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.     

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.     

Three‐Residue Turn in β‐Peptides Nucleated by a 12/10 Helix

A new three‐residue turn in β peptides nucleated by a 12/10‐mixed helix is presented. In this design, β peptides were derived from the 1:1 alternation of C‐linked carbo‐β‐amino acid ester [BocNH‐(R)‐β‐Caa_(r)‐OMe] (Boc=tert‐butyloxycarbonyl), which consisted of a D ‐ribo furanoside side chain, and β‐hGly residues. The hexapeptide with (R)‐β‐Caa_(r) at the N terminus showed the ‘turn’ stabilized by a 14‐membered NH(4) ??? CO(6) hydrogen bond at the C terminus nucleated by a robust 12/10‐mixed helix, thus providing a ‘helix‐turn’ (HT) motif. The turn and the helix were additionally stabilized by intraresidue electrostatic interaction between the furan oxygen in the carbohydrate side chain and NH in the backbone. However, the hexapeptide with a β‐hGly residue at the N terminus demonstrated the presence of a 10/12 helix through its entire length, which again showed the intraresidue interaction between NH and furan oxygen. The intraresidue NH ??? O? Me electrostatic interactions observed in the monomer, however, were absent in the peptides.     

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.     

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.     

Structural varieties of selectively mixed G‐ and C‐rich short DNA sequences studied with electrospray ionization mass spectrometry

Short guanine(G)‐repeat and cytosine(C)‐repeat DNA strands can self‐assemble to form four‐stranded G‐quadruplexes and i‐motifs, respectively. Herein, G‐rich and C‐rich strands with non‐G or non‐C terminal bases and different lengths of G‐ or C‐repeats are mixed selectively in pH 4.5 and 6.7 ammonium acetate buffer solutions and studied by electrospray ionization mass spectrometry (ESI‐MS). Various strand associations corresponding to bi‐, tri‐ and tetramolecular ions are observed in mass spectra, indicating that the formation of quadruplex structures is a random strand by strand association process. However, with increasing incubation time for the mixtures, initially associated hybrid tetramers will transform into self‐assembled conformations, which is mainly driven by the structural stability. The melting temperature values of self‐assembled quadruplexes suggest that the length of G‐repeats or C‐repeats shows more significant effect on the stability of quadruplex structures than that of terminal residues. Accordingly, we can obtain the self‐associated tetrameric species generated from the mixtures of various homologous G‐ or C‐strands efficiently by altering the length of G‐ or C‐repeats. Our studies demonstrate that ESI‐MS is a very direct, fast and sensitive tool to provide significant information on DNA strand associations and stoichiometric transitions, particularly for complex mixtures. Copyright © 2016 John Wiley & Sons, Ltd.     

Improved Stability and Tunable Functionalization of Parallel β‐Sheets via Multicomponent N‐Alkylation of the Turn Moiety

In contrast to the myriad of methods available to produce α‐helices and antiparallel β‐sheets in synthetic peptides, just a few are known for the construction of stable, non‐cyclic parallel β‐sheets. Herein, we report an efficient on‐resin approach for the assembly of parallel β‐sheet peptides in which the N‐alkylated turn moiety enhances the stability and gives access to a variety of functionalizations without modifying the parallel strands. The key synthetic step of this strategy is the multicomponent construction of an N‐alkylated turn using the Ugi reaction on varied isocyano‐resins. This four‐component process assembles the orthogonally protected turn fragment and incorporates handles serving for labeling/conjugation purposes or for reducing peptide aggregation. NMR and circular dichroism analyses confirm the better‐structured and more stable parallel β‐sheets in the N‐alkylated peptides compared to the non‐functionalized variants.     

Three‐Residue Turns in α/β‐Peptides and Their Application in the Design of Tertiary Structures

A new three‐residue turn was serendipitously discovered in α/β hybrid peptides derived from alternating C‐linked carbo‐β‐amino acids (β‐Caa) and L ‐Ala residues. The three‐residue β‐α‐β turn at the C termini, nucleated by a helix at the N termini, resulted in helix‐turn (HT) supersecondary structures in these peptides. The turn in the HT motif is stabilized by two H bonds—CO(i?2)–NH(i), with a seven‐membered pseudoring (γ turn) in the backward direction, and NH(i?2)–CO(i), with a 13‐membered pseudoring in the forward direction (i being the last residue)—at the C termini. The study was extended to generalize the new three‐residue turn (β‐α‐β) by using different α‐ and β‐amino acids. Furthermore, the HT motifs were efficiently converted, by an extension with helical oligomers at the C termini, into peptides with novel helix‐turn‐helix (HTH) tertiary structures. However, this resulted in the destabilization of the β‐α‐β turn with the concomitant nucleation of another three‐residue turn, α‐β‐β, which is stabilized by 11‐ and 15‐membered bifurcated H bonds. Extensive NMR spectroscopic studies were carried out to delineate the secondary and tertiary structures in these peptides, which are further supported by molecular dynamics (MD) investigations.     

β‐Strand Mimetic Foldamers Rigidified through Dipolar Repulsion

Many therapeutically relevant protein–protein interactions contain hot‐spot regions on secondary structural elements, which contribute disproportionately to binding enthalpy. Mimicry of such α‐helical regions has met with considerable success, however the analogous approach for the β‐strand has received less attention. Presented herein is a foldamer for strand mimicry in which dipolar repulsion is a central determinant of conformation. Computation as well as solution‐ and solid‐phase data are consistent with an ensemble weighted almost exclusively in favor of the desired conformation.     

Design of Stable β‐Sheet‐Based Cyclic Peptide Assemblies Assisted by Metal Coordination: Selective Homo‐ and Heterodimer Formation

Metal‐directed supramolecular construction represents one of the most powerful tools to prepare a large variety of structures and functions. The ability of metals to organize different numbers and types of ligands with a variety of geometries (linear, trigonal, octahedral, etc.) expands the supramolecular synthetic architecture. We describe here the precise construction of homo‐ and heterodimeric cyclic peptide entities through coordination of a metal (Pd, Au) and to β‐sheet‐type hydrogen‐bonding interactions. The selective coordination properties of the appropriate metal allow control over the cross‐strand interaction between the two‐peptide strands.     

Inherent Conformational Preferences of Ac‐Gln‐Gln‐NHBn: Sidechain Hydrogen Bonding Supports a β‐Turn in the Gas Phase

Gas‐phase single‐conformation spectroscopy is used to study Ac‐Gln‐Gln‐NHBn in order to probe the interplay between sidechain hydrogen bonding and backbone conformational preferences. This small, amide‐rich peptide offers many possibilities for backbone–backbone, sidechain–backbone, and sidechain–sidechain interactions. The major conformer observed experimentally features a type‐I β‐turn with a canonical 10‐membered ring C=O—H?N hydrogen bond between backbone amide groups. In addition, the C=O group of each Gln sidechain participates in a seven‐membered ring hydrogen bond with the backbone NH of the same residue. Thus, sidechain hydrogen‐bonding potential is satisfied in a manner that is consistent with and stabilizes the β‐turn secondary structure. This turn‐forming propensity may be relevant to pathogenic amyloid formation by polyglutamine segments in human proteins.     

Dynamic relaying properties of a β-turn peptide in long-range electron transfer

The relay stations play a significant role in long-range charge hopping transfer in proteins. Although studies have clarified that many more protein structural motifs can function as relays in charge hopping transfers by acting as intermediate charge carriers, the relaying properties are still poorly understood. In this work, taking a β-turn oligopeptide as an example, we report a dynamic character of a relay with tunable relaying properties using the density functional theory calculations. Our main finding is that a β-turn peptide can serve as an effective electron relay in facilitating long-range electron migration and its relay properties is vibration-tunable. The vibration-induced structural transient distortions remarkably affect the lowest occupied molecular orbital (LUMO) energy, vertical electron affinity and electron-binding mode of the β-turn oligopeptide and the singly occupied molecular orbital (SOMO) energy of the corresponding electron adduct and thus the relaying properties. Different vibration modes lead to different structural distortions and thus have different effects on the relaying properties and ability of the β-turn peptide. For the relaying properties, there approximately is a linear negative correlation of electron affinity with the LUMO energy of the β-turn or the SOMO energy of its electron adduct. Besides, such relaying properties also vary in the vibration evolution process, and the electron-binding modes may be tunable. As an important addition to the known static charge relaying properties occurring in various protein structural motifs, this work reports the dynamic electron-relaying characteristics of a β-turn oligopeptide with variable relaying properties governed by molecular vibrations which can be applied to different proteins in mediating long-range charge transfers. Clearly, this work reveals molecular vibration effects on the electron relaying properties of protein structural motifs and provides new insights into the dynamics of long-range charge transfers in proteins. © 2018 Wiley Periodicals, Inc.     

Prediction of membrane spanning segments and topology in β‐barrel membrane proteins at better accuracy

Prediction of membrane spanning segments in β‐barrel outer membrane proteins (OMP) and their topology is an important problem in structural and functional genomics. In this work, we propose a method based on radial basis networks for predicting the number of β‐strands in OMPs and identifying their membrane spanning segments. Our method showed a leave‐one‐out cross validation accuracy of 96% in a set of 28 OMPs, which have the range of 8–22 β‐strand segments. The β‐strand segments in OMPs and the residues in membrane spanning segments are correctly predicted with the accuracy of 96% and 87%, respectively. We have developed a web server, TMBETAPRED‐RBF for predicting the transmembrane β‐strands from amino acid sequence and it is available at http://rbf.bioinfo.tw/～sachen/tmrbf.html . We suggest that our method could be an effective tool for predicting the membrane spanning regions and topology of β‐barrel membrane proteins. © 2009 Wiley Periodicals, Inc. J Comput Chem 2010     

The N‐Terminal Domain of ALS‐Linked TDP‐43 Assembles without Misfolding

Transactivation response element (TAR) DNA‐binding protein 43 (TDP‐43) misfolding is implicated in several neurodegenerative diseases characterized by aggregated protein inclusions. Misfolding is believed to be mediated by both the N‐ and C‐terminus of TDP‐43; however, the mechanistic basis of the contribution of individual domains in the process remained elusive. Here, using single‐molecule fluorescence and ensemble biophysical techniques, and a wide range of pH and temperature conditions, we show that TDP‐43^NTD is thermodynamically stable, well‐folded and undergoes reversible oligomerization. We propose that, in full‐length TDP‐43, association between folded N‐terminal domains enhances the propensity of the intrinsically unfolded C‐terminal domains to drive pathological aggregation.     

12‐ to 22‐Membered Bridged β‐Lactams as Potential Penicillin‐Binding Protein Inhibitors

As potential inhibitors of penicillin‐binding proteins (PBPs), we focused our research on the synthesis of non‐traditional 1,3‐bridged β‐lactams embedded into macrocycles. We synthesized 12‐ to 22‐membered bicyclic β‐lactams by the ring‐closing metathesis (RCM) of bis‐ω‐alkenyl‐3(S)‐aminoazetidinone precursors. The reactivity of 1,3‐bridged β‐lactams was estimated by the determination of the energy barrier of a concerted nucleophilic attack and lactam ring‐opening process by using ab initio calculations. The results predicted that 16‐membered cycles should be more reactive. Biochemical evaluations against R39 DD‐peptidase and two resistant PBPs, namely, PBP2a and PBP5, revealed the inhibition effect of compound 4d , which featured a 16‐membered bridge and the N‐tert‐butyloxycarbonyl chain at the C3 position of the β‐lactam ring. Surprisingly, the corresponding bicycle, 12d , with the PhOCH₂CO side chain at C3 was inactive. Reaction models of the R39 active site gave a new insight into the geometric requirements of the conformation of potential ligands and their steric hindrance; this could help in the design of new compounds.     

O2‐Mediated Oxidation of Aminoboranes through 1,2‐N Migration

In analogy to the classical reaction of C?B bonds with peroxides, the first oxidative functionalization of aminoboranes through a 1,2‐N migration was realized. Readily available aliphatic nitro compounds are thereby transformed into N‐ and O‐functionalized hydroxylamines in a single synthetic operation. Addition of hazardous peroxides is avoided. Instead, the insertion of O₂, as the terminal oxidant, into Zn?C bonds provides the necessary peroxides. The required zinc organyls, in turn, are formed through a boron‐to‐zinc exchange, from an organoboronic ester byproduct of the nitro‐to‐aminoborane transformation.     

Investigation on the individual contributions of N?H···OC and C?H···OC interactions to the binding energies of β‐sheet models

In this article, the binding energies of 16 antiparallel and parallel β‐sheet models are estimated using the analytic potential energy function we proposed recently and the results are compared with those obtained from MP2, AMBER99, OPLSAA/L, and CHARMM27 calculations. The comparisons indicate that the analytic potential energy function can produce reasonable binding energies for β‐sheet models. Further comparisons suggest that the binding energy of the β‐sheet models might come mainly from dipole–dipole attractive and repulsive interactions and VDW interactions between the two strands. The dipole–dipole attractive and repulsive interactions are further obtained in this article. The total of N? H···H? N and C?O···O?C dipole–dipole repulsive interaction (the secondary electrostatic repulsive interaction) in the small ring of the antiparallel β‐sheet models is estimated to be about 6.0 kcal/mol. The individual N? H···O?C dipole–dipole attractive interaction is predicted to be ?6.2 ± 0.2 kcal/mol in the antiparallel β‐sheet models and ?5.2 ± 0.6 kcal/mol in the parallel β‐sheet models. The individual C^α? H···O?C attractive interaction is ?1.2 ± 0.2 kcal/mol in the antiparallel β‐sheet models and ?1.5 ± 0.2 kcal/mol in the parallel β‐sheet models. These values are important in understanding the interactions at protein–protein interfaces and developing a more accurate force field for peptides and proteins. © 2009 Wiley Periodicals, Inc. J Comput Chem 2010     

Effect of the π Bridge and Acceptor on Intramolecular Charge Transfer in Push–Pull Cationic Chromophores: An Ultrafast Spectroscopic and TD‐DFT Computational Study

Three (donor–π–acceptor)⁺ systems with a methyl pyridinium or quinolinium as the electron‐deficient group, a dimethyl amino as the electron‐donor group, and an ethylene or butadiene group as the spacer have been investigated in a joint spectroscopic and TD‐DFT computational study. A negative solvatochromism has been revealed in the absorption spectra, which implies a solution color change, and interpreted by considering the variation in the permanent dipole moment modulus and orientation upon photoexcitation. The fluorescence efficiency decreases upon increasing solvent polarity, in agreement with the excited‐state optimized geometries (planar in low‐polarity media and twisted in high‐polarity media). Femtosecond transient absorption has revealed the occurrence of a fast photoinduced intramolecular charge transfer (ICT) and the molecular factors that determine an efficient ICT. Considering the crucial role of the ICT in tuning the nonlinear optical (NLO) properties, these compounds can be considered promising NLO materials.     

Contributions of a Surface Hydrophobic Cluster to the Folding and Structural Stability of Ubiquitin

The role of the small exterior hydrophobic cluster (SEHC) in the strand region of the N‐terminal β‐hairpin of ubiquitin on the structural stability and the folding/unfolding kinetics of the protein have been examined. We introduce a Phe→Ala substitution at residue 4 in the strand region of the N‐terminal β‐hairpin of the ubiquitin. A peptide with the same amino acid sequence as the first 21 residues of the mutated ubiquitin has also been synthesized. The F4A mutation unfolds the hairpin structure of the peptide segment without disruption of the turn. The same mutation does not seem to affect the overall structure, but the stability of the mutated full‐length protein decreases by approx. 2 kcal/mol. Kinetically, the entire hairpin structure is implicated in the transition state during folding of the wild type protein. The rate of refolding is retarded by the F4A mutation in ～80% of the protein molecules. The F4A substitution also increases the unfolding rate of the protein by 10 fold. Thus the hydrophobic side‐chain of Phe‐4 not only contributes to the stability of the hairpin, but also to the stability of the entire protein by forming a cluster together with the hydrophobic residues on the C‐terminal strand.     

Electron Attachment to Hydrated Oligonucleotide Dimers: Guanylyl‐3′,5′‐Cytidine and Cytidylyl‐3′,5′‐Guanosine

The dinucleoside phosphate deoxycytidylyl‐3′,5′‐deoxyguanosine (dCpdG) and deoxyguanylyl‐3′,5′‐deoxycytidine (dGpdC) systems are among the largest to be studied by reliable theoretical methods. Exploring electron attachment to these subunits of DNA single strands provides significant progress toward definitive predictions of the electron affinities of DNA single strands. The adiabatic electron affinities of the oligonucleotides are found to be sequence dependent. Deoxycytidine (dC) on the 5′ end, dCpdG, has larger adiabatic electron affinity (AEA, 0.90 eV) than dC on the 3′ end of the oligomer (dGpdC, 0.66 eV). The geometric features, molecular orbital analyses, and charge distribution studies for the radical anions of the cytidine‐containing oligonucleotides demonstrate that the excess electron in these anionic systems is dominantly located on the cytosine nucleobase moiety. The π‐stacking interaction between nucleobases G and C seems unlikely to improve the electron‐capturing ability of the oligonucleotide dimers. The influence of the neighboring base on the electron‐capturing ability of cytosine should be attributed to the intensified proton accepting–donating interaction between the bases. The present investigation demonstrates that the vertical detachment energies (VDEs) of the radical anions of the oligonucleotides dGpdC and dCpdG are significantly larger than those of the corresponding nucleotides. Consequently, reactions with low activation barriers, such as those for O? C σ bond and N‐glycosidic bond breakage, might be expected for the radical anions of the guanosine–cytosine mixed oligonucleotides.