound and free state. Here we, first, provide a meta-review of recent reviews on NMR studies of ligand binding and, second, review recent progress towards NMR characterization of the ligand binding mode in stable protein-ligand complexes, with particular emphasis on the global positioning system (GPS) approach enabled by paramagnetic lanthanide tags.

The diversity of modern proteins arose through the combinatorial shuffling and differentiation of a limited number of autonomously folding domain prototypes, but the origin of these prototypes themselves has long remained poorly understood. In recent years, the proposal that they originated by repetition, accretion, and recombination from an ancestral set of peptides, which evolved as cofactors of RNA-based replication and catalysis, has gained wide acceptance, supported by the systematic identification of such ancestral peptides and the experimental recapitulation of the mechanisms by which they could have yielded the first folded proteins. Inspired by this evolutionary process, protein engineers have seized on design from pre-optimized peptide components as a powerful approach to generating proteins with novel topology and functionality.

This study investigated the roles of amino acid sequence and type of anions on short peptide amphiphiles by applying external electric field. We designed two synthetic peptides of FFKKFF and KFFFFK with amino acid sequence and charge distribution that varied systematically. Next, the effects of aqueous milieu to the self-assembled structure stability was investigated through the addition of anion (I- or ClO4-). Growth and structural transition of the self-assembled structures was followed by using circular dichroism (CD) and scanning electron microscope (SEM) for 48 hours on removal of DC electric field. Diverse micrometre-sized self-assembled structures can be obtained depending on different anion presence in the solution. Our results showed that all the peptides favoured a needle-like structure in solutions containing ClO4-, which mainly consists of α-helical and β-sheet conformations with or without the electric field. It was found that the application of electric field stimulate the formation of a higher extend of end-to-end integration or sideways association. The absence of electric field facilitated the formation of a stable and organised network of interwoven fibrillary structure at 48th hour in solutions containing I-. Interestingly, fine needle-like structure was observed when electric field was applied to the same solutions. Our findings suggested that electric field favours the change of the peptides from β-sheet to a mixture of α-helical and β-sheet conformations in solutions containing I-. Removing the electric field did not reinstate the original conformation

Optically transparent fibroin nanofiber (FNF) paper was obtained without using binders by passing a fibroin suspension through a mechanical nanofibrillation process using a water-jet. The optical transmittance of the nanofiber paper increased with the number of passes through the water-jet and reached 79.3% and 80.3% of light at 600 nm after 15 and 30 passes, respectively. The fibroin of the transparent nanopaper remained fibrous with a native β-sheet secondary structure. Therefore, the transparent nanopaper showed a high elastic modulus, high thermal durability, and low thermal expansion. This novel protein material, which can be fabricated without toxic chemicals or organic solvents, is bio-friendly and shows promise for new applications in biocompatible photonic devices.

Self-assembling peptides have become important building blocks for materials design (e.g. hydrogels) and play a crucial role in a range of diseases including Alzheimer and Parkinson. In this context, accessing the nanomechanical properties of ubiquitous β-sheet rich nanofibres (e.g.: amyloids) is key to the formulation of materials and design of therapies. Although the bulk mechanical properties of hydrogels can easily be accessed using common techniques and equipment, the mechanical properties of their constituent fibres, in particular if with radii in the nanometre scale, are more challenging to measure and estimate. In this work we show for the first time how the rapid nanomechanical mapping technique: amplitude modulation-frequency modulation (AM-FM), can be used to determine the heights, Young's moduli and viscosity coefficients of a series of β-sheet peptide nanofibres with high statistical confidence. Our results show how peptide sequence and in particular length, charge and interaction with the substrate affect the viscoelastic properties of the peptide fibres.

The NSP6 protein of SARS-CoV-2 is a transmembrane protein, with some regions lying outside the membrane. Besides a brief role of NSP6 in autophagosome formation, this is not studied significantly. Also, there is no structural information available to date. Based on the prediction by TMHMM server for transmembrane prediction, it is found that the N-terminal residues (Giri et al., 2020; Naqvi et al., 2020; Bojkova et al., 2020; Gordon et al., 2020; V’kovski et al., 2021; von Brunn et al., 2007; Benvenuto et al., 2020; Cottam et al., 2011; Oostra et al., 2008; Angelini et al., 2013; Schweizer et al., 2014) [1-11], middle region residues (91–112), and C-terminal residues (231–290) lies outside the membrane. Molecular Dynamics (MD) simulations showed that NSP6 consists of helical structures. In contrast, the membrane outside lying region (91–112) showed partial helicity, which was further used as a model and obtained disordered type conformation during 1.5 microseconds. Additionally, a 200ns simulation study of residues 231–290 have shown significant conformational changes. As compared to helical and beta-sheet conformations in its structure model, the 200ns simulation resulted in the loss of beta-sheet structures while helical regions remained intact. Further, we have experimentally characterized the residue 91–112 by using reductionist approaches. CD spectroscopy suggests that the NSP6 (91–112) is disordered-like region in isolation, which gains helical conformation in different biological mimic environmental conditions. These studies can be helpful to study NSP6 (91–112) interactions with host proteins, where different protein conformations might play a significant role. The present study adds up more information about the NSP6 protein aspect, which could be exploited for its host protein interaction and pathogenesis., Graphical abstract The schematic representation of NSP6 membrane topology and conformational dynamics of residue 91–112. The N-terminal and C-terminal are shown in cytoplasmic side based on the experimental evidence on coronaviruses reported by Oostra et al., 2008. The membrane anchoring domain are shown based on the TMHMM server prediction.Image 1

We report herein the uracil-di-aza-amino acid ( U r AA ) as a new family of molecular scaffold to induce β-hairpin structure with H-bonded β-sheet conformation in a short peptide. This has been demonstrated in two conceptual fluorescent pentapeptides wherein triazolylpyrenyl alanine and/or triazolylmethoxynapthyl alanine ( TPy Ala Do and/or TMNap Ala Do ) are embedded into two arms of the uracil-amino acid via an intervening leucine. Conformational analysis by CD, IR, variable temperature and 2D NMR spectroscopy reveals the β-hairpin structures for both the peptides. Study of photophysical property reveals that the pentapeptide containing fluorescent triazolyl unnatural amino acids TMNap Ala Do and TPy Ala Do at the two termini exhibits dual path entry to exciplex emission-either via FRET from TMNap Ala Do to TPy Ala Do or via direct excitation of a FRET acceptor, TPy Ala Do . The other pentapeptide with TPy Ala Do / TPy Ala Do pair shows excimer emission. Furthermore, both the peptides maintaining their fundamental photophysics are found to interact with BSA as only a test biomolecule.

Far-UV Circular Dichroism experiments and Atomic Force Microscopy tomography are employed to assess the impact of beta-sheet breakers on the A?1-40 peptide aggregation process in the presence of Cu2+ or Zn2+ transition metals. In this work we focus on two specific 5-amino acids long beta-sheet breakers, namely the LPFFD Soto peptide, already known in the literature, and the LPFFN peptide recently designed and studied by our team. We provide evidence that both ?-sheet breakers are effective in reducing the A?1-40 aggregation propensity, even in the presence of metal ions.

Aqueous solutions of lactoferrin (LF) were subjected to thermal processing (72, 80, 85, 95 °C for 15 s) using a rheometer. Despite the low protein concentration and low ionic strength of the LF solutions, analyses post-processing revealed the presence of heat-induced, covalently-linked, protein aggregates. Aggregation was accompanied by increased surface hydrophobicity and zeta-potential and changes to the secondary structure of the protein, including reduction in the proportion of alpha helix domains coupled with increased intermolecular beta sheet structures. The native red colour of the protein was lost upon thermal processing, not attributable to the release of LF-bound iron. Micro-differential scanning calorimetry showed LF to be irreversibly denatured on heating with endotherms observed at 58.7 °C (apo-LF) and 89.6 °C (holo-LF). Thermal processing at 72–95 °C resulted in irreversible changes to the structural and physicochemical properties of bovine LF, whereby the lowest heating intensity had the least impact on LF structure.

Alzheimer’s is a fatal neurodegenerative disease for which there is no cure at present. The disease is characterized by the presence of plaques in the brains of a patient, which are composed mainly of aggregates of the amyloid-β peptide in the form of β-sheet fibrils. Here, we investigated the possibility of exploiting the superior binding ability of aromatic amino acids to a particular model of the amyloid-β fibrils. which is a difficult target for drug design. The β-sheet breaker peptide LPFFD was modified with aromatic amino acids and its binding to these fibrils was studied. We found that the orientation and the electrostatic complementarity of the modified peptide with respect to the fibrils played a crucial role in determining whether its binding was improved by the aromatic amino acids. The modified LPFFD peptides were able to bind to those fibril residues. which are important in the aggregation of amyloid-β peptides and thus can potentially inhibit the further aggregation of the amyloid-beta peptides by blocking their interactions. We found that the tryptophan modified LPFFD peptides had the best binding affinities. In most cases, the aromatic amino acids in the N-terminus of the modified peptides made more contacts with the fibrils than those in the C-terminus. We also found that increasing the aromatic content did not significantly improve the binding of the LPFFD peptide to the fibrils. Our study can serve as a basis for the design of novel peptide-based drugs for Alzheimer’s disease in which aromatic interactions play an important role.

It is a challenging work to design novel responsive materials by controlling the secondary structure of polypeptide, especially β-sheet. We designed and synthesized a novel dual responsive diblock copolymer of methyl poly (ethylene glycol)-b-poly (O-benzyl- l -threonine) (mPEG-b-PBnLT). The copolymer solutions exhibited unusual and reversible gel-to-sol-to-gel transition behavior with temperature, and the transition temperature window could be easily adjusted by changing copolymer concentration or PBnLT block length. It was confirmed that low temperature gel-to-sol transition was due to the disassembly of the preliminary β-sheet layered nano-assemblies that induced the transformation of self-organized morphology from nanosized fibrils to spherical aggregates, while the high temperature sol-to-gel transition was ascribed to the dehydration of PEG segments. The disassembly of β-sheet structures was caused by the change in the polarity of the pendant benzyl ether bond of PBnLT block in water with temperature. This stimuli-responsiveness of secondary self-assembled nanostructures and the corresponding change in macroscopic phase transition inspires a brand-new strategy for structural design and functional control of polypeptide materials.

The folding process has been described as the output of main mechanical forces which generate from the specific chemical interplay among residues of an amino acid chain. Recently, we have reported that dihedral angle values in (poly)peptides can be calculated by means of a newly defined parameter referred as folding factor. This physical measure is directly linked to the determination of the degrees of freedom (η), namely the number of possible interactions that can occur between atoms (on the base of changes in interatomic distances). Precise rules to accurately define the degrees of freedom and, in turn, the related folding factors, have not been reported to date. We propose here an empirical analysis of possible physico-chemical principles for a careful evaluation of the degrees of freedom, as well as the threshold distances for the chemical interactions to occur. To validate our systematic approach, we have applied these rules to the calculation of dihedral angles of the α-helix Vps27p and the beta-sheet U(1–17)T9D, highlighting wide agreement between predicted and experimental values.

Summary The understanding and prediction of the solubility of biomolecules, even of the simplest ones, reflect an open question and unmet need. Short aromatic tripeptides are among the most highly aggregative biomolecules. However, in marked contrast, Ala-Phe-Ala (AFA) was surprisingly found to be non-aggregative and could be solubilized at millimolar concentrations. Here, aiming to uncover the underlying molecular basis of its high solubility, we explore in detail the solubility, aggregation propensity, and atomic-level structure of the tripeptide. We demonstrate an unexpectedly high water solubility of AFA reaching 672 mM, two orders of magnitude higher than reported previously. The single crystal structure reveals an anti-parallel β sheet conformation devoid of any aromatic interactions. This study provides clear mechanistic insight into the structural basis of solubility and suggests a simple and feasible tool for its estimation, bearing implications for design of peptide drugs, peptides materials, and advancement of peptide nanotechnology., Graphical abstract, Highlights Balance between solubility versus aggregation of aromatic peptides is examined Significant differences in solubility are observed for analogous tripeptides Solid-state packing is found to be underlying molecular basis for high solubility Revealing the importance of biology at the solid state in physiology and pathology, The solubility of biomolecules in aqueous solutions is a central issue with regard to their physiological and pathological activities. Bera et al. provide insight into the solid-state packing-solubility relationship of biomolecules. This work may further extend the importance of solid-state biology for basic and applied science.

The structural underpinnings for the higher toxicity of the oligomeric intermediates of amyloidogenic peptides, compared to the mature fibrils, remain unknown at present. The transient nature and heterogeneity of the oligomers make it difficult to follow their structure. Here, using vibrational and solid-state nuclear magnetic resonance spectroscopy, and molecular dynamics simulations, we show that freely aggregating Aβ40 oligomers in physiological solutions have an intramolecular antiparallel configuration that is distinct from the intermolecular parallel β-sheet structure observed in mature fibrils. The intramolecular hydrogen-bonding network flips nearly 90°, and the two β-strands of each monomeric unit move apart, to give rise to the well-known intermolecular in-register parallel β-sheet structure in the mature fibrils. Solid-state nuclear magnetic resonance distance measurements capture the interstrand separation within monomer units during the transition from the oligomer to the fibril form. We further find that the D23–K28 salt-bridge, a major feature of the Aβ40 fibrils and a focal point of mutations linked to early onset Alzheimer’s disease, is not detectable in the small oligomers. Molecular dynamics simulations capture the correlation between changes in the D23–K28 distance and the flipping of the monomer secondary structure between antiparallel and parallel β-sheet architectures. Overall, we propose interstrand separation and salt-bridge formation as key reaction coordinates describing the structural transition of the small Aβ40 oligomers to fibrils.

The temperature-induced α-helix to β-sheet transition in long-chain poly-l-lysine (PLL), accompanied by the gauche-to-trans isomerization of CH2 groups in the hydrocarbon side chains of Lys amino acid residues, and formation of β-sheet as well as α-helix fibrillar aggregates of PLL have been studied using Fourier-transform infrared (FT-IR) and vibrational circular dichroism (VCD) spectroscopy, and transmission electron microscopy (TEM). In a low-temperature alkaline water solution or in a methanol-rich water mixture, the secondary structure of PLL is represented by α-helical conformations with unordered and gauche-rich hydrocarbon side chains. Under these conditions, PLL molecules aggregate into α-helical fibrils. PLLs dominated by extended antiparallel β-sheet structures with highly ordered trans-rich hydrocarbon side chains are formed in a high-temperature range at alkaline pD and aggregate into fibrillar, protofibrillar, and spherical forms. Presented data support the idea that fibrillar aggregation is a varied phenomenon possible in repetitive structural elements with not only a β-sheet-rich conformation, but also an α-helical-rich conformation.

Glucoamylase (EC 3.2.1.3) from Aspergillus niger possesses 31% α-helix, 36% β structure and rest aperiodic structure. A transition of glucoamylase structure in the presence of varying concentrations of glyoxal (GO) and trifluoroethanol (TFE) was studied by using multi-methodological approaches. At 20% GO, glucoamylase exists as molten globule state as evident by high tryptophan and ANS fluorescence, retention of secondary structure and loss of native tertiary structure. This state precedes the onset of the aggregation process and maximum is achieved at the highest concentration i.e. at 90% of GO. In parallel study TFE, on increasing concentration up to 25% induces secondary structure transformation leading to accumulation of intermolecular β sheets, altered tryptophan environment, high ANS and ThT fluorescence resulting in the formation of glucoamylase aggregates. Isothermal titration calorimetric curve is sigmoidal, indicating the weak binding of GO/TFE and glucoamylase. TEM studies showed that glucoamylase exists as globular and amorphous aggregates at 90% glyoxal and 25% TFE respectively. Further, TFE at 70% causes inhibition of enzyme aggregates; the majority of secondary structures observed at this concentration are α helices. Alpha helices being the main key player relocates glucoamylase native environment as evident by CD, FTIR and TEM. Hence induction of β sheet promotes protein aggregation and α helices inhibits protein aggregation.

The feasibility of using Fourier transform infrared spectroscopy (FTIR) to detect heat induced conformational rearrangements of proteins, protein–protein and protein–lipid interactions was studied with accelerated shelf-life protocols. Ultra-high temperature treated whole (WM) and skim milk (SM) were stored at 20, 30, 40 and 50 °C for 28 days. The changes leading to increased sedimentation in SM and WM at higher temperatures (≥40 °C) were observed during first 14 days of the storage period. Milk samples stored at 40 and 50 °C showed marked changes in the bands corresponding to conformations of milk lipids and formation of intermolecular β sheet of proteins, indicating protein–lipid interactions and aggregation. Dried sediment contained fat confirming protein–lipid participation in the sedimentation. FTIR was also able to detect changes that led to increased sedimentation in SM at temperatures lower than 40 °C, but only after 28 days.

We measured the thermal conductivity of Araneus ventricosus' spider dragline silk using a suspended microdevice. The thermal conductivity of the silk fiber was approximately 0.4Wm-1K-1 at room temperature and gradually increased with an increasing temperature in a manner similar to that of other disordered crystals or proteins. In order to elucidate the effect of β-sheet crystals in the silk, thermal denaturation was used to reduce the quantity of the β-sheet crystals. A calculation with an effective medium approximation supported this measurement result showing that the thermal conductivity of β-sheet crystals had an insignificant effect on the thermal conductivity of SDS. Additionally, the enhancement of bonding strength in a glycine-rich matrix by atomic layer deposition did not increase the thermal conductivity. Thus, this study suggests that the disordered part of the glycine-rich matrix prevented the peptide chains from being coaxially extended via the cross-linking covalent bonds.

Sup35 is a prion-like protein from yeast and shares the ability to transmit its aberrant fold and to aggregate into amyloid fibrils. 7GNNQQNY13 from the prion-determining domain of Sup35 was reported to form an amyloid. We first investigated the self-aggregation transition behavior of GNNQQNY to the β-sheet amyloid state under various conditions. Mechanical stirring using a magnetic bar resulted in accelerated aggregation of the GNNQQNY. The aggregation rate of GNNQQNY was also dependent on its concentration; the higher the GNNQQNY concentration, the faster the aggregation. Circular dichroism and Fourier transform-infrared spectral data indicated the formation of the β-sheet structure in the GNNQQNY aggregates. The fluorescence experiments using an amyloid-specific thioflavin T also demonstrated that the GNNQQNY aggregates formed the amyloid structures. The amyloid structure of the GNNQQNY aggregates served as seeds for the elongation of the monomeric GNNQQNY in the solution state. We further studied the ability of the GNNQQNY amyloid fibrils to act as seeds for the elongation of the amyloid-forming monomeric proteins (albumin, lysozyme and insulin). The cross-seeding experiments suggested that the GNNQQNY aggregate could possibly promote the amyloid fibril formation of heterogeneous insulin. The inverse monomeric GNNQQNY would have a binding capacity for the heterogeneous already-formed amyloid-β fibrils on a mice brain section. These basic data could be informative for elucidating the pathogenic and/or propagation mechanisms of prion agents and developing effective therapeutics and/or diagnosis for prion diseases.

Although conventional enzyme-linked immunosorbent assays (ELISA) and related assays have been widely applied for the diagnosis of diseases, many of them suffer from large error variance for monitoring the concentration of targets over time, and insufficient limit of detection (LOD) for assaying dilute targets. We herein report a readout mode of ELISA based on the binding between peptidic β-sheet structure and Congo Red. The formation of peptidic β-sheet structure is triggered by alkaline phosphatase (ALP). For the detection of P-Selectin which is a crucial indicator for evaluating thrombus diseases in clinic, the ‘β-sheet and Congo Red’ mode significantly decreases both the error variance and the LOD (from 9.7 ng/ml to 1.1 ng/ml) of detection, compared with commercial ELISA (an existing gold-standard method for detecting P-Selectin in clinic). Considering the wide range of ALP-based antibodies for immunoassays, such novel method could be applicable to the analysis of many types of targets.

The amyloidogenic peptide VDSWNVLVAG from Bgl2p–glucantransferase of Saccharomyces cerevisiae cell wall and its modifying analog VESWNVLVAG were taken for the construction of four types of bilayers which differ by orientation of the peptides in the layers and of the layers relative to each other. These bilayers were used as starting models for the molecular dynamics (MD) at three charge states (neutral, pH3, and pH5). The changes of the fraction of secondary structure during 1ns simulations were received for 96 MD trajectories. The data article contains the necessary information for the construction of models of β-strands organization in the oligomer structure. These results were used in the associated research article “Structural model of amyloid fibrils for amyloidogenic peptide from Bgl2p–glucantransferase of S. cerevisiae cell wall and its modifying analog. New morphology of amyloid fibrils” (Selivanova et al., 2016) [1].

In cardiac and skeletal muscle, the troponin complex turns muscle contraction on and off in a calcium-dependent manner. Many small molecules are known to bind to the troponin complex to modulate its calcium binding affinity, and this may be useful in a broad range of conditions in which striated muscle function is compromised, such as congestive heart failure. As a tool for developing drugs specific for the cardiac isoform of troponin, we have designed a chimeric construct (cChimera) consisting of the regulatory N-terminal domain of cardiac troponin C (cNTnC) fused to the switch region of cardiac troponin I (cTnI), mimicking the key binding event that turns on muscle contraction. We demonstrate by solution NMR spectroscopy that cChimera faithfully reproduces the native interface between cTnI and cNTnC. We determined that small molecules based on diphenylamine can bind to cChimera with a K D as low as 10 μM. Solution NMR structures show that minimal structural perturbations in cChimera are needed to accommodate 3-methyldiphenylamine (3-mDPA), which is probably why it binds with higher affinity than previously studied compounds like bepridil, despite its significantly smaller size. The unsubstituted aromatic ring of 3-mDPA binds to an inner hydrophobic pocket adjacent to the central beta sheet of cNTnC. However, the methyl-substituted ring is able to bind in two different orientations, either inserting into the cNTnC-cTnI interface or “flipping out” to form contacts primarily with helix C of cNTnC. Our work suggests that preservation of the native interaction between cNTnC and cTnI is key to the development of a high affinity cardiac troponin-specific drug.

Prior published papers has been reanalyzed in this retrospective study of β-lactoglobulin (β-LG) coerced into forming amyloids through assays using both light scattering and Thioflavin T (ThT) fluorescence. The published datasets were extracted and analyzed by a sigmoidal time-dependent model, which yields two time constants which allow comparison between various concentration levels of protein in solution, denaturing temperature, some other additives and the presence of mechanical agitation, however that was accomplished. Shorter aggregation times are observed with increased protein concentration in solution and increased denaturing temperature. The presence of agitation seems to have a large influence and future efforts should be clear to identify the type of agitation if standards are to be developed. Finally comparing scattering and ThT fluorescence, as indicators, it appears that scattering occurs at an earlier time point relative to ThT fluorescence linked with β sheet formation.