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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

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.

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.

Cyclic peptides with disc-shaped structures have emerged as potent building blocks for the preparation of new biomaterials in fields ranging from biological to material science. In this work, we analyze in depth the self-assembling properties of a new type of cyclic peptides based on the alternation of α-residues and cyclic δ-amino acids (α,δ-CPs). To examine the preferred stacking properties adopted by cyclic peptides bearing this type of amino acids, we carried out a synergistic in vitro/in silico approximation by using simple dimeric models and then extended to nanotubes. Although these new cyclic peptides (α,δ-CPs) can interact either in a parallel or antiparallel fashion, our results confirm that although the parallel β-sheet is more stable, it can be switched to the antiparallel stacking by choosing residues that can establish favorable cross-strand interactions. Moreover, the subsequent comparison by using the same methodology but applied to α,γ-CPs models, up to the moment assumed as antiparallel-like d,l-α-CPs, led to unforeseen conclusions that put into question preliminary conjectures about these systems. Surprisingly, they tend to adopt a parallel β-sheet directed by the skeleton interactions. These results imply a change of paradigm with respect to cyclic peptide designs that should be considered for dimers and nanotubes.

Peptide-protein docking is challenging due to the considerable conformational freedom of the peptide. CAPRI rounds 38-45 included two peptide-protein interactions, both characterized by a peptide forming an additional beta strand of a beta sheet in the receptor. Using the Rosetta FlexPepDock peptide docking protocol we generated top-performing, high-accuracy models for targets 134 and 135, involving an interaction between a peptide derived from L-MAG with DLC8. In addition, we were able to generate the only medium-accuracy models for a particularly challenging target, T121. In contrast to the classical peptide-mediated interaction, in which receptor side chains contact both peptide backbone and side chains, beta-sheet complementation involves a major contribution to binding by hydrogen bonds between main chain atoms. To establish how binding affinity and specificity are established in this special class of peptide-protein interactions, we extracted PeptiDBeta, a benchmark of solved structures of different protein domains that are bound by peptides via beta-sheet complementation, and tested our protocol for global peptide-docking PIPER-FlexPepDock on this dataset. We find that the beta-strand part of the peptide is sufficient to generate approximate and even high resolution models of many interactions, but inclusion of adjacent motif residues often provides additional information necessary to achieve high resolution model quality.

Despite the fundamental clinical importance of amyloid fibril formation, its mechanism is still enigmatic. Crystallography of minimal amyloid models was a milestone in the understanding of the architecture and biological activities of amyloid fibers. However, the crystal structure of ultimate dipeptide-based amyloids is not yet reported. Herein, we present the crystal structure of a typical amyloid-forming minimal dipeptide, Ac-Phe-Phe-NH2 (Ac-FF-NH2 ), showing a canonical β-sheet structure at the atomic level. The simplicity of the structure helped in investigating amyloid-inhibition using crystallography, never previously reported for larger peptide models. Interestingly, in the presence of an inhibitor, the supramolecular packing of Ac-FF-NH2 molecules rearranged into a supramolecular 2-fold helix (21 helix). This study promotes our understanding of the mechanism of amyloid formation and of the structural transitions that occur during the inhibition process in a most fundamental model.

The present study was aimed to get insights on the role of calcium ions on the thermodynamic stability, substrate specificity, and organic solvent compatibility of the extracellular protease produced by Bacillus aquimaris VITP4. Presence of Ca2+ enhanced the activity of the enzyme in the temperature range of 30-60 °C and increased the half-life from 164 to 234 Min. Circular dichroism experiments indicated that the temperature of half-denaturation (Tm ) of the protease increased from 76 to 86 °C. As judged by fluorescence emission profiles, the overall fold of the enzyme around the tryptophan residues could be similar. Further, thermal inactivation experiments revealed that the enzyme followed first order kinetics, with increase in energy for inactivation (Eai ) by 24.2 ± 1.2 kJ mol -1 in the presence of Ca2+ . Studies with synthetic peptides as well as with bovine serum albumin signified preferential hydrolysis of the peptide bonds at the C-terminus of alanine residues (with a kcat /KM of 141,400 M-1 Sec-1 ) and at the C-terminus of arginine residues with a lower specificity (72,400 M-1 Sec-1 ), indicating bisubstrate specificity of the enzyme. The enzyme was found to be compatible with organic solvents (50%, v/v) such as acetonitrile and butanol, indicating possible application under demanding nonaqueous conditions.

The β-sheet breaker (BSB) peptides interfere with amyloid fibril assembly and used as therapeutic agents in the treatment of Alzheimer's disease (AD). In this regard, a simple yet effective in silico screening methodology was applied in the present study to evaluate a potential 867 pentapeptide library based on known BSB peptide, LPFFD, for destabilizing Aβ42 protofibrils. The molecular docking based virtual screening was used to filter out pentapeptides having binding affinities stronger than LPFFD. In the next step, binding free energies of the top 10 pentapeptides were evaluated using the MM-PBSA method. The residue-wise binding free energy analysis reveals that two pentapeptides, PVFFE, and PPFYE, bind to the surface of Aβ42 protofibril and another pentapeptide, PPFFE, bind in the core region of Aβ42 protofibril. By employing molecular dynamics simulation as a post filter for the top-hit peptides from MM-PBSA, the pentapeptides, PPFFE, PVFFE, and PPFYE, have been identified as potential BSB peptides for destabilizing Aβ42 protofibril structure. The conformational microstate analysis, a significant decrease in the β-sheet content of Aβ42 protofibril, a loss in the total number of hydrogen bonds in Aβ42 protofibril, Asp23-Lys28 salt bridge destabilization and analysis of the free energy surfaces highlight Aβ42 protofibril structure destabilization in presence of pentapeptides. Among three top-hit pentapeptides, PPFFE displayed the most potent Aβ42 protofibril destabilization effect that shifted the energy minima toward lowest value of β-sheet content as well as lowest number of hydrogen bonds in Aβ42 protofibril. The in silico screening workflow presented in the study highlight an alternative tool for designing novel peptides with enhanced BSB ability as potential therapeutic agents for AD.

Many protein and peptide sequences are self-assembled into β-sheet-rich fibrous structures called amyloids. Their atomic details provide insights into fundamental knowledge related to amyloid diseases. To study the detailed structure of the amyloid, we have developed a model system that mimics the self-assembling process of the amyloid within a water-soluble protein, termed peptide self-assembly mimic (PSAM). PSAM enables capturing of a peptide sequence within a water-soluble protein, thus making structural and energetics-related studies possible. In this work, we extend our PSAM approach to a naturally occurring chameleon sequence from αB crystallin. We chose "Val-Leu-Gly-Asp-Val (VLGDV)", a five amino-acid sequence, which forms a β-turn in the native structure and a β-barrel in the amyloid oligomer cylindrin, as a grafting sequence to the PSAM scaffold. The crystal structure revealed that the sequence grafting induced β-sheet bending at the grafted site. We further investigated the role of the central glycine residue and found that its role in the β-sheet bending is dependent on the neighboring residues. The ability of PSAM to observe the structural alterations induced by the grafted sequence provides an opportunity to evaluate the structural impact of a sequence from the peptide self-assembly.

Intermolecular forces constrain peptide conformation. However, the role of each intermolecular force in constraining peptide conformation remains poorly understood. In this work, we show that aromatic-aromatic interactions drive peptides into β-sheets, and the hydrophobic effect determines the assembly speed of peptides. By using intermolecular forces to artificially control the assembly of β-sheets, a multi-modal analytical system was developed that allows five readouts and dual qualitative-quantitative analysis, and satisfies both point-of-care testing (POCT) and laboratory-based testing. For Mycoplasma Pneumoniae diagnosis, this system eradicates misdiagnosis (from 30 % to 0 %) and broadens the linear range by three-fold, both of which are critical for guiding therapy. This work not only illustrates exact roles of intermolecular forces in driving the formation of β-sheets, but also provides a guideline for the construction of a multi-modal analytical system for disease diagnosis.

Amphipathic peptides with amino acids arranged in alternating patterns of hydrophobic and hydrophilic residues efficiently self‐assemble into β‐sheet bilayer nanoribbons. Hydrophobic side chain functionality is effectively buried in the interior of the putative bilayer of these nanoribbons. This study investigates consequences on self‐assembly of increasing the surface area of aromatic side chain groups that reside in the hydrophobic core of nanoribbons derived from Ac‐(XKXE)2‐NH2 peptides (X = hydrophobic residue). A series of Ac‐(XKXE)2‐NH2 peptides incorporating aromatic amino acids of increasing molecular volume and steric profile (X = phenylalanine [Phe], homophenylalanine [Hph], tryptophan [Trp], 1‐naphthylalanine [1‐Nal], 2‐naphthylalanine [2‐Nal], or biphenylalanine [Bip]) were assessed to determine substitution effects on self‐assembly propensity and on morphology of the resulting nanoribbon structures. Additional studies were conducted to determine the effects of incorporating amino acids of differing steric profile in the hydrophobic core (Ac‐X1KFEFKFE‐NH2 and Ac‐(X1,5KFE)‐NH2 peptides, X = Trp or Bip). Spectroscopic analysis by circular dichroism (CD) and Fourier transform infrared (FT‐IR) spectroscopy indicated β‐sheet formation for all variants. Self‐assembly rate increased with peptide hydrophobicity; increased molecular volume of the hydrophobic side chain groups did not appear to induce kinetic penalties on self‐assembly rates. Transmission electron microscopy (TEM) imaging indicated variation in fibril morphology as a function of amino acid in the X positions. This study confirms that hydrophobicity of amphipathic Ac‐(XKXE)2‐NH2 peptides correlates to self‐assembly propensity and that the hydrophobic core of the resulting nanoribbon bilayers has a significant capacity to accommodate sterically demanding functional groups. These findings provide insight that may be used to guide the exploitation of self‐assembled amphipathic peptides as functional biomaterials., Self‐assembling peptide β‐sheet nanoribbons were synthesized with varying side aromatic chain surface areas using natural and non‐natural residues. It was found that the nanoribbon bilayers can accommodate packing of very large side chain groups. The self‐assembly kinetics was accelerated as the hydrophobicity of the amino acids increased.

N-myc downstream-regulated gene 1 (NDRG1) is a tumour suppressor involved in vesicular trafficking and stress response. NDRG1 participates in peripheral nerve myelination, and mutations in the NDRG1 gene lead to Charcot-Marie-Tooth neuropathy. The 43-kDa NDRG1 is considered as an inactive member of the α/β hydrolase superfamily. In addition to a central α/β hydrolase fold domain, NDRG1 consists of a short N terminus and a C-terminal region with three 10-residue repeats. We determined the crystal structure of the α/β hydrolase domain of human NDRG1 and characterised the structure and dynamics of full-length NDRG1. The structure of the α/β hydrolase domain resembles the canonical α/β hydrolase fold with a central β sheet surrounded by α helices. Small-angle X-ray scattering and CD spectroscopy indicated a variable conformation for the N- and C-terminal regions. NDRG1 binds to various types of lipid vesicles, and the conformation of the C-terminal region is modulated upon lipid interaction. Intriguingly, NDRG1 interacts with metal ions, such as nickel, but is prone to aggregation in their presence. Our results uncover the structural and dynamic features of NDRG1, as well as elucidate its interactions with metals and lipids, and encourage studies to identify a putative hydrolase activity of NDRG1. DATABASES: The coordinates and structure factors for the crystal structure of human NDRG1 were deposited to PDB (PDB ID: 6ZMM).

Some disulfide bonds perform important structural roles in proteins, but another group has functional roles via redox reactions. Forbidden disulfides are stressed disulfides found in recognizable protein contexts, which currently constitute more than 10% of all disulfides in the PDB. They likely have functional redox roles and constitute a major subset of all redox-active disulfides. The torsional strain of forbidden disulfides is typically higher than for structural disulfides, but not so high as to render them immediately susceptible to reduction under physionormal conditions. Previously we characterized the most abundant forbidden disulfide in the Protein Data Bank, the aCSDn: a canonical motif in which disulfide-bonded cysteine residues are positioned directly opposite each other on adjacent anti-parallel β-strands such that the backbone hydrogen-bonded moieties are directed away from each other. Here we perform a similar analysis for the aCSDh, a less common motif in which the opposed cysteine residues are backbone hydrogen bonded. Oxidation of two Cys in this context places significant strain on the protein system, with the β-chains tilting toward each other to allow disulfide formation. Only left-handed aCSDh conformations are compatible with the inherent right-handed twist of β-sheets. aCSDhs tend to be more highly strained than aCSDns, particularly when both hydrogen bonds are formed. We discuss characterized roles of aCSDh motifs in proteins of the dataset, which include catalytic disulfides in ribonucleotide reductase and ahpC peroxidase as well as a redox-active disulfide in P1 lysozyme, involved in a major conformation change. The dataset also includes many binding proteins.

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

Secondary structures such as α-helix and β-sheet are the major structural motifs within the three-dimensional geometry of proteins. Therefore, structure transitions from β-sheet to α-helix not only can serve as an effective strategy for the therapy of neurological diseases through the inhibition of β-sheet aggregation but also extend the application of α-helix fibrils in biomedicine. Herein, we present a charge-induced secondary structure transition of amyloid-derived dipeptide assemblies from β-sheet to α-helix. We unravel that the electrostatic (charge) repulsion between the C-terminal charges of the dipeptide molecules are responsible for the conversion of the secondary structure. This finding provides a new perspective to understanding the secondary structure formation and transformation in the supramolecular organization and life activity.

The sensor histidine kinases of two-component signal-transduction systems (TCSs) are essential for bacteria to adapt to variable environmental conditions. The two-component regulatory system BaeS/R increases multidrug and metal resistance in Salmonella and Escherichia coli. In this study, we report the X-ray structure of the periplasmic sensor domain of BaeS from Serratia marcescens FS14. The BaeS sensor domain (34-160) adopts a mixed α/β-fold containing a central four-stranded antiparallel β-sheet flanked by a long N-terminal α-helix and additional loops and a short C-terminal α-helix on each side. Structural comparisons revealed that it belongs to the PDC family with a remarkable difference in the orientation of the helix α2. In the BaeS sensor domain, this helix is situated perpendicular to the long helix α1 and holds helix α1 in the middle with the beta sheet, whereas in other PDC domains, helix α2 is parallel to helix α1. Because the helices α1 and α2 is involved in the dimeric interface, this difference implies that BaeS uses a different dimeric interface compared with other PDC domains. Proteins 2017; 85:1784-1790. © 2017 Wiley Periodicals, Inc.

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

The structure and function of naturally occurring proteins are governed by a large number of amino acids (≥100). The design of miniature proteins with desired structures and functions not only substantiates our knowledge about proteins but can also contribute to the development of novel applications. Excellent progress has been made towards the design of helical proteins with diverse functions. However, the development of functional β-sheet proteins remains challenging. Herein, we describe the construction and characterization of four-stranded β-sheet miniproteins made up of about 19 amino acids that bind heme inside a hydrophobic binding pocket or "heme cage" by bis-histidine coordination in an aqueous environment. The designed miniproteins bound to heme with high affinity comparable to that of native heme proteins. Atomic-resolution structures confirmed the presence of a four-stranded β-sheet fold. The heme-protein complexes also exhibited high stability against thermal and chaotrope-induced unfolding.

Aminopeptidases are ubiquitous hydrolases that cleave the N-terminal residues of proteins and oligopeptides. They are broadly distributed throughout all kingdoms of life and have been implicated in a wide variety of physiological processes, including viral infection, parasite metabolism, protein processing, regulation of peptide hormones, and cancer cell proliferation. Members of the M1 family, also termed gluzincins, are defined by two highly conserved motifs in the catalytic domain: a zinc-binding motif, HEXXH-(X18)-E; and an exopeptidase motif, GXMEN. We report the high-resolution X-ray structures of E. coli aminopeptidase N (PepN) in complex with three aminobenzosuberone scaffolds that display various Ki values (50, 0.33, and 0.034 microM) and provide a compelling view of the outstanding selectivity of these chemical entities for the M1 aminopeptidases. This series of inhibitors interacts as transition state mimics with highly conserved residues of the catalytic machinery and substrate recognition sites. Structural comparisons and model-building studies allowed a deep interpretation of the SAR observed for bacterial, as well as mammalian enzymes. Proteins 2017; 85:1413-1421. (c) 2017 Wiley Periodicals, Inc.

Alpha-synuclein (α-syn) is a 140 residue protein that plays a central role in Parkinson's disease and other neurological disorders. The precise function and pathological properties of α-syn remain however poorly understood. While α-syn is considered to be a flexible and disordered protein under native conditions, its ability to adopt a variety of conformational ensembles depending on the environment is considered to be related to its pathology. Raman optical activity (ROA) is a chiroptical spectroscopic technique that is uniquely sensitive to the secondary structure of proteins in solution and was used here for the first time to study the different conformational ensembles of α-syn. In this paper, the Raman and ROA spectral characteristics of these different conformations of α-syn are investigated. We show that Raman and ROA spectroscopy are sensitive enough not only to detect transitions from a disordered to an α-helical or a β-sheet rich ensemble but also to differentiate between the α-helical forms of wild-type and C-terminal truncated α-syn 107. Using increasing concentrations of fluorinated alcohols, we induce the aggregation pathway of α-syn and identify a molten globule intermediate structure and β-sheet rich oligomers. Taken together, these results demonstrate the power of Raman and ROA spectroscopies for the structural elucidation of proteins that are challenging to characterise. Copyright © 2017 John Wiley & Sons, Ltd.

The effect of citrate-stabilized gold nanoparticles (AuNPs) on the secondary structure of an artificial β-sheet-forming cationic peptide has been studied. The AuNPs inhibited β-sheet formation and led to fragmented fibrils and spherical oligomers with assembled AuNPs on their surface. Besides this structural change, the functional properties of the peptide are also different. Whereas the peptide was unable to act as a vector for gene delivery, formation of a complex with AuNPs allowed successful gene delivery into cells.

The folding processes of mini-proteins (FSD-EY/FBPWW28 domain) were computationally investigated by an enhanced conformational sampling method. Through the analyses of trajectories, these mini-proteins had multiple folding pathways different from a simple two-state folding, and the multiple folding processes were initiated by partial formations of secondary structures. These findings can be used to understand the folding of large proteins, that is, which secondary structures are partially folded in the early process, and how the remaining parts are sequentially folded. It is found that FSD-EY (α/β topology) folds by a simple diffusion-collision mechanism, while the folding process of the FBPWW28 domain (all-β topology) requires a modification of the diffusion-collision theory to adequately treat the coil-sheet transition for the β sheet formation. © 2017 Wiley Periodicals, Inc.

Biological gels generally require polymeric chains that produce long‐lived physical entanglements. Low molecular weight colloids offer an alternative to macromolecular gels, but often require ad‐hoc synthetic procedures. Here, a short biomimetic peptide composed of eight amino acid residues derived from squid sucker ring teeth proteins is demonstrated to form hydrogel in water without any cross‐linking agent or chemical modification and exhibits a stiffness on par with the stiffest peptide hydrogels. Combining solution and solid‐state NMR, circular dichroism, infrared spectroscopy, and X‐ray scattering, the peptide is shown to form a supramolecular, semiflexible gel assembled from unusual right‐handed 3_10‐helices stabilized in solution by π–π stacking. During gelation, the 3_10‐helices undergo conformational transition into antiparallel β‐sheets with formation of new interpeptide hydrophobic interactions, and molecular dynamic simulations corroborate stabilization by cross β‐sheet oligomerization. The current study broadens the range of secondary structures available to create supramolecular hydrogels, and introduces 3_10‐helices as transient building blocks for gelation via a 3_10‐to‐β‐sheet conformational transition., Advanced Science, 6 (21), ISSN:2198-3844

Free‐standing single‐layer β‐sheets are extremely rare in naturally occurring proteins, even though β‐sheet motifs are ubiquitous. Here we report the crystal structures of three homologous, single‐layer, anti‐parallel β‐sheet proteins, comprised of three or four twisted β‐hairpin repeats. The structures reveal that, in addition to the hydrogen bond network characteristic of β‐sheets, additional hydrophobic interactions mediated by small clusters of residues adjacent to the turns likely play a significant role in the structural stability and compensate for the lack of a compact hydrophobic core. These structures enabled identification of a family of secreted proteins that are broadly distributed in bacteria from the human gut microbiome and are putatively involved in the metabolism of complex carbohydrates. A conserved surface patch, rich in solvent‐exposed tyrosine residues, was identified on the concave surface of the β‐sheet. These new modular single‐layer β‐sheet proteins may serve as a new model system for studying folding and design of β‐rich proteins.

Protegrin-4 (PG-4) is a member of the porcine leukocyte protegrins family of cysteine-rich antimicrobial peptides (AMPs) isolated from Sus scrofa. It consists of 18 amino acid residues and works as a part of innate immune system. In this study, we examined the intrinsic aggregation propensity of this AMP using multiple computational algorithms, namely, TANGO, AGGRESCAN, FOLDAMYLOID, AMYLPRED, and ZYGGREGATOR, and found that the peptide is predicted to have a high propensity for the β sheet formation that disposes this peptide to be amyloidogenic. Under in vitro conditions, PG-4 formed visible aggregates and displayed the hallmark properties of typical amyloids such as enhanced binding of Congo red, increased fluorescence with Thioflavin-T, and fibrillar morphology under transmission electron microscopy. Then we examined its antimicrobial activity against Bacillus subtilis and found that the aggregated peptide retained its antimicrobial activity. Additionally, the aggregates remain non-toxic to the HEK293 and Caco2 cells. Our study suggests that the inherent aggregation properties of AMP can rationally be explored as a potential source of peptide-based antimicrobials with enhanced stability.

Fluorescence (FL) is a basic optical phenomenon that is widely used in molecular spectroscopy, high-resolution microscopy, and bioimaging. It has exciting and emission spectra defined by an electronic structure of particular fluorochrome molecules. In this work, another type of FL is investigated. This FL phenomenon is observed both in biological amyloid and bioinspired amyloidogenic assemblies, folded into specific biomolecular β-sheet secondary structure. A fine mechanism of this fold-sensitive FL effect, arising at the earliest stages of seeding and nucleation of β-sheet nanofibers, and its growth under thermally activated conformational refolding of helical to β-sheet state in bioinspired ultrashort peptide structures are studied. This FL effect demonstrates different properties compared to the classical FL from non-folded biomolecules or molecular dyes. Its excitation and emission spectra are similar in any β-sheet peptide/protein assemblies irrespective of their biochemical composition, primary structure, and origin. It is shown that this fold-sensitive FL effect exhibits a wideband spectrum, covering the entire visible region (400–650 nm), high quantum yield, and tunable FL wavelength. This research paves the way for advanced FL optical nanomaterials, for new methods of bioimaging and diagnostics as well as for the development of biocompatible nanoscale FL sources.

Three PEGylated β-sheet breaker peptides are designed as new inhibitors of β-amyloid fibrillization. The peptide Ac-Leu-Pro-Phe-Phe-Asp-NH2 , considered the lead compound, and hexamers in which taurine and β-alanine substitute the acetyl group, are conjugated to poly(ethylene glycol); this conjugates self-assemble into nanoparticles. The activity of the PEGylated peptides as inhibitors of amyloid fibrillization are tested in vitro using circular dichroism spectroscopy and scanning electron microscopy. The experimental results indicate that PEGylation does not impair the ability of the β-sheet breaker peptides to inhibit fibrillogenesis in vitro. Moreover, microscopy images of β-amyloid incubated for 6 days with the taurine-containing peptide, suggest that this conjugate has major anti-fibrillogenesis activity and demonstrate the important role of the sulfonamide function against the amyloid aggregation.

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

Amyloid fibrils are supramolecular polymers with β-sheet-rich structures formed by polymerization of protein/peptide with intermolecular interaction. Amyloid fibrils have been miscast as toxic villains since they have historically been studied as pathogens associated with serious diseases, including Alzheimer's and Parkinson's disease. However, recent studies on their toxicity and formation mechanism and discovery of their functionality in nature correct the misconception and strongly facilitate the possible use of β-sheet-forming peptides in designing novel nanomaterials. Self-assembly based on β-sheet-forming peptides can provide highly ordered nanostructures under certain conditions. Therefore, ingenious design of the building block peptides allows the construction of nano-assemblies, which contain large quantities of bio-functional molecules, including drugs and bioactive peptides, and exhibit unique properties, such as assembly or disassembly in response to external stimulus or specific molecules. These properties provide a novel strategy for the creation of innovative nanomaterials, especially for biomedical applications. Here, we describe recent progress in the biomedical application of fibrous assemblies based on β-sheet-forming peptides, such as the suppression of aberrant protein aggregation, controlled release, tissue engineering and other applications. This review focuses not only on the function of the nanofibrous assemblies but also on the functions of component molecules, namely amyloidogenic peptides. © 2016 Society of Chemical Industry

Mechanisms of beta sheet formation by the human prion protein are not clear yet. In this work, we clarified the role of the region containing C-half of the second helix and N-half of the third helix of that protein in the process of alpha helix to beta sheet transition. Solid phase automatic synthesis of the original peptide (CC36: Cys179-Cys214) failed because of the beta hairpin formation in the region 206-MERVVEQMC-214 with a high beta strand potential. Using Met206Arg and Val210Arg substitutions, we increased the probability of alpha helix formation by that sequence. After that modification, the complete CC36 peptide with disulfide bond has been synthesized. Modified peptide has been studied by circular dichroism (CD) and fluorescence spectrography. According to the CD spectra analysis, the CC36 peptide contains 37% of residues in beta sheet and just 15% in helix. Thermal analysis under the control of CD shows that the secondary structure content of the peptide is stable from 5°C to 80°C. Dissociation of oligomers of the CC36 peptide finishes at 37°C according to the fluorescence analysis. The CC36 peptide is able to bind Mn(2+) cations, which causes small temperature-associated structural shifts at concentrations of 2 - 10·10(-6) M. Predicted beta hairpin of the CC36 peptide (two beta strands are: 184-IKQHTVT-190 and 197-TETDVKM-205) should be the part of a longer beta hairpin from the scrapie form of the prion protein (PrPSc). Analogs of the CC36 peptide may be considered as antigens for the future development of a vaccine against PrPSc. Proteins 2016; 84:1462-1479. © 2016 Wiley Periodicals, Inc.

The metal ions Zn(2+) , Cu(2+) , and Fe(2+) play a significant role in the aggregation mechanism of Aβ peptides. However, the nature of binding between metal and peptide has remained elusive; the detailed information on this from the experimental study is very difficult. Density functional theory (dft) (M06-2X/6-311++G (2df,2pd) +LANL2DZ) has employed to determine the force field resulting due to metal and histidine interaction. We performed 200 ns molecular dynamics (MD) simulation on Aβ1-42 -Zn(2+) , Aβ1-42 -Cu(2+) , and Aβ1-42 -Fe(2+) systems in explicit water with different combination of coordinating residues including the three Histidine residues in the N-terminal. The present investigation, the Aβ1-42 -Zn(2+) system possess three turn conformations separated by coil structure. Zn(2+) binding caused the loss of the helical structure of N-terminal residues which transformed into the S-shaped conformation. Zn(2+) has reduced the coil and increases the turn content of the peptide compared with experimental study. On the other hand, the Cu(2+) binds with peptide, β sheet formation is observed at the N-terminal residues of the peptide. Fe(2+) binding is to promote the formation of Glu22-Lys28 salt-bridge which stabilized the turn conformation in the Phe19-Gly25 residues, subsequently β sheets were observed at His13-Lys18 and Gly29-Gly37 residues. The turn conformation facilitates the β sheets are arranged in parallel by enhancing the hydrophobic contact between Gly25 and Met35, Lys16 and Met35, Leu17 and Leu34, Val18 and Leu34 residues. The Fe(2+) binding reduced the helix structure and increases the β sheet content in the peptide, which suggested, Fe(2+) promotes the oligomerization by enhancing the peptide-peptide interaction. Proteins 2016; 84:1257-1274. © 2016 Wiley Periodicals, Inc.

Determining the cause of human calcitonin (hCT) aggregation could be of help in the effort to utilize hCT for treatment of hypercalcemia. Here we report that a dimer model of hCT13-32 aggregated to a greater degree than native hCT under aqueous 2,2,2-trifluoroethanol conditions. Analyses using circular dichroism spectroscopy, thioflavine-T binding assays and atomic force microscopy suggest that the α-helical portion of hCT is important for initiation of the aggregation process, which yields long fibrils. Dimerization, which stabilizes the β-sheet structure of hCT, enhances aggregation potency. Dimerization of hCT stabilizes the α-helix under aqueous TFE conditions, leading to the long fibril formation. Up to now, there have been no reports of using a dimer model to investigate the properties of hCT aggregation. Our findings could potentially serve as the basis for development of novel hCT derivatives that could be utilized for treatment of hypercalcemia, as well as for development of novel therapeutics for other ailments caused by amyloid peptides. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.

The study of intermediates in the protein folding pathway provides a wealth of information about the energy landscape. The intermediates also frequently initiate pathogenic fibril formations. While observing the intermediates is difficult due to their transient nature, extreme conditions can partially unfold the proteins and provide a glimpse of the intermediate states. Here, we observe the high resolution structure of a hydrophobic core mutant of Ubiquitin at an extreme acidic pH by nuclear magnetic resonance (NMR) spectroscopy. In the structure, the native secondary and tertiary structure is conserved for a major part of the protein. However, a long loop between the beta strands β3 and β5 is partially unfolded. The altered structure is supported by fluorescence data and the difference in free energies between the native state and the intermediate is reflected in the denaturant induced melting curves. The unfolded region includes amino acids that are critical for interaction with cofactors as well as for assembly of poly-Ubiquitin chains. The structure at acidic pH resembles a late folding intermediate of Ubiquitin and indicates that upon stabilization of the protein's core, the long loop converges on the core in the final step of the folding process.

Protein spin labeling to yield the nitroxide-based R1 side chain is a powerful method to measure protein dynamics and structure by electron spin resonance. However, R1 measurements are complicated by the flexibility of the side chain. While analysis approaches for solvent-exposed α-helical environment have been developed to partially account for flexibility, similar work in β-sheets is lacking. The goal of this study is to provide the first essential steps for understanding the conformational preferences of R1 within edge β-strands using X-ray crystallography and double electron electron resonance (DEER) distance measurements. Crystal structures yielded seven rotamers for a non-hydrogen-bonded site and three rotamers for a hydrogen-bonded site. The observed rotamers indicate contextual differences in R1 conformational preferences compared to other solvent-exposed environments. For the DEER measurements, each strand site was paired with the same α-helical site elsewhere on the protein. The most probable distance observed by DEER is rationalized based on the rotamers observed in the crystal structure. Additionally, the appropriateness of common molecular modeling methods that account for R1 conformational preferences are assessed for the β-sheet environment. These results show that interpretation of R1 behavior in β-sheets is difficult and indicate further development is needed for these computational methods to correctly relate DEER distances to protein structure at edge β-strand sites.

Elastin is a fibrous structural protein of the extracellular matrix that provides reversible elastic recoil to vertebrate tissues such as arterial vessels, lung, and skin. The elastin monomer, tropoelastin, contains a large proportion of intrinsically disordered and flexible hydrophobic sequences that collectively are responsible for the initial phase separation of monomers during assembly, and are essential for driving elastic recoil. While structural disorder of hydrophobic sequences is controlled by a high proline and glycine residue composition, hydrophobic domain 30 of human tropoelastin is atypically proline-poor, and forms β-sheet amyloid-like fibrils as an individual peptide. We explored the contribution of confined regions of secondary structure at the location of domain 30 in human tropoelastin to fiber assembly and mechanical properties using a set of mutations designed to inhibit or enhance the propensity of β-sheet formation at this location. Our data support a dual role for confined β-sheet secondary structure in domain 30 of tropoelastin in guiding the formation of fibers, and as a determinant of stiffness and viscoelastic properties of cross-linked materials. Together, these results suggest a mechanism for specificity in fiber assembly, and elucidate structure-function relationships for the rational design of elastomeric biomaterials with defined mechanical properties.

The crystal structure of a tripeptide Boc-Leu-Val-Ac12 c-OMe (1) is determined, which incorporates a bulky 1-aminocyclododecane-1-carboxylic acid (Ac12 c) side chain. The peptide adopts a semi-extended backbone conformation for Leu and Val residues, while the backbone torsion angles of the C(α,α) -dialkylated residue Ac12 c are in the helical region of the Ramachandran map. The molecular packing of 1 revealed a unique supramolecular twisted parallel β-sheet coiling into a helical architecture in crystals, with the bulky hydrophobic Ac12 c side chains projecting outward the helical column. This arrangement resembles the packing of peptide helices in crystal structures. Although short oligopeptides often assemble as parallel or anti-parallel β-sheet in crystals, twisted or helical β-sheet formation has been observed in a few examples of dipeptide crystal structures. Peptide 1 presents the first example of a tripeptide showing twisted β-sheet assembly in crystals.

Effects of exposure of 4 h to mobile phones microwaves at 1765 MHz at a power density around 940 mW/m(2) on four typical proteins (hemoglobin in H2 O solution, and myoglobin, bovine serum albumin, and lysozyme in D2 O solution) were studied by means of Fourier Transform Infrared spectroscopy and Fourier self-deconvolution analysis. Increase in intensity of parallel β-sheet component around 1635 cm(-1) was observed after exposure of hemoglobin, myoglobin, and bovine serum albumin, showing that a mechanism of unfolding occurred after exposure, whereas no appreciable change in the amide I region occurred after lysozyme exposure. In addition, a relationship between protein dipole moment and protein unfolding rate was demonstrated with a correlation coefficient r = 0.973 and 95% confidence interval.

β‐sheets often have one face packed against the core of the protein and the other facing solvent. Mutational studies have indicated that the solvent‐facing residues can contribute significantly to protein stability, and that the preferred amino acid at each sequence position is dependent on the precise structure of the protein backbone and the identity of the neighboring amino acids. This suggests that the most advantageous methods for designing β‐sheet surfaces will be approaches that take into account the multiple energetic factors at play including side chain rotamer preferences, van der Waals forces, electrostatics, and desolvation effects. Here, we show that the protein design software Rosetta, which models these energetic factors, can be used to dramatically increase protein stability by optimizing interactions on the surfaces of small β‐sheet proteins. Two design variants of the β‐sandwich protein from tenascin were made with 7 and 14 mutations respectively on its β‐sheet surfaces. These changes raised the thermal midpoint for unfolding from 45°C to 64°C and 74°C. Additionally, we tested an empirical approach based on increasing the number of potential salt bridges on the surfaces of the β‐sheets. This was not a robust strategy for increasing stability, as three of the four variants tested were unfolded.

The secondary structure formation of polypeptides not only governs folding and solution self-assembly but also affects the nucleophilic ring-opening polymerization of alpha-amino acid-N-carboxyanhydrides (NCAs). Whereby helical structures are known to enhance polymerization rates, beta-sheet-like assemblies reduce the propagation rate or may even terminate chain growth by precipitation or gelation. To overcome these unfavorable properties, racemic mixtures of NCAs can be applied. In this work, racemicS-(ethylsulfonyl)-dl-cysteine NCA is investigated for the synthesis of polypeptides, diblock and triblock copolypept(o)ides. In contrast to the polymerization of stereoregularS-(ethylsulfonyl)-l-cysteine NCA, the reaction ofS-(ethylsulfonyl)-dl-cysteine NCA proceeds with a rate constant of up tok(p) = 1.70 x 10(-3)L mol(-1)s(-1)and is slightly faster than the enatiopure polymerization. While the polymerization ofS-(ethylsulfonyl)-l-cysteine NCA suffers from incomplete monomer conversion and degrees of polymerization (DPs) limited to 30-40, racemic mixtures yield polypeptides with DPs of up to 102 with high conversion rates and well-defined dispersities (1.2-1.3). The controlled living nature of the ring-opening polymerization ofS-(ethylsulfonyl)-dl-cysteine NCA thus enables the synthesis of triblock copolymers by sequential monomer addition. This methodology allows for precise control over DPs of individual blocks and yields uniform triblock copolymers with symmetric molecular weight distributions at a reduced synthetic effort.