An artificial beta-sheet that dimerizes through parallel beta-sheet interactions

This Article introduces a simple chemical model of a beta-sheet (artificial beta-sheet) that dimerizes by parallel beta-sheet formation in chloroform solution. The artificial beta-sheet consists of two N-terminally linked peptide strands that are linked with succinic or fumaric acid and blocked along one edge with a hydrogen-bonding template composed of 5-aminoanisic acid hydrazide. The template is connected to one of the peptide strands by a turn unit composed of (S)-2-aminoadipic acid (Aaa). 1H NMR spectroscopic studies show that these artificial beta-sheets fold in CDCl3 solution to form well-defined beta-sheet structures that dimerize through parallel beta-sheet interactions. Most notably, all of these compounds show a rich network of NOEs associated with folding and dimerization. The compounds also exhibit chemical shifts and coupling constants consistent with the formation of folded dimeric beta-sheet structures. The aminoadipic acid unit shows patterns of NOEs and coupling constants consistent with a well-defined turn conformation. The present system represents a significant step toward modeling the type of parallel beta-sheet interactions that occur in protein aggregation.     

Synthesis of functionalized de novo designed 8-16 kDa model proteins towards metal ion-binding and esterase activity

De novo design and total chemical synthesis of proteins provides a powerful approach for biological and biophysical studies with the ability to prepare artificial proteins with tailored properties, potentially of importance for biophysical studies, material science, nanobioscience, and as molecular probes. In this paper, the previously developed concept of carbohydrates as templates is employed in the de novo design of model proteins (artificial helix bundles) termed 'carboproteins'. The 4-alpha-helix bundle is a macromolecular structure, where four amphiphilic alpha-helical peptide strands form a hydrophobic core. Here this structure is modified towards achieving metal ion-binding and catalytic activity. We report: (i) test of directional effects from different tetravalent carbohydrate templates, (ii) synthesis and evaluation of carboproteins functionalized with phenol, pyridyl or imidazolyl moieties as potential ligands for metal ion-binding as well as for catalysis. Our results include: (i) support of our previous 'controversial' finding that for some carboproteins the degree of alpha-helicity depends on the template, i.e., that there is, to some extent, a controlling effect from the template, (ii) demonstration of binding of Cu(ii) to tetra-functional carboproteins by electrospray ionization-time of flight-mass spectrometry (ESI-TOF-MS), UV-VIS absorption spectroscopy and size exclusion chromatography-inductively coupled plasma-mass spectrometry (SEC-ICP-MS); (iii) a kinetic investigation of the esterase activity.     

Monosaccharide templates for de novo designed 4-alpha-helix bundle proteins: template effects in carboproteins

De novo design and total chemical synthesis of proteins provide powerful approaches to critically test our understanding of protein folding, structure, and stability. The 4-alpha-helix bundle is a frequently studied structure in which four amphiphilic alpha-helical peptide strands form a hydrophobic core. Assembly of protein models on a template has been suggested as a way to reduce the entropy of folding. We have previously developed the concept of carbohydrates as templates in the de novo design of protein models termed 'carboproteins'. Here we present the chemical synthesis of three 8.1 kDa 4-alpha-helix bundles by oxime ligation of tetra-aminooxyacetyl functionalized D-galacto-, D-gluco-, and D-altropyranoside templates with an amphiphilic C-terminal hexadecapeptide aldehyde sequence. CD spectroscopy indicated that the choice of template has an effect on the overall structure of the carboprotein, as the altro-based carboprotein was found to be more alpha-helical than the corresponding galacto- and gluco-carboproteins. However, an influence on stability could not be detected in the present experiments, as the three carboproteins gave similar free energy of foldings (deltaG(F)H2O) and melting points in chemical and thermal denaturation experiments.     

Cholic acid as template for multivalent peptide assembly

Cholic acid, an amphiphilic steroid containing several selectively addressable functionalities, was exploited as a rigid template for multivalent peptide assembly. Thus, cholic acid-based templates suitable for chemoselective peptide ligation were synthesized, in which maleimide or bromoacetyl moieties were selectively introduced at the 3alpha, 7alpha, 12alpha-positions of cholic acid with varied length of linkers. Three peptides were chosen and tested for the chemoselective ligation. These include the HIV-1 peptide inhibitor DP178, the universal T-helper epitope derived from tetanus toxoid (830-844), and the minimum epitope sequence of the HIV-neutralizing antibody 2F5. It was found that the maleimide-functionalized templates are highly efficient for the ligation of all the peptides, while bromoacetyl templates led to low yield of ligation. Circular dichroism (CD) spectroscopic studies of the multivalent peptides (10a and 11a) containing three strands of peptide DP178 indicate that the template-assembled peptides form three alpha-helix bundles with significantly enhanced alpha-helix contents than the single peptide. The results suggest that cholic acid is a valuable template for constructing alpha-helix bundles that may be useful as mimics of conformational epitopes for vaccine development.     

Template-directed oligonucleotide strand ligation, covalent intramolecular DNA circularization and catenation using click chemistry

The copper-catalyzed azide-alkyne cycloaddition reaction has been used for the template-mediated chemical ligation of two oligonucleotide strands, one with a 5'-alkyne and the other with a 3'-azide, to produce a DNA strand with an unnatural backbone at the ligation point. A template-free click-ligation reaction has been used for the intramolecular circularization of a single stranded oligonucleotide which was used as a template for the synthesis of a covalently closed DNA catenane.     

An unnatural amino acid that induces beta-sheet folding and interaction in peptides

This paper introduces a unique amino acid that can readily be incorporated into peptides to make them fold into beta-sheetlike structures that dimerize through beta-sheet interactions. This new amino acid, Orn(i-PrCO-Hao), consists of an ornithine residue with the beta-strand-mimicking amino acid Hao [J. Am. Chem. Soc. 2000, 122, 7654-7661] attached to its side chain. When Orn(i-PrCO-Hao) is incorporated into a peptide, or appended to its N-terminus, the Hao group hydrogen bonds to the three subsequent residues to form a beta-sheetlike structure. The amino acid Orn(i-PrCO-Hao) is readily used in peptide synthesis as its Fmoc derivative, Fmoc-Orn(i-PrCO-Hao)-OH (3). Fmoc-Orn(i-PrCO-Hao)-OH behaves like a regular amino acid in peptide synthesis and was uneventfully incorporated into the peptide o-anisoyl-Val-Orn(i-PrCO-Hao)-Phe-Ile-Leu-NHMe (4) through standard automated Fmoc solid-phase peptide synthesis, with DIC and HOAt as the coupling agent for Fmoc-Orn(i-PrCO-Hao)-OH and o-anisic acid and HATU as the coupling agent for all other couplings. A second synthetic strategy was developed to facilitate the preparation of peptides with N-terminal Orn(i-PrCO-Hao) residues, which avoids the need for the preparation of Fmoc-Orn(i-PrCO-Hao)-OH. In this strategy, Boc-Orn(Fmoc)-OH is used as the penultimate amino acid in the peptide synthesis, and i-PrCO-Hao-OH (2) is used as the final amino acid. N-Terminal Orn(i-PrCO-Hao) peptide H-Orn(i-PrCO-Hao)-Phe-Ile-Leu-NHMe.TFA (5) was prepared in a fashion similar to that for 4, using DIC and HOAt as the coupling agent for i-PrCO-Hao-OH and HATU as the coupling agent for all other couplings. 1H NMR transverse-ROESY, coupling constant, and chemical shift studies establish that peptide 4 forms a dimeric beta-sheetlike structure in CDCl3 solution. The 1H NMR studies also suggest that the ornithine unit adopts a well-defined turn conformation. Analogous 1H NMR studies of peptide 5 indicate that this TFA salt folds but does not dimerize in CD3OD solution. Collectively, these synthetic and spectroscopic studies establish that the amino acid Orn(i-PrCO-Hao) induces beta-sheet structure and interactions in peptides in suitable organic solvents. Unlike the Hao amino acid, which acts as a prosthetic to replace three residues of the peptide strand, the Orn(i-PrCO-Hao) amino acid acts as a splint that helps enforce a beta-sheetlike structure without replacing the residues and their side chains. This feature of Orn(i-PrCO-Hao) is important, because it allows the creation of beta-sheet structure with minimal perturbation of the peptide sequence.     

Controlling the morphology of cross beta-sheet assemblies by rational design

Low molecular weight peptidomimetics with simple amphiphilic sequences can help to elucidate the structures of cross beta-sheet assemblies, such as amyloid fibrils. The peptidomimetics described herein comprise a dibenzofuran template, two peptide strands made up of alternating hydrophilic and hydrophobic residues, and carboxyl termini, each of which can be varied to probe the structural requirements for beta-sheet self-assembly processes. The dibenzofuran template positions the strands approximately 10 A apart, allowing corresponding hydrophobic side chains in the strands to pack into a collapsed U-shaped structure. This conformation is stabilized by hydrophobic interactions, not intramolecular hydrogen bonds. Intermolecular stacking of the collapsed peptidomimetics, enabled by intermolecular hydrogen bonding and hydrophobic interactions, affords 25-27 A wide protofilaments having a cross beta-sheet structure. Association of protofilaments, mediated by the dibenzofuran substructures and driven by the hydrophobic effect, affords 50-60 A wide filaments. These widths can be controlled by changing the length of the peptide strands. Further assembly of the filaments into fibrils or ribbons can be controlled by modification of the template, C-terminus, and buffer ion composition.     

Design and construction of an open multistranded beta-sheet polypeptide stabilized by a disulfide bridge

The design and characterization of an open eight-stranded beta-sheet in a synthetic, 2-fold symmetric 70-residue peptide is described. The design strategy involves the generation of a 35-residue four-stranded beta-sheet peptide in which successive hairpins are nucleated by appropriately positioned (D)Pro-Xxx sequences. Oxidative dimerization using a single Cys residue positioned at the center of the C-terminal strand results in a disulfide-bridged eight-stranded structure. Nuclear Overhauser effects firmly establish an eight-stranded beta-sheet in methanol. In water, the outer strands are frayed, but a well-defined four-stranded beta-sheet stabilized by a disulfide bridge and a hydrophobic cluster is determined from NMR data. Comparison of the precursor peptide with the disulfide-bridged dimer reveals considerable enhancement of beta-sheet content in the latter, suggesting that the disulfide cross-link is an effective strategy for the stabilization of beta-sheets.     

The organization and assembly of a beta-sheet formed by a prion peptide in solution: an isotope-edited FTIR study

Insight into the details of protein misfolding diseases requires a detailed understanding of the conformation and dynamics of multistrand beta-sheet aggregates. Here, we report an isotope-edited FTIR study of a model peptide directed at the elucidation of residue-level details of the structure and mechanism of a beta-sheet aggregate. A series of specifically isotope-labeled derivatives of a short peptide (H1) derived from residues 109 through 122 of the prion protein PrPC have been synthesized and characterized by FTIR. On the basis of the analysis of variable temperature FTIR spectra of these peptides in solution, the organization of strands within the beta-sheets has been determined; at equilibrium, the strands form a beta-sheet in which the hydrophobic core (112-122) participates in the sheet structure, resulting in the alignment of residue 117 in all of the strands. The peptides initially form a kinetically trapped intermediate beta-sheet, with a distribution of strand alignments, which can be rearranged into the stable equilibrium conformation by an annealing cycle. These observations are discussed in terms of the biological significance of residue 117 of the prion protein and the mechanism of beta-aggregate nucleation in prion proteins.     

DNA-directed formation of peptide bond: a model study toward DNA-programmed peptide ligation

A model study of DNA-directed peptide ligation has been developed by transferring fluorescent reporting group from small molecule thioester to a DNA strand (template DNA) in the presence of a thiol-functionalized DNA strand (auxiliary DNA), mimicking the Native Chemical Ligation (NCL) reaction. This DNA-directed transfer shows dependence on the sequence complementarity of the two DNA strands, with in situ generated 4-thiolphenylmethyl functionalized oligonucleotide as the auxiliary DNA strand, under mild basic condition (pH=8.5), and with tris(2-carboxyethyl) phosphine hydrochloride (TCEP) as a reducing agent. Reactions with different amino acid α-thioesters resulted in varied transfer efficiencies from glycine to α-substituted amino acids. This study has provided the basic foundation to use DNA-programmed chemistry toward the chemical synthesis or unnatural modification of protein molecules.     

Hierarchically structured porous cadmium selenide polycrystals using polystyrene bilayer templates

In this study, a novel approach is demonstrated to fabricate hierarchically structured cadmium selenide (CdSe) layers with size-tunable nano/microporous morphologies achieved using polystyrene (PS) bilayered templates (top layer: colloidal template) via potentiostatic electrochemical deposition. The PS bilayer template is made in two steps. First, various PS patterns (stripes, ellipsoids, and circles) are prepared as the bottom layers through imprint lithography. In a second step, a top template is deposited that consists of a self-assembled layer of colloidal 2D packed PS particles. Electrochemical growth of CdSe crystals in the voids and selective removal of the PS bilayered templates give rise to hierarchically patterned 2D hexagonal porous CdSe structures. This simple and facile technique provides various unconventional porous CdSe films, arising from the effect of the PS bottom templates.     

A noncovalent approach to antiparallel beta-sheet formation

Four tripeptide chains, when attached to the same end of a hydrogen-bonded duplex (1.2) with the unsymmetrical, complementary sequences of ADAA/DADD, have been brought into proximity, leading to the formation of four hybrid duplexes, 1a.2a, 1a.2b, 1b.2a, and 1b.2b, each of which contains a two-stranded beta-sheet segment. The extended conformations of the peptide chains were confirmed by 1D and 2D NMR. The peptide strands stay registered through hydrogen bonding and the beta-sheets are stabilized by side chain interactions. Two-dimensional NMR data also indicate that the duplex template prevents further aggregation in the peptide segment. When the peptide chains are attached to the two different termini of the duplex template, NMR studies show the presence of a mixture with no clearly defined conformations. In the absence of the duplex template, the tripeptides are found to associate randomly. Finally, isothermal titration calorimetry studies revealed that the hybrid duplex 1a.2a was more stable than either the duplex template or the peptides alone.     

Thermodynamic analysis of autonomous parallel beta-sheet formation in water

We report the first thermodynamic analysis of parallel beta-sheet formation in a model system that folds in aqueous solution. NMR chemical shifts were used to determine beta-sheet population, and van't Hoff anaysis provided thermodynamic parameters. Our approach relies upon the d-prolyl-1,1-dimethyl-1,2-diaminoethane unit to promote parallel beta-sheet formation between attached peptide strands. The development of a macrocyclic reference molecule to provide chemical shift data for the fully folded state was crucial to the quantitative anaylsis.     

Impact of Strand Number on Parallel β‐Sheet Stability

We have examined whether parallel β‐sheet secondary structure becomes more stable as the number of β‐strands increases, via comparisons among peptides designed to adopt two‐ or three‐stranded parallel β‐sheet conformations in aqueous solution. Our three‐strand design is the first experimental model of a triple‐stranded parallel β‐sheet. Analysis of the designed peptides by nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy supports the hypothesis that increasing the number of β‐strands, from two to three, increases the stability of the parallel β‐sheet. We present the first experimental evidence for cooperativity in the folding of a triple‐stranded parallel β‐sheet, and we show how minimal model systems may enable experimental documentation of characteristic properties, such as CD spectra, of parallel β‐sheets.     

Cyclic modular beta-sheets

The development of peptide beta-hairpins is problematic, because folding depends on the amino acid sequence and changes to the sequence can significantly decrease folding. Robust beta-hairpins that can tolerate such changes are attractive tools for studying interactions involving protein beta-sheets and developing inhibitors of these interactions. This paper introduces a new class of peptide models of protein beta-sheets that addresses the problem of separating folding from the sequence. These model beta-sheets are macrocyclic peptides that fold in water to present a pentapeptide beta-strand along one edge; the other edge contains the tripeptide beta-strand mimic Hao [JACS 2000, 122, 7654] and two additional amino acids. The pentapeptide and Hao-containing peptide strands are connected by two delta-linked ornithine (deltaOrn) turns [JACS 2003, 125, 876]. Each deltaOrn turn contains a free alpha-amino group that permits the linking of individual modules to form divalent beta-sheets. These "cyclic modular beta-sheets" are synthesized by standard solid-phase peptide synthesis of a linear precursor followed by solution-phase cyclization. Eight cyclic modular beta-sheets 1a-1h containing sequences based on beta-amyloid and macrophage inflammatory protein 2 were synthesized and characterized by 1H NMR. Linked cyclic modular beta-sheet 2, which contains two modules of 1b, was also synthesized and characterized. 1H NMR studies show downfield alpha-proton chemical shifts, deltaOrn delta-proton magnetic anisotropy, and NOE cross-peaks that establish all compounds but 1c and 1g to be moderately or well folded into a conformation that resembles a beta-sheet. Pulsed-field gradient NMR diffusion experiments show little or no self-association at low (相似文献   

Role of locked nucleic acid modified complementary strand in quadruplex/Watson-Crick duplex equilibrium

In the human genome, the G-rich sequences that form quadruplexes are present along with their C-rich complementary strands; this suggests the existence of equilibrium between a quadruplex and a Watson-Crick duplex which allows the execution of their respective biological functions. We have investigated the sensitivity of this equilibrium to pharmacological agents by employing locked nucleic acid (LNA) modified complementary strands, and demonstrated successful invasion of the stable telomeric quadruplex d[(G(3)TTA)(3)G(3)]. Fluorescence, UV, ITC, and SPR studies were performed to understand the binding process involving the preformed quadruplex and LNA-modified complementary strands compared with that involving the unmodified complementary strand. Our data indicate that LNA modifications in the complementary strand shift the equilibrium toward the duplex state. These modifications confer increased thermodynamic stability to the duplex and increase the magnitude of relative free energy (DeltaDeltaG degrees) difference between duplex and quadruplex, thus favoring the predominance of duplex population over quadruplex. This superior ability of LNA-modified complementary strand can be exploited to pave an exploratory approach in which it hybridizes to a telomeric quadruplex and drives duplex formation, and inhibits the recognition of 3' G-rich overhang by RNA template of telomerase which guides telomere extension.     

Unwinding DNA and RNA with Synthetic Complexes: On the Way to Artificial Helicases

Synthetic helicases can be designed on the basis of ligands that bind more strongly to single‐stranded nucleic acids than to double‐stranded nucleic acids. This can be achieved with ligands containing phenyl groups, which intercalate into single strands, but due to their small size not into double strands. Moreover, two phenyl rings are combined with a distance that allows bis‐intercalation with only single strands and not double strands. In this respect, such ligands also mimic single‐strand binding (SSB) proteins. Exploration with more than 23 ligands, mostly newly synthesised, shows that the distance between the phenyl rings and between those and the linker influence the DNA unwinding efficiency, which can reach a melting point decrease of almost ΔT_m=50 °C at much lower concentrations than that with any other known artificial helicases. Conformational pre‐organisation of the ligand plays a decisive role in optimal efficiency. Substituents at the phenyl rings have a large effect, and increase, for example, in the order of H<F<Cl<Br, which illustrates the strong role of dispersive interactions in intercalation. Studies with homopolymers revealed significant selectivity: for example, with a ligand concentration of 40 μM at 35 °C, only GC double strands melt (ΔT_m=48 °C), whereas the AT strand remains untouched, and with poly(rA)–poly(rU) as an RNA model one observes unfolding at 29 °C with a concentration of only 30 μM .     

General synthesis of unsymmetrical norbornane scaffolds as inducers for hydrogen bond interactions in peptides

Starting from readily accessible endo-cis-(2S,3R)-norbornene dicarboxylic acid benzyl monoester, a general and efficient synthetic approach toward unsymmetrical two-stranded peptidic structures was developed. In these structures the peptide strands are oriented in a parallel geometry. Their synthesis is easily applicable to a variety of amino acids and peptides. Specifically, a norbornane template as molecular scaffold induces hydrogen bonding between the adjacent peptide strands. The specific hydrogen bonding patterns between these strands were revealed by detailed NMR analysis including TOCSY/NOE experiments.     

Anomeric DNA Strand Displacement with α-D Oligonucleotides as Invaders and Ethidium Bromide as Fluorescence Sensor for Duplexes with α/β-, β/β- and α/α-D Configuration

DNA strand displacement is a technique to exchange one strand of a double stranded DNA by another strand (invader). It is an isothermal, enzyme free method driven by single stranded overhangs (toeholds) and is employed in DNA amplification, mismatch detection and nanotechnology. We discovered that anomeric (α/β) DNA can be used for heterochiral strand displacement. Homochiral DNA in β-D configuration was transformed to heterochiral DNA in α-D/β-D configuration and further to homochiral DNA with both strands in α-D configuration. Single stranded α-D DNA acts as invader. Herein, new anomeric displacement systems with and without toeholds were designed. Due to their resistance against enzymatic degradation, the systems are applicable to living cells. The light-up intercalator ethidium bromide is used as fluorescence sensor to follow the progress of displacement. Anomeric DNA displacement shows benefits over canonical DNA in view of toehold free displacement and simple detection by ethidium bromide.