1,2,3‐Triazole Bridge as Conformational Constrain in β‐Hairpin Peptides: Analysis of Hydrogen‐Bonded Positions

Conformational constrained β‐hairpin peptides are useful tool to modulate protein–protein interactions. A triazole bridge in hydrogen‐bonded positions between two antiparallel strands induces a conformational stabilization of the β‐hairpin peptide. The entity of the stability of the β‐hairpin peptide depends on the length of the bridge.     

Micelle‐Triggered β‐Hairpin to α‐Helix Transition in a 14‐Residue Peptide from a Choline‐Binding Repeat of the Pneumococcal Autolysin LytA

Choline‐binding modules (CBMs) have a ββ‐solenoid structure composed of choline‐binding repeats (CBR), which consist of a β‐hairpin followed by a short linker. To find minimal peptides that are able to maintain the CBR native structure and to evaluate their remaining choline‐binding ability, we have analysed the third β‐hairpin of the CBM from the pneumococcal LytA autolysin. Circular dichroism and NMR data reveal that this peptide forms a highly stable native‐like β‐hairpin both in aqueous solution and in the presence of trifluoroethanol, but, strikingly, the peptide structure is a stable amphipathic α‐helix in both zwitterionic (dodecylphosphocholine) and anionic (sodium dodecylsulfate) detergent micelles, as well as in small unilamellar vesicles. This β‐hairpin to α‐helix conversion is reversible. Given that the β‐hairpin and α‐helix differ greatly in the distribution of hydrophobic and hydrophilic side chains, we propose that the amphipathicity is a requirement for a peptide structure to interact and to be stable in micelles or lipid vesicles. To our knowledge, this “chameleonic” behaviour is the only described case of a micelle‐induced structural transition between two ordered peptide structures.     

A Designed Three‐Stranded β‐Sheet in an α/β Hybrid Peptide

The incorporation of β‐amino acid residues into the antiparallel β‐strand segments of a multi‐stranded β‐sheet peptide is demonstrated for a 19‐residue peptide, Boc‐LV^βFV^DPGL^βFVVL^DPGLVL^βFVV‐OMe (BBH19). Two centrally positioned ^DPro–Gly segments facilitate formation of a stable three‐stranded β‐sheet, in which β‐phenylalanine (^βPhe) residues occur at facing positions 3, 8 and 17. Structure determination in methanol solution is accomplished by using NMR‐derived restraints obtained from NOEs, temperature dependence of amide NH chemical shifts, rates of H/D exchange of amide protons and vicinal coupling constants. The data are consistent with a conformationally well‐defined three‐stranded β‐sheet structure in solution. Cross‐strand interactions between ^βPhe3/^βPhe17 and ^βPhe3/Val15 residues define orientations of these side‐chains. The observation of close contact distances between the side‐chains on the N‐ and C‐terminal strands of the three‐stranded β‐sheet provides strong support for the designed structure. Evidence is presented for multiple side‐chain conformations from an analysis of NOE data. An unusual observation of the disappearance of the Gly NH resonances upon prolonged storage in methanol is rationalised on the basis of a slow aggregation step, resulting in stacking of three‐stranded β‐sheet structures, which in turn influences the conformational interconversion between type I′ and type II′ β‐turns at the two ^DPro–Gly segments. Experimental evidence for these processes is presented. The decapeptide fragment Boc‐LV^βFV^DPGL^βFVV‐OMe (BBH10), which has been previously characterized as a type I′ β‐turn nucleated hairpin, is shown to favour a type II′ β‐turn conformation in solution, supporting the occurrence of conformational interconversion at the turn segments in these hairpin and sheet structures.     

Folding Dynamics of 10‐Residue β‐Hairpin Peptide Chignolin

Short peptides that fold into β‐hairpins are ideal model systems for investigating the mechanism of protein folding because their folding process shows dynamics typical of proteins. We performed folding, unfolding, and refolding molecular dynamics simulations (total of 2.7 μs) of the 10‐residue β‐hairpin peptide chignolin, which is the smallest β‐hairpin structure known to be stable in solution. Our results revealed the folding mechanism of chignolin, which comprises three steps. First, the folding begins with hydrophobic assembly. It brings the main chain together; subsequently, a nascent turn structure is formed. The second step is the conversion of the nascent turn into a tight turn structure along with interconversion of the hydrophobic packing and interstrand hydrogen bonds. Finally, the formation of the hydrogen‐bond network and the complete hydrophobic core as well as the arrangement of side‐chain–side‐chain interactions occur at approximately the same time. This three‐step mechanism appropriately interprets the folding process as involving a combination of previous inconsistent explanations of the folding mechanism of the β‐hairpin, that the first event of the folding is formation of hydrogen bonds and the second is that of the hydrophobic core, or vice versa.     

Stability and Unfolding Mechanism of the N‐terminal β‐Hairpin from [2Fe‐2S] Ferredoxin I by Molecular Dynamics Simulations

The stability and unfolding mechanism of the N‐terminal β‐hairpin of the [2Fe‐2S] ferredoxin I from the blue‐green alga Aphanothece sacrum in pure methanol, 40% (v/v) methanol‐water, and pure water systems were investigated by 10 ns molecular dynamics simulations under periodic boundary conditions. The β‐hairpin was mostly in its native‐like state in pure methanol, whereas it unfolds dramatically following the ‘zip‐up’ mechanism when it was placed in pure water. Both interstrand and inside‐turn hydrogen bonds account for the stability of the β‐hairpin in its native‐like conformation, whereas hydrophobic interactions among nonpolar side chains are responsible for maintaining its stable loop‐like intermediate structures in 40% (v/v) methanol‐water. Reducing solvent polarity seems to increase the stability of the β‐hairpin in its native‐like structure. Methanol is likely to mimic the partially hydrophobic environment around the N‐terminal β‐hairpin by the subsequent α‐helix.     

Engineering a β‐Helical d,l‐Peptide for Folding in Polar Media

β Helices—helices formed by alternating d,l ‐peptides and stabilized by β‐sheet hydrogen bonding—are found naturally in only a handful of highly hydrophobic peptides. This paper explores the scope of β‐helical structure by presenting the first design and biophysical characterization of a hydrophilic d,l ‐peptide, 1 , that forms a β helix in methanol. The design of 1 is based on the β‐hairpin/β helix—a new supersecondary that had been characterized previously only for hydrophobic peptides in nonpolar solvents. Incorporating polar residues in 1 provided solubility in methanol, in which the peptide adopts the expected β‐hairpin/β‐helical structure, as evidenced by CD, analytical ultracentrifugation (AUC), NMR spectroscopy, and NMR‐based structure calculations. Upon titration with water (at constant peptide concentration), the structure in methanol ( 1 m ) transitions cooperatively to an extended conformation ( 1 w ) resembling a cyclic β‐hairpin; observation of an isodichroic point in the solvent‐dependent CD spectra indicates that this transition is a two‐state process. In contrast, neither 1 m nor 1 w show cooperative thermal melting; instead, their structures appear intact at temperatures as high as 65 °C; this observation suggests that steric constraint is dominant in stabilizing these structures. Finally, the ¹H NMR CαH spectroscopic resonances of 1 m are downfield‐shifted with respect to random‐coil values, a hitherto unreported property for β helices that appears to be a general feature of these structures. These results show for the first time that an appropriately designed β‐helical peptide can fold stably in a polar solvent; furthermore, the structural and spectroscopic data reported should prove useful in the future design and characterization of water‐soluble β helices.     

Beta‐hairpin restraint potentials for calculations of potentials of mean force as a function of beta‐hairpin tilt,rotation, and distance

We have developed a set of restraint potentials for β‐hairpin tilt relative to the membrane normal, β‐hairpin rotation around the β‐hairpin axis, and hairpin–hairpin distance. Such restraint potentials enable us to characterize the molecular basis of specific β‐hairpin tilt and rotation in membranes and hairpin–hairpin interactions at the atomic level by sampling their conformational space along these degrees of freedom, i.e., reaction coordinates, during molecular dynamics simulations. We illustrate the efficacy of the β‐hairpin restraint potentials by calculating the potentials of mean force (PMFs) as a function of tilt and rotation angles of protegrin‐1 (PG‐1), a β‐hairpin antimicrobial peptide, in an implicit membrane model. The peptide association in the membrane is also examined by calculating the PMFs as a function of distance between two PG‐1 peptides in various dimer interfaces. These novel restraint potentials are found to perform well in each of these cases and are expected to be a useful means to study the microscopic driving forces of insertion, tilting, and rotation of β‐hairpin peptides in membranes as well as their association in aqueous solvent or membrane environments particularly when combined with explicit solvent models. © 2008 Wiley Periodicals, Inc. J Comput Chem, 2009     

Structural Studies and Anticancer Activity of a Novel Class of β‐Peptides

Functionalized oligomeric organic compounds with well‐defined β‐proline scaffold have been synthesized by a cycloadditive oligomerization approach in racemic and enantiopure forms. The structure of the novel β‐peptides was investigated by NMR spectroscopic and X‐ray methods determining the conformational shapes of the β‐proline oligomers in solution and solid states. The main structural elements subject to conformational switches are β‐peptide bonds between 5‐arylpyrrolidine‐2‐carboxylic acid units existing in Z/E configurations. The whole library of short β‐peptides and intermediate acrylamides has been tested on antiproliferative activity towards the hormone‐refractory prostate cancer cell line PC‐3 revealing several oligomeric compounds with low micromolar and submicromolar activities. Bromine‐substituted dimeric and trimeric acrylamides induced caspase‐dependent apoptosis of PC‐3 cells through cell‐cycle arrest and mitochondrial damage.     

Filaggrin Peptides with β‐Hairpin Structure Bind Rheumatoid Arthritis Antibodies

In the early detection of rheumatoid arthritis (RA) synthetic filaggrin peptides serve as antigens for rheumatoid‐specific autoantibodies (anti‐citrullinated peptide antibody, ACPA) in ELISA tests. In this work we present a peptide that exhibits the binding epitope of ACPA in the form of a stable folding β‐hairpin. The homogeneity of the peptide folding was confirmed by NMR spectroscopy and might lead to the first proposed structure of the antibody‐bound conformation of the epitope.     

A pH Switch for β‐Sheet Protein Folding

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 (T_m) was observed at pH 2.5: ΔΔG_F ≥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.     

1,2,3‐Trimethoxy‐4‐[(E)‐2‐phenylvinyl]benzene and (E,E)‐1,4‐bis(2,3,4‐trimethoxyphenyl)buta‐1,3‐diene

The stilbene derivative 1,2,3‐trimethoxy‐4‐[(E)‐2‐phenylvinyl]benzene, C₁₇H₁₈O₃, (I), and its homocoupling co‐product (E,E)‐1,4‐bis(2,3,4‐trimethoxyphenyl)buta‐1,3‐diene, C₂₂H₂₆O₆, (II), both have double bonds in trans conformations in their conjugated linkages. In the structure of stilbene (I), the aromatic rings deviate significantly from coplanarity, in contrast with coproduct (II), the core of which is rigorously planar. The deviation in stilbene (I) seems to be driven by intermolecular electrostatic interactions. Diene (II) sits on a crystallographic inversion centre, which bisects the conjugated linkage.     

Monitoring Backbone Hydrogen‐Bond Formation in β‐Barrel Membrane Protein Folding

β‐barrel membrane proteins are key components of the outer membrane of bacteria, mitochondria and chloroplasts. Their three‐dimensional structure is defined by a network of backbone hydrogen bonds between adjacent β‐strands. Here, we employ hydrogen–deuterium (H/D) exchange in combination with NMR spectroscopy and mass spectrometry to monitor backbone hydrogen bond formation during folding of the outer membrane protein X (OmpX) from E. coli in detergent micelles. Residue‐specific kinetics of interstrand hydrogen‐bond formation were found to be uniform in the entire β‐barrel and synchronized to formation of the tertiary structure. OmpX folding thus propagates via a long‐lived conformational ensemble state in which all backbone amide protons exchange with the solvent and engage in hydrogen bonds only transiently. Stable formation of the entire OmpX hydrogen bond network occurs downhill of the rate‐limiting transition state and thus appears cooperative on the overall folding time scale.     

Synthesis and Conformational Characterisation of Hexameric β‐Peptide Foldamers by Using Double POAC Spin Labelling and cw‐EPR

A selected set of terminally protected β‐hexapeptides, each containing two nitroxide‐based (3R,4R)‐4‐amino‐1‐oxyl‐2,2,5,5‐tetramethylpyrrolidine‐3‐carboxylic acid (POAC) residues combined with four (1S,2S)‐2‐aminocyclopentane‐1‐carboxylic acid (ACPC) residues, was synthesised by using solution methods and was fully characterised. The two POAC residues are separated in the sequences by different numbers of intervening ACPC residues. The conformational features of the doubly spin‐labelled β‐hexapeptides were examined in chloroform by FTIR absorption and continuous‐wave electron paramagnetic resonance spectroscopic techniques. In particular, the biradical exchange coupling (J) between two POAC residues within each peptide indicates unambiguously that the secondary structure overwhelmingly adopted is the 12‐helix. Taken together, these results support the view that POAC is an excellent β‐amino acid for exploring this type of helical conformation in doubly labelled β‐peptides.     

Gramicidin S Derivatives Containing cis‐ and trans‐Morpholine Amino Acids (MAAs) as Turn Mimetics

The cyclic decapeptide gramicidin S (GS) was used as a model for the evaluation of four turn mimetics. For this purpose, one of the D ‐Phe‐Pro two‐residue turn motifs in the rigid cyclic β‐hairpin structure of GS was replaced with morpholine amino acids (MAA 2 – 5 ), differing in stereochemistry and length of the side‐chain. The conformational properties of the thus obtained GS analogues ( 6 – 9 ) was assessed by using NMR spectroscopy and X‐ray crystallography, and correlated with their biological properties (antimicrobial and hemolytic activity). We show that compound 8 , containing the dipeptide isostere trans‐MAA 4 , has an apparent high structural resemblance with GS and that its antibacterial activity against a panel of Gram positive and ‐negative bacterial strains is better than the derivatives 6 , 7 and 9 .     

Sequestration of a β‐Hairpin for Control of α‐Synuclein Aggregation

The misfolding and aggregation of the protein α‐synuclein (α‐syn), which results in the formation of amyloid fibrils, is involved in the pathogenesis of Parkinson’s disease and other synucleinopathies. The emergence of amyloid toxicity is associated with the formation of partially folded aggregation intermediates. Here, we engineered a class of binding proteins termed β‐wrapins (β‐wrap proteins) with affinity for α‐synuclein (α‐syn). The NMR structure of an α‐syn:β‐wrapin complex reveals a β‐hairpin of α‐syn comprising the sequence region α‐syn(37–54). The β‐wrapin inhibits α‐syn aggregation and toxicity at substoichiometric concentrations, demonstrating that it interferes with the nucleation of aggregation.     

Isomerization‐ and Temperature‐Jump‐Induced Dynamics of a Photoswitchable β‐Hairpin

Conformational changes in proteins and peptides can be initiated by diverse processes. This raises the question how the variation of initiation mechanisms is connected to differences in folding or unfolding processes. In this work structural dynamics of a photoswitchable β‐hairpin model peptide were initiated by two different mechanisms: temperature jump (T‐jump) and isomerization of a backbone element. In both experiments the structural changes were followed by time‐resolved IR spectroscopy in the nanosecond to microsecond range. When the photoisomerization of the azobenzene backbone switch initiated the folding reaction, pronounced absorption changes related to folding into the hairpin structure were found with a time constant of about 16 μs. In the T‐jump experiment kinetics with the same time constant were observed. For both initiation processes the reaction dynamics revealed the same strong dependence of the reaction time on temperature. The highly similar transients in the microsecond range show that the peptide dynamics induced by T‐jump and isomerization are both determined by the same mechanism and exclude a downhill‐folding process. Furthermore, the combination of the two techniques allows a detailed model for folding and unfolding to be presented: The isomerization‐induced folding process ends in a transition‐state reaction scheme, in which a high energetic barrier of 48 kJ mol^?1 separates unfolded and folded structures.     

Stereoselective Organocatalyzed Synthesis of α‐Fluorinated β‐Amino Thioesters and Their Application in Peptide Synthesis

α‐Fluorinated β‐amino thioesters were obtained in high yields and stereoselectivities by organocatalyzed addition reactions of α‐fluorinated monothiomalonates (F‐MTMs) to N‐Cbz‐ and N‐Boc‐protected imines. The transformation requires catalyst loadings of only 1 mol % and proceeds under mild reaction conditions. The obtained addition products were readily used for coupling‐reagent‐free peptide synthesis in solution and on solid phase. The α‐fluoro‐β‐(carb)amido moiety showed distinct conformational preferences, as determined by crystal structure and NMR spectroscopic analysis.     

Chirality and Template‐Mediated Induction of Helical Preferences in Achiral β‐Peptides

This study describes chirality‐ or template‐mediated helical induction in achiral β‐peptides for the first time. A strategy of end capping β‐peptides derived from β‐hGly (the smallest achiral β‐amino acid) with a chiral β‐amino acid that possesses a carbohydrate side chain (β‐Caa; C‐linked carbo β‐amino acid) or a small, robust helical template derived from β‐Caas, was adopted to investigate folding propensity. A single chiral (R)‐β‐Caa residue at the C‐ or N‐terminus in these oligomers led to a preponderance of right‐handed 12/10‐helical folds, which was reiterated more strongly in peptides capped at both the C‐ and N‐terminus. Likewise, the presence of a template (a 12/10‐helical trimer) at both the C‐ and N‐terminus resulted in a very robust helix. The propagation of the helical fold and its sustenance was found in a homo‐oligomeric sequence with as many as seven β‐hGly residues. In both cases, the induction of helicity was stronger from the N terminus, whereas an anchor at the C terminus resulted in reduced helical propensity. Although these oligomers have been theoretically predicted to favor a 12/10‐mixed helix in apolar solvents, this study provides the first experimental evidence for their existence. Diastereotopicity was found in both the methylene groups of the β‐hGly moieties due to chirality. Additionally, the β‐hGly units have shown split behavior in the conformational space to accommodate the 12/10‐helix. Thus, end capping to assist chiralty‐ or template‐mediated helical induction and stabilization in achiral β‐peptides is a very attractive strategy.     

Antiparallel Self‐Association of a γ,α‐Hybrid Peptide: More Relevance of Weak Interactions

To learn how a preorganized peptide‐based molecular template, together with diverse weak non‐covalent interactions, leads to an effective self‐association, we investigated the conformational characteristics of a simple γ,α‐hybrid model peptide, Boc‐γ‐Abz‐Gly‐OMe. The single‐crystal X‐ray diffraction analysis revealed the existence of a fully extended β‐strand‐like structure stabilized by two non‐conventional C?H???O=C intramolecular H‐bonds. The 2D ¹H NMR ROESY experiment led us to propose that the flat topology of the urethane‐γ‐Abz‐amide moiety is predominantly preserved in a non‐polar environment. The self‐association of the energetically more favorable antiparallel β‐strand‐mimic in solid‐state engenders an unusual ‘flight of stairs’ fabricated through face‐to‐face and edge‐to‐edge Ar???Ar interactions. In conjunction with FT‐IR spectroscopic analysis in chloroform, we highlight that conformationally semi‐rigid γ‐Abz foldamer in appositely designed peptides may encourage unusual β‐strand or β‐sheet‐like self‐association and supramolecular organization stabilized via weak attractive forces.