Supramolecular helix and β-sheet through self-assembly of two isomeric tetrapeptides in crystals and formation of filaments and ribbons in the solid state

Single crystal X-ray diffraction studies show that the β-turn structure of tetrapeptide I, Boc-Gly-Phe-Aib-Leu-OMe (Aib: α-amino isobutyric acid) self-assembles to a supramolecular helix through intermolecular hydrogen bonding along the crystallographic a axis. By contrast the β-turn structure of an isomeric tetrapeptide II, Boc-Gly-Leu-Aib-Phe-OMe self-assembles to a supramolecular β-sheet-like structure via a two-dimensional (a, b axis) intermolecular hydrogen bonding network and π-π interactions. FT-IR studies of the peptides revealed that both of them form intermolecularly hydrogen bonded supramolecular structures in the solid state. Field emission scanning electron micrographs (FE-SEM) of the dried fibrous materials of the peptides show different morphologies, non-twisted filaments in case of peptide I and non-twisted filaments and ribbon-like structures in case of peptide II.     

Tunable self-assembled peptide amphiphile nanostructures

Peptide amphiphiles are capable of self-assembly into a diverse array of nanostructures including ribbons, tubes, and vesicles. However, the ability to select the morphology of the resulting structure is not well developed. We examined the influence of systematic changes in the number and type of hydrophobic and hydrophilic amino acids on the self-assembly of amphiphilic peptides. Variations in the morphology of self-assembled peptides of the form X(6)K(n) (X = alanine, valine, or leucine; K = lysine; n = 1-5) are investigated using a combination of transmission electron microscopy and dynamic light scattering measurements. The secondary structures of the peptides are determined using circular dichroism. Self-assembly is controlled through a combination of interactions between the hydrophobic segments of the peptide molecules and repulsive forces between the charged segments. Increasing the hydrophobicity of the peptide by changing X to a more lipophilic amino acid or decreasing the number of hydrophilic amino acids transforms the self-assembled nanostructures from vesicles to tubes and ribbons. Changes in the hydrophobicity of the peptides are reflected in changes in the critical micelle concentration observed using pyrene probe fluorescence analysis. Self-assembled materials formed from cationic peptide amphiphiles of this type display promise as carriers for insoluble molecules or negatively charged nucleic acids in drug or gene delivery applications.     

Effect of the peptide secondary structure on the peptide amphiphile supramolecular structure and interactions

Bottom-up fabrication of self-assembled nanomaterials requires control over forces and interactions between building blocks. We report here on the formation and architecture of supramolecular structures constructed from two different peptide amphiphiles. Inclusion of four alanines between a 16-mer peptide and a 16 carbon long aliphatic tail resulted in a secondary structure shift of the peptide headgroups from α helices to β sheets. A concomitant shift in self-assembled morphology from nanoribbons to core-shell worm-like micelles was observed by cryogenic transmission electron microscopy (cryo-TEM) and atomic force microscopy (AFM). In the presence of divalent magnesium ions, these a priori formed supramolecular structures interacted in distinct manners, highlighting the importance of peptide amphiphile design in self-assembly.     

Understanding self-assembled amphiphilic peptide supramolecular structures from primary structure helix propensity

Small amphiphilic peptides are attractive building blocks to design biocompatible supramolecular structures via self-assembly, with applications in, for example, drug delivery, tissue engineering, and nanotemplating. We address the influence of systematical changes in the amino acid sequence of such peptides on the self-assembled macromolecular structures. For cationic-head surfactant-like eight-residue peptides, the apolar tail amino acids were chosen to systematically vary the propensity to form an alpha-helical secondary structure while conserving the overall hydrophobicity of the sequence. Characterization of the supramolecular structures indicates that for short peptides a beta-sheet secondary structure correlates with ribbonlike assemblies while random-coil and alpha-helical secondary structures correlate with assembly of rods.     

The application of a hypothesis-driven strategy to the sensitive detection and location of acetylated lysine residues

The application of a hypothesis-driven method for the sensitive determination of lysine acetylation sites on enzymatically digested proteins is described. Comparative sensitivity tests were carried out using serial dilution of an acetylated bovine serum albumin (AcBSA) digest to assess the performance of a multiple reaction monitoring (MRM)-based approach as compared to a more conventional precursor scanning (PS) method. Both methods were capable of selectively detecting an acetylated peptide at the low femtomole level when spiked into a background of 500 fmol six-protein tryptic digest. The MRM approach was roughly tenfold more sensitive than precursor scanning with one acetylated peptide detected and sequenced at the level of 2 fmol on-column. The technique was subsequently applied to a gel-derived sample of cytokeratin-8 (CK8) shown to contain acetylated lysine residues by Western blot analysis. The strategy applied herein, termed MRM-initiated detection and sequencing (MIDAS), resulted in the facile identification of novel sites of acetylation on this protein.     

Global proteomic analysis of lysine acetylation in zebrafish (Danio rerio) embryos

Lysine acetylation is an important post‐translational modification (PTM). Since the development of MS‐based proteomics technology, important roles of lysine acetylation beyond histones have focused on chromatin remodeling during the cell cycle and regulation of nuclear transport, metabolism, and translation. Zebrafish (Danio rerio) is a widely used vertebrate model in genetics and biologic studies. Although studies in several mammalian species have been performed, the mechanism of lysine acetylation in D. rerio embryos is incompletely understood. Here, we investigated the global acetylome in D. rerio embryos by using an MS‐based proteomics approach. We identified 351 acetylated peptides and 377 nonredundant acetylation sites on 189 lysine‐acetylated proteins in 5‐day postfertilization (hpf) embryos of D. rerio. Among lysine‐acetylated peptides, 40.2% indicated three motifs: (ac)KxxxK, (ac)KxxxxK, and Lx(ac)K. Of 190 acetylated proteins, 81 (42.6%) were mainly distributed in the cytoplasm. Gene ontology enrichment and Kyoto Encyclopedia of Genes and Genomes pathway analyses showed that lysine acetylation in D. rerio was enriched in metabolic pathways. Additionally, 17 of 30 acetylated ribosomal proteins were evolutionarily conserved between zebrafish and humans. Our results indicate that acetyllysine might have regulatory effects on ribosomal proteins involved in protein biosynthesis.     

Acetylation of the HIV-1 Tat protein: an in vitro study

In the last few years, the understanding of lysine acetylation as a regulatory post-translational modification of proteins in cell signalling cascades has increased. It is now known that not only histones but also non-histone factors can serve as substrates of different acetyltransferase enzymes. Acetylated lysine residues in non-histone factors are often identified using radioactive labelling experiments and immunochemical analysis of synthetic peptides. In this study of the human immunodeficiency virus 1 (HIV-1) Tat protein, we demonstrate the benefits of matrix-assisted laser desorption/ionisation mass spectrometry, proteolytic digestion and Edman sequencing for the mapping of acetylation sites. We confirmed that the HIV-1 Tat protein is acetylated in vitro by the acetyltransferase p300 at a specific lysine residue at position 50 in its RNA binding region. Furthermore, we showed that the Tat cysteine-rich region is acetylated at multiple cysteine residues in the absence of enzyme. Since this non-enzymatic cysteine acetylation occurs independently from the surrounding peptide sequence, we consider the presence of cysteine residues in acetylated peptides an important factor for the interpretation of in vitro acetylation assays in general.Abbreviations aa Amino acid - AcCoA Acetyl coenzyme A - acm Acetamidomethyl - ARM Arginine-rich motif - CRR Cysteine-rich region - HAT Histone acetyltransferaseThis article is dedicated to Harald zur Hausen on the occasion of his retirement as head of the German Cancer Research Center (Deutsches Krebsforschungszentrum) with gratitude and appreciation for 20 years of leadership     

Structural Investigation of Hybrid Peptide Foldamers Composed of α-Dipeptide Equivalent β-Oxy-δ5-amino Acids

Due to their equivalent lengths, δ-amino acids can serve as surrogates of α-dipeptides. However, δ-amino acids with proteinogenic side chains have not been well studied because of synthetic difficulties and because of their insolubility in organic solvents. Recently we reported the spontaneous supramolecular gelation of δ-peptides composed of β(O)-δ⁵-amino acids. Here, we report the incorporation of β(O)-δ⁵-amino acids as guests into the host α-helix, α,γ-hybrid peptide 12-helix and their single-crystal conformations. In addition, we studied the solution conformations of hybrid peptides composed of 1:1 alternating α and β(O)-δ⁵-amino acids. In contrast to the control α-helix structures, the crystal structure of peptides with β(O)-δ⁵-amino acids exhibit α-helical conformations consisting of both 13- and 10-membered H-bonds. The α,δ-hybrid peptide adopted mixed 13/11-helix conformation in solution with alternating H-bond directionality. Crystal-structure analysis revealed that the α,γ⁴-hybrid peptide accommodated the guest β(O)-δ⁵-amino acid without significant deviation to the overall helix folding. The results reported here emphasize that β(O)-δ⁵-amino acids with proteinogenic side chains can be accommodated into regular α-helix or 12-helix as guests without much deviation of the overall helix folding of the peptides.     

Analysis of protein-protein interaction surfaces using a combination of efficient lysine acetylation and nanoLC-MALDI-MS/MS applied to the E9:Im9 bacteriotoxin—immunity protein complex

To understand how proteins perform their function, knowledge about their structure and dynamics is essential. Here we use a combination of an efficient chemical lysine acetylation reaction and nanoLC-MALDI tandem mass spectrometry to probe the accessibility of every lysine residue in a protein complex. To demonstrate the applicability of this approach, we studied the interaction between the DNase domain of Colicin E9 (E9) and its immunity protein Im9. Free E9 and E9 in complex with Im9 were rapidly acetylated, followed by proteolytic digestion and analysis by LC-MALDI-TOF/TOF MS/MS. Acetylated peptides could be filtered out of the complex peptide mixtures using selective ion chromatograms of the specific immonium marker ions. Additionally, isobaric acetylated peptides, acetylated at different sites, could be separated by their LC retention times. The combination of LC and MALDI-TOF/TOF MS/MS provided information about the amount of acetylation on each individual lysine even for peptides containing several lysine residues. In general, our data agree well with those derived from the crystal structure of E9 and the E9:Im9 complex. Interestingly, next to in the binding interface expected lysines, K89 and K97, two from the crystal structure data unexpected lysines, K81 and K76, were observed to become less exposed upon Im9 binding. Moreover, K55 and K63, positioned in the predicted DNA binding region, were also found to be less accessible upon Im9 binding. These findings may illustrate some of the described differences in the solution-phase structure of the E9:Im9 complex compared with the crystal structure.     

Playing with peptides: how to build a supramolecular peptide nanostructure by exploiting helix···helix macrodipole interactions

A novel method to build bicomponent peptide self-assembled monolayers (SAMs) has been developed, by exploiting helix···helix macrodipole interactions. In this work, a peptide-based self-assembled monolayer composed of two helical peptides was immobilized on a gold surface. Specifically, a pyrene-containing octapeptide, devoid of any sulfur atom (A8Pyr), and a hexapeptide, functionalized at the N-terminus with (S,R) lipoic acid, for binding to gold substrates (SSA4WA) via a Au-S linkage, have been employed. Both peptides investigated attain a helical structure, because they are almost exclusively formed by strongly folding inducer C(α)-tetrasubstituted α-amino acids. We demonstrate that the two peptides generate a stable supramolecular nanostructure (a densely packed bicomponent peptide monolayer), where A8Pyr is incorporated into the SSA4WA palisade by exploiting helix···helix macrodipole interactions. The presence of both peptides on the gold surface was investigated by spectroscopic and electrochemical techniques, while the morphology of the monolayer was analyzed by ultra high-vacuum scanning tunnelling microscopy. The composition of the bicomponent SAM on the surface was studied by a combination of electrochemical and spectroscopic techniques. In particular, the amount of Au-S linkages from the sulfur-containing peptides was quantified from reductive desorption of the peptide-based SAM, while the amount of A8Pyr was estimated by fluorescence spectroscopy. The antiparallel orientation of the A8Pyr and SSA4WA peptide chains minimizes the interaction energy between the helix dipoles, suggesting that this kind of electrostatic phenomenon is the driving force that stabilizes the bicomponent SAM.     

Selection of Secondary Structures of Heterotypic Supramolecular Peptide Assemblies by an Enzymatic Reaction

In a model study to investigate the consequence of reactions of intrinsically disordered regions (IDRs) of proteins in the context of the formation of highly ordered structures, we found that enzymatic reactions control the secondary structures of peptides during assembly. Specifically, phosphorylation of an α‐helix‐dominant peptide results in mostly disordered conformations, which become β‐strand‐dominant after enzymatic dephosphorylation to regenerate the peptide. In the presence of another peptide largely with a β‐strand conformation, direct coassembly of the peptides results in amorphous aggregates consisting of α‐helix and β‐strand peptides, but the enzymatically generated peptide coassemblies (from the phosphopeptide) mainly adopt a β‐strand conformation and form ordered structures (e.g., nanofibers). These results indicate that enzymatic dephosphorylation instructs conformationally flexible peptides to adopt thermodynamically favorable conformations in homotypic or heterotypic supramolecular assemblies.     

Effects of peptide acetylation and dimethylation on electrospray ionization efficiency

Peptide acetylation and dimethylation have been widely used to derivatize primary amino groups (peptide N‐termini and the ε‐amino group of lysines) for chemical isotope labeling of quantitative proteomics or for affinity tag labeling for selection and enrichment of labeled peptides. However, peptide acetylation results in signal suppression during electrospray ionization (ESI) due to charge neutralization. In contrast, dimethylated peptides show increased ionization efficiency after derivatization, since dimethylation increases hydrophobicity and maintains a positive charge on the peptide under common LC conditions. In this study, we quantitatively compared the ESI efficiencies of acetylated and dimethylated model peptides and tryptic peptides of BSA. Dimethylated peptides showed higher ionization efficiency than acetylated peptides for both model peptides and tryptic BSA peptides. At the proteome level, peptide dimethylation led to better protein identification than peptide acetylation when tryptic peptides of mouse brain lysate were analyzed with LC‐ESI‐MS/MS. These results demonstrate that dimethylation of tryptic peptides enhanced ESI efficiency and provided up to two‐fold improved protein identification sensitivity in comparison with acetylation. Copyright © 2016 John Wiley & Sons, Ltd.     

Azalysine analogues as probes for protein lysine deacetylation and demethylation

Reversible lysine acetylation and methylation regulate the function of a wide variety of proteins, including histones. Here, we have synthesized azalysine-containing peptides in acetylated and unacetylated forms as chemical probes of the histone deacetylases (HDAC8, Sir2Tm, and SIRT1) and the histone demethylase, LSD1. We have shown that the acetyl-azalysine modification is a fairly efficient substrate for the sirtuins, but a weaker substrate for HDAC8, a classical HDAC. In addition to deacetylation by sirtuins, the acetyl-azalysine analogue generates a novel ADP-ribose adduct that was characterized by mass spectrometry, Western blot analysis, and nuclear magnetic resonance spectroscopy. This peptide-ADP-ribose adduct is proposed to correspond to a derailed reaction intermediate, providing unique evidence for the direct 2'-hydroxyl attack on the O-alkylimidate intermediate that is formed in the course of sirtuin catalyzed deacetylation. An unacetylated azalysine-containing H3 peptide proved to be a potent inhibitor of the LSD1 demethylase, forming an FAD adduct characteristic of previously reported related structures, providing a new chemical probe for mechanistic analysis.     

Distance dependence of electron transfer across peptides with different secondary structures: the role of Peptide energetics and electronic coupling

The charge-transfer transition energies and the electronic-coupling matrix element, |H(DA)|, for electron transfer from aminopyridine (ap) to the 4-carbonyl-2,2'-bipyridine (cbpy) in cbpy-(gly)(n)-ap (gly = glycine, n = 0-6) molecules were calculated using the Zerner's INDO/S, together with the Cave and Newton methods. The oligopeptide linkages used were those of the idealized protein secondary structures, the alpha-helix, 3(10)-helix, beta-strand, and polyproline I- and II-helices. The charge-transfer transition energies are influenced by the magnitude and direction of the dipole generated by the peptide secondary structure. The electronic coupling |H(DA)| between (cbpy) and (ap) is also dependent on the nature of the secondary structure of the peptide. A plot of 2.ln|H(DA)| versus the charge-transfer distance (assumed to be the dipole moment change between the ground state and the charge-transfer states) showed that the polyproline II structure is a more efficient bridge for long-distance electron-transfer reactions (beta = 0.7 A(-1)) than the other secondary structures (beta approximately 1.3 A(-1)). Similar calculations on charged dipeptide derivatives, [CH(3)CONHCH(2)CONHCH(3)](+/)(-), showed that peptide-peptide interaction is more dependent on conformation in the cationic than in the anionic dipeptides. The alpha-helix and polyproline II-helix both have large peptide-peptide interactions (|H(DA)| > 800 cm(-1)) which arise from the angular dependence of their pi-orbitals. Such an interaction is much weaker than in the beta-strand peptides. These combined results were found to be consistent with electron-transfer rates experimentally observed across short peptide bridges in polyproline II (n = 1-3). These results can also account for directional electron transfer observed in an alpha-helical structure (different ET rates versus the direction of the molecular dipole).     

Reactivity characteristics of squid β-chitin as compared with those of shrimp chitin: High potentials of squid chitin as a starting material for facile chemical modifications

Chemical reactivity of β-chitin isolated from squid pens has been examined in various reactions to elucidate the possibility of facile modifications in simple manners leading to the preparation of derivatives with well-defined structures. β-Chitin swelled in common solvents such as methanol and pyridine unlike the ordinary α-chitin and exhibited much higher reactivity than β-chitin. Free amino groups present in β-chitin were easily and selectively acetylated with acetic anhydride in methanol to give chitin with a uniform structure, poly(N-acetyl-D-glucosamine). When acetylation reaction was carried out in pyridine, O-acetylation proceeded smoothly besides N-acetylation. In the presence of 4-dimethylaminopyridine as the catalyst, even full acetylation was achieved under mild conditions. Tosylation was also quite efficient in pyridine without side reactions such as N-deacetylation which is unavoidable in the tosylation of α-chitin. β-Chitin also enabled direct tritylation in pyridine in the presence of 4-dimethylaminopyridine. All these reactions were quite sluggish with β-chitin, and no reactions or only very low extents of substitution were observed, indicating the high potential of β-chitin as a versatile starting material for facile modification reactions. © 1994 John Wiley & Sons, Inc.     

Amphiphilic peptides as models for protein-membrane interactions: interfacial behaviour of sequential Lys- and Leu-based peptides and their penetration into lipid monolayers

Synthetic peptides constructed with doublets of hydrophobic residues tandemly repeated with doublets of positively charged residues, (Leu-Lys-Lys-Leu)_n, were used as models for the study of protein-membrane interactions. Their behaviour has been compared with that of their strictly alternating iso peptides, (Leu-Lys)_n. Both peptides present a random coil structure in pure water. In saline solutions, (Leu-Lys-Lys-Leu)_n peptides adopt an α-helical structure whereas (Leu-Lys)_n transit into a β-sheet structure. These peptides form multilayer assemblies on a pure water subphase but they are organized in monomolecular films on a saline aqueous subphase. The stability of these films increases with the peptide length. Structured peptides (α helices and β sheets) penetrate readily into lipid monolayers, whereas the penetration of unordered peptides is very slow. We have not observed any significant difference between the behaviour of a helices and β-sheet structures.     

Chiral Fibers Formation Upon Assembly of Tetraphenylalanine Peptide Conjugated to a PNA Dimer

Self-assembly of biomolecules such as peptides, nucleic acids or their analogues affords supramolecular objects, exhibiting structures and physical properties dependent on the amino-acid or nucleobase composition. Conjugation of the peptide diphenylalanine (FF) to peptide nucleic acids triggers formation of self-assembled structures, mainly stabilized by interactions between FF. In this work we report formation of homogeneous chiral fibers upon self-assembly of the hybrid composed of the tetraphenylalanine peptide (4F) conjugated to the PNA dimer adenine-thymine (at). In this case nucleobases seem to play a key role in determining the morphology and chirality of the fibers. When the PNA “at” is replaced by guanine-cytosine dimer “gc”, disordered structures are observed. Spectroscopic characterization of the self-assembled hybrids, along with AFM and SEM studies is reported. Finally, a structural model consistent with the experimental evidence has also been obtained, showing how the building blocks of 4Fat arrange to give helical fibers.     

Glycine Substitution Effects on the Supramolecular Morphology and Rigidity of Cell-Adhesive Amphiphilic Peptides

Self-assembling peptides that are capable of adopting β-sheet structures can generate nanofibers that lead to hydrogel formation. Herein, to tune the supramolecular morphologies, mechanical properties, and stimuli responses of the hydrogels, we investigated glycine substitution in a β-sheet-forming amphiphilic peptide. Glycine substitution generally enhances conformational flexibility. Indeed, glycine substitution in an amphiphilic peptide weakened the hydrogels or even inhibited the gelation. However, unexpectedly, glycine substitution at the center of the peptide molecule significantly enhanced the hydrogel stiffness. The central glycine substitution affected the molecular packing and led to twisted β-sheet structures and to nanofiber bundling, which likely led to the stiffened hydrogel. Importantly, the supramolecular structures were accurately predicted by molecular dynamics simulations, demonstrating the helpfulness of these techniques for the identification of self-assembling peptides. The hydrogel formed by the amphiphilic peptide with the central glycine substitution had cell adhesive function, and showed a reversible thermal gel-to-sol transition. Thus, glycine substitution is effective in modulating self-assembling structures, rheological properties, and dynamics of biofunctional self-assembling peptides.     

Triggers for β-sheet formation at the hydrophobic-hydrophilic interface: high concentration, in-plane orientational order, and metal ion complexation

Amyloid formation plays a causative role in neurodegenerative diseases such as Alzheimer's disease or Parkinson's disease. Soluble peptides form β-sheets that subsequently rearrange into fibrils and deposit as amyloid plaques. Many parameters trigger and influence the onset of the β-sheet formation. Early stages are recently discussed to be cell-toxic. Aiming at understanding various triggers such as interactions with hydrophobic-hydrophilic interfaces and metal ion complexation and their interplay, we investigated a set of model peptides at the air-water interface. We are using a general approach to a variety of diseases such as Alzheimer's disease, Parkinson's disease, and type II diabetes that are connected to amyloid formation. Surface sensitive techniques combined with film balance measurements have been used to assess the conformation of the peptides and their orientation at the air-water interface (IR reflection-absorption spectroscopy). Additionally, the structures of the peptide layers were characterized by grazing incidence X-ray diffraction and X-ray reflectivity. The peptides adsorb to the air-water interface and immediately adopt an α-helical conformation. This helical intermediate transforms into β-sheets upon further triggering. The factors that result in β-sheet formation are dependent on the peptide sequence. In general, the interface has the strongest effect on peptide conformation compared to high concentrations or metal ions. Metal ions are able to prevent aggregation in bulk but not at the interface. At the interface, metal ion complexation has only minor effects on the peptide secondary structure, influencing the in-plane structure that is formed in two dimensions. At the air-water interface, increased concentrations or a parallel arrangement of the α-helical intermediates are the most effective triggers. This study reveals the role of various triggers for β-sheet formation and their complex interplay. Our main finding is that the hydrophobic-hydrophilic interface largely governs the conformation of peptides. Therefore, the present study implies that special care is needed when interpreting data that may be affected by different amounts or types of interfaces during experimentation.     

Supramolecular bidentate ligands by metal-directed in situ formation of antiparallel beta-sheet structures and application in asymmetric catalysis

The principles of protein structure design, molecular recognition, and supramolecular and combinatorial chemistry have been applied to develop a convergent metal-ion-assisted self-assembly approach that is a very simple and effective method for the de novo design and the construction of topologically predetermined antiparallel beta-sheet structures and self-assembled catalysts. A new concept of in situ generation of bidentate P-ligands for transition-metal catalysis, in which two complementary, monodentate, peptide-based ligands are brought together by employing peptide secondary structure motif as constructing tool to direct the self-assembly process, is achieved through formation of stable beta-sheet motifs and subsequent control of selectivity. The supramolecular structures were studied by (1)H, (31)P, and (13)C NMR spectroscopy, ESI mass spectrometry, X-ray structure analysis, and theoretical calculations. Our initial catalysis results confirm the close relationship between the self-assembled sheet conformations and the catalytic activity of these metallopeptides in the asymmetric rhodium-catalyzed hydroformylation. Good catalyst activity and moderate enantioselectivity were observed for the selected combination of catalyst and substrate, but most importantly the concept of this new methodology was successfully proven. This work presents a perspective interface between protein design and supramolecular catalysis for the design of beta-sheet mimetics and screening of libraries of self-organizing supramolecular catalysts.