Self-assembly of amphipathic β-sheet peptides: insights and applications

Amphipathic peptides composed of alternating polar and nonpolar residues have a strong tendency to self-assemble into one-dimensional, amyloid-like fibril structures. Fibrils derived from peptides of general (XZXZ)(n) sequence in which X is hydrophobic and Z is hydrophilic adopt a putative β-sheet bilayer. The bilayer configuration allows burial of the hydrophobic X side chain groups in the core of the fibril and leaves the polar Z side chains exposed to solvent. This architectural arrangement provides fibrils that maintain high solubility in water and has facilitated the recent exploitation of self-assembled amphipathic peptide fibrils as functional biomaterials. This article is a critical review of the development and application of self-assembling amphipathic peptides with a focus on the fundamental insight these types of peptides provide into peptide self-assembly phenomena.     

Conjugation of β-sheet peptides to modify the rheological properties of hyaluronic acid

Hyaluronic acid (HA) is a naturally occurring polysaccharide that is commonly used in cosmetic, wound healing, and tissue regeneration applications because of its biocompatibility and intrinsic biological activities. However, the rheological behavior of unmodified HA is not ideal for many of these. In particular, whereas chain entanglements result in an increase in viscosity, they do not prevent flow from delivery sites under zero-shear conditions. It would be of significant benefit if strategies could be developed in which robust but reversible cross-links could be incorporated within the material to allow the formation of a gel under static conditions. In developing a modification strategy, the extent of functionalization should be low to preserve the biological activities of HA. Therefore, this study focused on attaching peptides that self-assemble into β-sheets to HA to modify the viscosity at low shear rates. It was found that the peptide sequence (LS)(4) forms β-sheets in aqueous media and when reacted with HA using EDC/HOBt coupling to give 6.0 ± 1.5% modification the peptide-modified HA exhibits significant increases in low-shear viscosities in comparison with the unmodified HA. However, this increase in viscosity was observed only at lower polymer concentrations and at low shear rates, suggesting that network formation is sensitive to external forces and may change at high concentrations. At higher shear rates and at higher polymer concentrations the viscosity profile of the modified HA matches that of the unmodified HA, indicating that the peptide interactions were disrupted or ineffective under these conditions. The rheology of the peptide-modified HA was also compared with samples of HA reacted with the same molar ratio of aniline, but we found that the aniline-modified HA displayed behavior comparable to that of the unmodified HA, which demonstrates that the β-sheet peptide modification technique is superior to the technique used in commercial products, such as Hyaff, at low degrees of functionalization.     

Synthesis and antimicrobial activity of β-carboline derivatives with N2-alkyl modifications

β-Carbolines constitute a vast group of indole alkaloids and exhibit various biological actions. The objective of this study was to investigate the structure–activity relationships of β-carboline derivatives on in vitro inhibitory effects against clinically relevant microorganisms. A series of β-carboline dimers and their N²-alkylated analogues were therefore prepared and evaluated for their antimicrobial effects. Among these, a dimeric 6-chlorocarboline N²-benzylated salt exerted potent activity against Staphylococcus aureus at MICs of 0.01–0.05?μmol/mL. Our work highlights that N¹-N¹ dimerization and N²-benzylation significantly enhanced the antimicrobial effects of compounds.     

Computational design and evaluation of β-sheet breaker peptides for destabilizing Alzheimer's amyloid-β42 protofibrils

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.     

Denaturation of β-sheet structure

A two-dimensional Ising model is used to study the thermal denaturation of parallel β-sheet structures in biomolecules.The fraction of intact hydrogen bonds and the excess heat capacity are evaluated as a function of the temperature.     

Self-assembly and Self-replication of Short Amphiphilic β-sheet Peptides

Most self-replicating peptide systems are made of α-helix forming sequences. However, it has been postulated that shorter and simpler peptides may also serve as templates for replication when arranged into well-defined structures. We describe here the design and characterization of new peptides that form soluble β-sheet aggregates that serve to significantly accelerate their ligation and self-replication. We then discuss the relevance of these phenomena to early molecular evolution, in light of additional functionality associated with β-sheet assemblies.     

Effect of site-specific amino acid D-isomerization on β-sheet transition and fibril formation profiles of Tau microtubule-binding repeat peptides

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.     

Design and biological activity of β-sheet breaker peptide conjugates

The sequence LPFFD (iAβ₅) prevents amyloid-β peptide (Aβ) fibrillogenesis and neurotoxicity, hallmarks of Alzheimer’s disease (AD), as previously demonstrated. In this study iAβ₅ was covalently linked to poly(ethylene glycol) (PEG) and the activity of conjugates was assessed and compared to the activity of the peptide alone by in vitro studies. The conjugates were characterized by MALDI-TOF. Competition binding assays established that conjugates retained the ability to bind Aβ with similar strength as iAβ₅. Transmission electron microscopy analysis showed that iAβ₅ conjugates inhibited amyloid fibril formation, which is in agreement with binding properties observed for the conjugates towards Aβ. The conjugates were also able to prevent amyloid-induced cell death, as evaluated by activation of caspase 3. These results demonstrated that the biological activity of iAβ₅ is not affected by the pegylation process.     

Protein dynamics of a β-sheet protein

Rhodnius prolixus Nitrophorin 4 (abbreviated NP4) is an almost pure β-sheet heme protein. Its dynamics is investigated by X-ray structure determination at eight different temperatures from 122 to 304 K and by means of Mössbauer spectroscopy. A comparison of this β-sheet protein with the pure α-helical protein myoglobin (abbreviated Mbmet) is performed. The mean square displacement derived from the Mössbauer spectra increases linearly with temperature below a characteristic temperature T _c. It is about 10 K larger than that of myoglobin. Above T _c the mean square displacements increase dramatically. The Mössbauer spectra are analyzed by a two state model. The increased mean square displacements are caused by very slow motions occurring on a time scale faster than 140 ns. With respect to these motions NP4 shows the same protein specific modes as Mbmet. There is, however, a difference in the fast vibration regime. The B values found in the X-ray structures vary linearly over the entire temperature range. The mean square displacements in NP4 increase with slopes which are 60% larger than those observed for Mbmet. This indicates that nitrophorin has a larger structural distribution which makes it more flexible than myoglobin.     

Computational design and experimental testing of the fastest-folding β-sheet protein

One of the most important and elusive goals of molecular biology is the formulation of a detailed, atomic-level understanding of the process of protein folding. Fast-folding proteins with low free-energy barriers have proved to be particularly productive objects of investigation in this context, but the design of fast-folding proteins was previously driven largely by experiment. Dramatic advances in the attainable length of molecular dynamics simulations have allowed us to characterize in atomic-level detail the folding mechanism of the fast-folding all-β WW domain FiP35. In the work reported here, we applied the biophysical insights gained from these studies to computationally design an even faster-folding variant of FiP35 containing only naturally occurring amino acids. The increased stability and high folding rate predicted by our simulations were subsequently validated by temperature-jump experiments. The experimentally measured folding time was 4.3 μs at 80 °C—about three times faster than the fastest previously known protein with β-sheet content and in good agreement with our prediction. These results provide a compelling demonstration of the potential utility of very long molecular dynamics simulations in redesigning proteins well beyond their evolved stability and folding speed.     

Synthesis and characterization of quaternized β-chitin

Water-soluble 2′-O-hydroxypropyltrimethylammoniumchitin chloride (2′-O-HTACCt) was prepared directly from β-chitin and 3-chloro-2-hydroxypropyltrimethylammonium chloride (CTA) in basic medium. The effect of alkali concentration, reaction temperature, reaction time, and dosage of CTA on yield and degree of substitution (DS) of 2′-O-HTACCt were studied. These quaternized chitin derivatives were characterized by FTIR and ¹H NMR spectroscopy, conductometric titration, and elemental analysis methods. Research results indicate that β-chitin can react directly with CTA to produce a water-soluble 2′-O-HTACCt derivative with a high DS. The optimal preparation conditions were determined to be 35-40 wt % (aq NaOH), 40 °C (reaction temperature), 6 h (reaction time), and 4 (molar ratio of CTA to β-chitin unit).     

Synthesis of α-aspartyl-containing cyclic peptides

Summary α-Aspartyl-containing cyclic pentapeptides were synthesised in high yields using a strategy that maintained fluorenylmethyl protection on the aspartic acid side chain during chain assembly, resin cleavage and cyclisation of the linear precursors. Tetra-n-butylammonium fluoride treatment of the fluorenylmethyl-protected cyclic peptides catalysed imide formation, whereas piperidine-induced deprotection resulted in good yields of the target cyclic peptides.     

Synthesis of methyl α- and β-maltotriosides and aryl β-maltotriosides

Reaction of β-maltotriose hendecaacetate with phosphorus pentachloride gave 2′,2″,3,3′,3″,4″,6,6′,6″,-nona-O-acetyl-(2)-O-trichloroacetyl-β-maltotriosyl chloride (2) which was isomerized into the corresponding α anomer (8). Selective ammonolysis of 2 and 8 afforded the 2-hydroxy derivatives 3 and 9, respectively; 3 was isomerized into the α anomer 9. Methanolysis of 2 and 3 in the presence of pyridine and silver nitrate and subsequent deacetylation gave methyl α-maltotrioside. Likewise, methanolysis and O-deacetylation of 9 gave methyl β-maltotrioside which was identical with the compound prepared by the Koenigs—Knorr reaction of 2,2′,2″,3,3′,3″,4″,6,6′,6″-deca-O-acetyl-α-maltotriosyl bromide (12) with methanol followed by O-deacetylation. Several substituted phenyl β-glycosides of maltotriose were also obtained by condensation of phenols with 12 in an alkaline medium. Alkaline degradation of the o-chlorophenyl β-glycoside decaacetate readily gave a high yield of 1,6-anhydro-β-maltotriose.     

Synthesis,characterization, and wound healing properties of α/γ hybrid peptides

The present work describes the synthesis, characterization, and wound healing properties of α/γ hybrid peptides: Boc-Phe-γ⁴-Phe-Val-OMe ( S1 ), Boc-^DPhe-γ⁴-Phe-Val-OMe ( S2 ), Boc-Ala-γ⁴-Phe-Val-OMe ( S3 ), Boc-^DAla-γ⁴-Phe-Val-OMe ( S4 ), Boc-Leu-γ⁴-Phe-Val-OMe ( S5 ), and Boc-^DLeu-γ⁴-Phe-Val-OMe ( S6 ). Peptides S1–S6 were screened against human keratinocytes (HaCaT) and RAW 264.7 cells. Among all, S1 - and S2 -treated cells exhibited high cell viability; S1 and S2 induced keratinocyte migration and inhibited the production of the cytokines IL-6 and TNF-α. In vivo results demonstrated that the hybrid peptides S1 and S2 accelerate wound healing in Wistar rats with 83% and 88% at 50 μg/ml, and 74% and 76% at 25 μg/ml, respectively.     

Characteristics and effects of specific peptides on heat-induced aggregation of β-lactoglobulin

A bovine β-lactoglobulin hydrolysate, obtained by the hydrolysis by the Glu specific enzyme Bacillus licheniformis protease (BLP), was fractionated at pH 7.0 into a soluble and an insoluble fraction and characterized by LC-MS. From the 26 peptides identified in the soluble fraction, five peptides (A[f97-112] = [f115-128], AB[f1-45], AB[f135-157], AB[f135-158], and AB[f138-162]) bound to β-lactoglobulin at room temperature. After heating of β-lactoglobulin in the presence of peptides, eight peptides were identified in the pellet formed, three of them belonging to the previously mentioned peptides. Principle component analysis revealed that the binding at room temperature (to β-lactoglobulin) was related to the total hydrophobicity and the total charge of the peptides. The binding to the unfolded protein could not be attributed to distinct properties of the peptides. The presence of the peptides caused a 50% decrease in denaturation enthalpy (from 148 ± 3 kJ/mol for the protein alone to 74 ± 2 kJ/mol in the presence of peptides), while no change in secondary structure or denaturation temperature was observed. At temperatures <85 °C, the addition of peptides resulted in a 30-40% increase of precipitated β-lactoglobulin. At pH < 6, no differences in the amount of aggregated β-lactoglobulin were observed, which indicates the lack of binding of peptides to β-lactoglobulin at those pH values as was also observed by SELDI-TOF-MS. Although only a few peptides were found to participate in aggregation, suggesting specificity, principal component analysis was unable to identify specific properties responsible for this.     

Nonnative intermediate state of acid-stable β-sheet protein

Cell adhesion molecule, CD2, from the immunoglobulin superfamily, is comprised of antibodies and Ig-like domains and plays a fundamental role, not only in the immune system, but also in the interactions between cells, specifically in cell-cell adhesion. This study examines the N-terminal domain 1 of CD2 (CD2-1) at different pHs, and in 2,2,2-trifluoroethanol (TFE), using nears- and far-UV circular dichroism (CD), fluorescence, and 1H nuclear magnetic resonance to elucidate factors contributing to the Ig beta-structure. Contrary to the complete unfolding induced by guanidinehydrochloride, CD2-1 retains its native tertiary structure at pHs from 1.0 to 10.0. Like the effects of high temperatures that have previously been observed, TFE reduces the integrity of the tertiary structure, while reorganizing the secondary structure from a native all-beta-sheet to a significantly alpha-helical conformation. The induced helicity of CD2-1 correlates with the helicity inherent in its primary sequence. Our results suggest that electrostatic interactions are less important for the formation of the native secondary and tertiary structure of CD2-1, although they are crucial for CD2's adhesion function. Interference with the protein's hydrophobic interactions and hydrogen-bonding networks, however, causes significant changes in its conformation. Residues of CD2-1, with high conformational flexibility, may contribute for the formation of a metastable dimer by domain-swapping.     

Synthesis of β-Tubanol

Synthesis of β-tubanol (VIa) was achieved by the bromination of dihydro-β-tubanol acetate (VIIb) followed by dehydrobromination and the subsequent hydrolysis. 2,2-Dimethyl-5-hydroxy-chromanone as well as its derivatives (IV) and the corresponding chromanols (V) and chromans (VII) were also prepared.     

The paradox of MHC-DRB exon/intron evolution: α-helix and β-sheet encoding regions diverge while hypervariable intronic simple repeats coevolve with β-sheet codons

Twenty-one different caprine and 13 ovine MHC-DRB exon 2 sequences were determined including part of the adjacent introns containing simple repetitive (gt)_n(ga)_m elements. The positions for highly polymorphic DRB amino acids vary slightly among ungulates and other mammals. From man and mouse to ungulates the basic (gt)_n(ga)_m structure is fixed in evolution for 7 × 10⁷ years whereas ample variations exist in the tandem (gt)_n and (ga)_m dinucleotides and especially their degenerated derivatives. Phylogenetic trees for the -helices and -pleated sheets of the ungulate DRB sequences suggest different evolutionary histories. In hoofed animals as well as in humans DRB -sheet encoding sequences and adjacent intronic repeats can be assembled into virtually identical groups suggesting coevolution of noncoding as well as coding DNA. In contrast a-helices and C-terminal parts of the first DRB domain evolve distinctly. In the absence of a defined mechanism causing specific, site-directed mutations, double-recombination or gene-conversion-like events would readily explain this fact. The role of the intronic simple (gt)_n(ga)_m repeat is discussed with respect to these genetic exchange mechanisms during evolution.     

Molecular dynamics simulations of three protegrin-type antimicrobial peptides: interplay between charges at the termini, β-sheet structure and amphiphilic interactions

We have carried out molecular dynamics simulations of the naturally occurring protegrin PG-1 peptide and two of its mutants, PC-9 and PC-13 in the presence of a dodecyl-phosphocholine (DPC) micelle. The effects of mutations that disrupt the β-sheet structure in the case of PC-9 and reduce the charge at the C-terminus in the case of PC-13 are analyzed. It is found that the surface-bound conformations of the peptides are severely affected by both mutations. PG-1 exhibits a conformation in which the C-terminus and the β-hairpin turn interact strongly with the micelle lipid head groups, while its N-terminal strand bends away from the micelle and resides in the aqueous region; PC-13 exhibits strong interactions with the micelle at its N-terminus as well as the β-hairpin turn region, while retaining a much more compact conformation than PG-1; PC-9 achieves a highly distorted conformation relative to the homologous PG-1 structure, which allows both its termini and the β-hairpin region to interact with the micelle. These significant differences observed as a result of seemingly minor mutations to the sequences of the three peptides are explained in terms of the interplay between residue charges, structural rigidity and amphiphilic interactions. Conservative inferences are made bridging these biophysical interactions and the pharmacological profiles of the peptides.     

Cardiovascular effects of peptides related to the enkephalins and β-casomorphin

In the urethane-anesthetized rat, intravenous injections of morphine produced a short-lasting fall in heart rate and blood pressure. The fall in heart rate (which is vagal in origin) was used here to bioassay peptides related to the enkephalins [-enk] and β-casomorphin [β-C, H-Tyr-Pro-Phe-Pro-Gly-Pro-Ile-OH]. A > 10% decrease in heart rate was used as a quantal index of response and the intravenous ED50 [μmol/kg] were estimated as: methionine-enk, 1.3 ± .24; leucine-enk, 1.5 ± .20; [D-Ala², D-Leu⁵]-enk, 0.45 ± .05; [D-Ala², NMePhe⁴, Met(0)⁵-ol]-enk, 0.0011 ± .0002; H-Tyr-Arg-OH, > 2.0; β-C (1–7), > 2.0; β-C(1–5), > 2.0; β-C(1–4), > 4.0; β-C(1–3), > 2.0; morphine sulfate, 0.11 ± .03; and human β-endorphin,, 0.07 ± .01. One β-C derivative, H-Tyr-Pro-Phe-Pro-NH₂ [β-C(1–4)-NH₂], was active at 0.32 ± .08. Naloxone pretreatment blocked the bradycardia produced by the enkephalins and β-C(1–4)-NH₂. The bioassay described here, based on heart rate, may prove to be useful for the rapid detection and estimation of the $\text{in vivo}$ pharmacological activities of new opioid peptides.