Folding and Unfolding of Two Mixed α/β Peptides

We present a molecular dynamics simulation study of two peptides containing α‐ and β‐amino acid residues. According to experiment, the two peptides differ in the dominant fold when solvated in methanol: one shows a helical fold, the other a β hairpin. The simulations at 300 and 340 K were done by starting from a NMR spectroscopic model structure and from an extended (denatured) structure. The typical structural features of the two peptides are reproduced and a folding/unfolding equilibrium is observed on the nanosecond timescale at 300 K. Analysis of proton–proton NOE distance bounds and backbone ³J coupling constants gives results consistent with the experimental data. We conclude that our simulations are complementary to the experiments by providing detailed information on the conformational distributions.     

Design of Lanthanide Fingers: Compact Lanthanide‐Binding Metalloproteins

Lanthanides have interesting chemical properties; these include luminescent, magnetic, and catalytic functions. Toward the development of proteins incorporating novel functions, we have designed a new lanthanide‐binding motif, lanthanide fingers. These were designed based on the Zif268 zinc finger, which exhibits a ββα structural motif. Lanthanide fingers utilize an Asp₂Glu₂ metal‐coordination environment to bind lanthanides through a tetracarboxylate peptide ligand. The iterative design of a general lanthanide‐binding peptide incorporated the following key elements: 1) residues with high α‐helix and β‐sheet propensities in the respective secondary structures; 2) an optimized big box α‐helix N‐cap; 3) a Schellman α‐helix C‐cap motif; and 4) an optional D ‐Pro‐Ser type II’ β‐turn in the β‐hairpin. The peptides were characterized for lanthanide binding by circular dichroism (CD), NMR, and fluorescence spectroscopy. In all instances, stabilization of the peptide secondary structures resulted in an increase in metal affinity. The optimized protein design was a 25‐residue peptide that was a general lanthanide‐binding motif; this binds all lanthanides examined in a competitive aqueous environment, with a dissociation constant of 9.3 μM for binding Er³⁺. CD spectra of the peptide‐lanthanide complexes are similar to those of zinc fingers and other ββα proteins. Metal binding involves residues from the N‐terminal β‐hairpin and the C terminal α‐helical segments of the peptide. NMR data indicated that metal binding induced a global change in the peptide structure. The D ‐Pro‐Ser type II’ β‐turn motif could be replaced by Thr–Ile to generate genetically encodable lanthanide fingers. Replacement of the central Phe with Trp generated genetically encodable lanthanide fingers that exhibited terbium luminescence greater than that of an EF‐hand peptide.     

Conformational Flexibility in the Binding Surface of the Potassium Channel Blocker ShK

ShK is a 35‐residue peptide that binds with high affinity to human voltage‐gated potassium channels through a conserved K‐Y dyad. Here we have employed NMR measurements of backbone‐amide ¹⁵N spin‐relaxation rates to investigate motions of the ShK backbone. Although ShK is rigid on the ps to ns timescale, increased linewidths observed for 11 backbone‐amide ¹⁵N resonances identify chemical or conformational exchange contributions to the spin relaxation. Relaxation dispersion profiles indicate that exchange between major and minor conformers occurs on the sub‐millisecond timescale. Affected residues are mostly clustered around the central helix‐kink‐helix structure and the critical K22–Y23 motif. We suggest that the less structured minor conformer increases the exposure of Y23, known to contribute to binding affinity and selectivity, thereby facilitating its interaction with potassium channels. These findings have potential implications for the design of new channel blockers based on ShK.     

Solution‐NMR Characterization of Outer‐Membrane Protein A from E. coli in Lipid Bilayer Nanodiscs and Detergent Micelles

X‐ray crystallography and solution NMR of detergent‐reconstituted OmpA (outer membrane protein A from E. coli) had shown that this protein forms an eight‐stranded transmembrane β‐barrel, but only limited information was obtained for the extracellular loops. In NMR studies of OmpA in two different detergent micelles, “NMR‐invisible” amino acid residues in‐between the extracellular loops and the β‐barrel prevented complete structural characterization. Here, we show that this NMR‐invisible ring around the β‐barrel of OmpA is also present in lipid bilayer nanodiscs and in mixed micelles with a third detergent, thus suggesting that the implicated rate processes have a functional role rather than representing an artifact of the protein reconstitution. In addition to sequence‐specific NMR assignments for OmpA in the nanodiscs, the present results are based on a protocol of micro‐coil TROSY‐ and CRINEPT‐type NMR diffusion measurements for studying the hydrodynamic properties and the foldedness of [²H,¹⁵N]‐labeled membrane proteins in nanodiscs. This protocol can be applied under conditions closely similar to those used for NMR structure determinations or crystallization trials.     

Arginine Arrangement of Bacteriophage λ N‐Peptide Plays a Role as a Core Motif in GNRA Tetraloop RNA Binding

A simple α‐helical N‐model‐peptide was designed to investigate the role of the arginine‐rich motif of bacteriophage λ N‐peptide in selective binding with boxB RNA. The five‐arginine arrangement of native N‐peptide was retained; all other residues were replaced with alanine. In vitro selection of RNA (30 random‐nucleotide region) was carried out with N‐model‐peptide immobilized on a 27 MHz quartz‐crystal microbalance (QCM). Selected RNAs were evaluated on the same QCM plate to obtain binding constants (K_a=10⁷–10⁸ M ^?1). Many selected RNAs contained GNR(N)A‐type loops (similar to the boxB RNA motif recognized by the native N‐peptide). Fragments and minimal RNAs containing the GNRA‐type loop also bound to N‐model‐peptide (K_a=10⁶–10⁷ M ^?1). The RNA recognition specificity of the peptide was studied by changing the “closing” U–A base pair and one base in the tetraloop of the RNA aptamers, and by peptide mutations (18th residue of N‐model‐peptide). It was concluded that the five‐arginine arrangement of the peptide performs selective recognition of the GNRA tetraloop and GNR(N)A pentaloop RNA structures, and that substitution of another functional amino acid residue at the 18th position in N‐peptide adds the recognition ability for a loop‐RNA sequence.     

Dissociation of Antimicrobial and Hemolytic Activities of Gramicidin S through N‐Methylation Modification

β‐Sheet antimicrobial peptides (AMPs) are well recognized as promising candidates for the treatment of multidrug‐resistant bacterial infections. To dissociate antimicrobial activity and hemolytic effect of β‐sheet AMPs, we hypothesize that N‐methylation of the intramolecular hydrogen bond(s)‐forming amides could improve their specificities for microbial cells over human erythrocytes. We utilized a model β‐sheet antimicrobial peptide, gramicidin S (GS), to study the N‐methylation effects on the antimicrobial and hemolytic activities. We synthesized twelve N‐methylated GS analogues by replacement of residues at the β‐strand and β‐turn regions with N‐methyl amino acids, and tested their antimicrobial and hemolytic activities. Our experiments showed that the HC₅₀ values increased fivefold compared with that of GS, when the internal hydrogen‐bonded leucine residue was methylated. Neither hemolytic effect nor antimicrobial activity changed when proline alone was replaced with N‐methylalanine in the β‐turn region. However, analogues containing N‐methylleucine at β‐strand and N‐methylalanine at β‐turn regions exhibited a fourfold increase in selectivity index compared to GS. We also examined the conformation of these N‐methylated GS analogues using ¹H NMR and circular dichroism (CD) spectroscopy in aqueous solution, and visualized the backbone structures and residue orientations using molecular dynamics simulations. The results show that N‐methylation of the internal hydrogen bond‐forming amide affected the conformation, backbone shape, and side chain orientation of GS.     

Carnosine Inhibits Aβ42 Aggregation by Perturbing the H‐Bond Network in and around the Central Hydrophobic Cluster

Aggregation of the amyloid‐β peptide (Aβ) into fibrillar structures is a hallmark of Alzheimer's disease. Thus, preventing self‐assembly of the Aβ peptide is an attractive therapeutic strategy. Here, we used experimental techniques and atomistic simulations to investigate the influence of carnosine, a dipeptide naturally occurring in the brain, on Aβ aggregation. Scanning force microscopy, circular dichroism and thioflavin T fluorescence experiments showed that carnosine does not modify the conformational features of Aβ42 but nonetheless inhibits amyloid growth. Molecular dynamics (MD) simulations indicated that carnosine interacts transiently with monomeric Aβ42 by salt bridges with charged side chains, and van der Waals contacts with residues in and around the central hydrophobic cluster (¹⁷LVFFA²¹). NMR experiments on the nonaggregative fragment Aβ12–28 did not evidence specific intermolecular interactions between the peptide and carnosine, in agreement with MD simulations. However, a close inspection of the spectra revealed that carnosine interferes with the local propensity of the peptide to form backbone hydrogen bonds close to the central hydrophobic cluster (residues E22, S26 and N27). Finally, MD simulations of aggregation‐prone Aβ heptapeptide segments show that carnosine reduces the propensity to form intermolecular backbone hydrogen bonds in the region 18–24. Taken together, the experimental and simulation results (cumulative MD sampling of 0.2 ms) suggest that, despite the inability of carnosine to form stable contacts with Aβ, it might block the pathway toward toxic aggregates by perturbing the hydrogen bond network near residues with key roles in fibrillogenesis.     

Tumor-Suppressor p53TAD1–60 Forms a Fuzzy Complex with Metastasis-Associated S100A4: Structural Insights and Dynamics by an NMR/MD Approach

Conformationally flexible protein complexes represent a major challenge for structural and dynamical studies. We present herein a method based on a hybrid NMR/MD approach to characterize the complex formed between the disordered p53TAD^1–60 and the metastasis-associated S100A4. Disorder-to-order transitions of both TAD1 and TAD2 subdomains upon interaction is detected. Still, p53TAD^1–60 remains highly flexible in the bound form, with residues L26, M40, and W53 being anchored to identical hydrophobic pockets of the S100A4 monomer chains. In the resulting “fuzzy” complex, the clamp-like binding of p53TAD^1–60 relies on specific hydrophobic anchors and on the existence of extended flexible segments. Our results demonstrate that structural and dynamical NMR parameters (cumulative Δδ, SSP, temperature coefficients, relaxation time, hetNOE) combined with MD simulations can be used to build a structural model even if, due to high flexibility, the classical solution structure calculation is not possible.     

OCRE Domains of Splicing Factors RBM5 and RBM10: Tyrosine Ring-Flip Frequencies Determined by Integrated Use of 1H NMR Spectroscopy and Molecular Dynamics Simulations

The 55-residue OCRE domains of the splicing factors RBM5 and RBM10 contain 15 tyrosines in compact, globular folds. At 25 °C, all 15 tyrosines show symmetric ¹H NMR spectra, with averaged signals for the pairs of δ- and ϵ-ring hydrogens. At 4 °C, two tyrosines were identified as showing ¹H NMR line-broadening due to lowered frequency of the ring-flipping. For the other 13 tyrosine rings, it was not evident, from the ¹H NMR data alone, whether they were either all flipping at high frequencies, or whether slowed flipping went undetected due to small chemical-shift differences between pairs of exchanging ring hydrogen atoms. Here, we integrate ¹H NMR spectroscopy and molecular dynamics (MD) simulations to determine the tyrosine ring-flip frequencies. In the RBM10-OCRE domain, we found that, for 11 of the 15 tyrosines, these frequencies are in the range 2.0×10⁶ to 1.3×10⁸ s⁻¹, and we established an upper limit of <1.0×10⁶ s⁻¹ for the remaining four residues. The experimental data and the MD simulation are mutually supportive, and their combined use extends the analysis of aromatic ring-flip events beyond the limitations of routine ¹H NMR line-shape analysis into the nanosecond frequency range.     

Biocompatible,stimuli‐responsive hydrogel of chemically crosslinked β‐cyclodextrin as amoxicillin carrier

A novel pH‐responsive, chemically crosslinked hydrogelator (cl‐β‐CD/pVP) has been fabricated using β‐cyclodextrin (β‐CD) and N‐vinyl pyrollidone (N‐VP) in presence of diurethane dimethacrylate (DUDMA) crosslinker/azobisisobutyronitrile initiator through free radical polymerization. Various grades of cl‐β‐CD/pVP have been synthesized and the best grade has been considered with higher crosslinking density, higher gel strength, and lower % swelling ratio. The hydrogelator has been characterized by FTIR, ¹H and ¹³C NMR spectroscopy, TGA, and FESEM analyses. The hydrogelator demonstrates pH‐responsive behavior, which has been confirmed by swelling behavior and gel characteristics at various pH (at 37 °C). Using hen egg lysozyme, degradation experiment has been performed, which confirms the biodegradable nature of the hydrogel. The in vitro cytotoxicity study and live–dead assay suggest that the hydrogelator is cytocompatible toward MG‐63 cells. Finally, the hydrogelator shows excellent efficacy as an antibiotic (amoxicillin) carrier. © 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2018 , 135, 45939.     

Solid‐state NMR characterization of cyclomaltoheptaose (β‐cyclodextrin) polymers using high‐resolution magic angle spinning with gradients

Solid‐state nuclear magnetic resonance (NMR) techniques were used to characterize cyclomaltoheptaose (β‐cyclodextrin, β‐CD) polymers. These insoluble materials have been investigated by cross‐polarization magic angle spinning with dipolar decoupling (CP/MAS), magic angle spinning without dipolar decoupling (MAS), and high‐resolution magic angle spinning with gradients (HRMAS). These NMR spectra allow the assignment of the principal ¹H and ¹³C signals. The presence of two distinct components (cross‐linked β‐CD and polymerized epichlorohydrin) in the materials was clearly demonstrated. These polymers were used as sorbents and the resulting NMR spectra are presented and discussed. © 2000 John Wiley & Sons, Inc. J Appl Polym Sci 75: 1288–1295, 2000     

PPII Helical Peptidomimetics Templated by Cation–π Interactions

Poly‐proline type II (PPII) helical PXXP motifs are the recognition elements for a variety of protein–protein interactions that are critical for cellular signaling. Despite development of protocols for locking peptides into α‐helical and β‐strand conformations, there remains a lack of analogous methods for generating mimics of PPII helical structures. We describe herein a strategy to enforce PPII helical secondary structure in the 19‐residue TrpPlexus miniature protein. Through sequence variation, we showed that a network of cation–π interactions could drive the formation of PPII helical conformations for both peptide and N‐substituted glycine peptoid residues. The achievement of chemically diverse PPII helical scaffolds provides a new route towards discovering peptidomimetic inhibitors of protein–protein interactions mediated by PXXP motifs.     

Study of novel biodegradable thermo‐sensitive hydrogels of methoxy‐poly(ethylene glycol)‐block‐polyester diblock copolymers

A series of biodegradable thermo‐sensitive hydrogels were synthesized by ring‐opening polymerization of methoxy‐poly(ethylene glycol) (mPEG) and various ester monomers, i.e. D ,L ‐lactide, glycolide, β‐propiolactone, δ‐valerolactone and ε‐caprolactone. The copolymers were characterized using ¹H NMR spectroscopy and gel permeation chromatography. The micelle properties were also measured. The results indicated that the diblock copolymers formed nano‐micelles at low concentrations in aqueous phase. The lower critical solution temperatures of the diblock copolymers were above 35 °C at 1 wt%. As the temperature increased above room temperature, the diblock copolymer solutions underwent a sol‐to‐gel phase transition, which was manifested in viscosity increases, indicative of the formation of a gel. The mPEG–polyester diblock copolymer solutions exhibited sol‐gel transition behavior as a function of temperature and polymer concentration. Copyright © 2010 Society of Chemical Industry     

Galectin‐1–Asialofetuin Interaction Is Inhibited by Peptides Containing the Tyr‐Xxx‐Tyr Motif Acting on the Glycoprotein

Galectin‐1 (Gal‐1), a ubiquitous β‐galactoside‐binding protein expressed by various normal and pathological tissues, has been implicated in cancer and autoimmune/inflammatory diseases in consequence of its regulatory role in adhesion, cell viability, proliferation, and angiogenesis. The functions of Gal‐1 depend on its affinity for β‐galactoside‐containing glycoconjugates; accordingly, the inhibition of sugar binding blocks its functions, hence promising potential therapeutic tools. The Tyr‐Xxx‐Tyr peptide motifs have been reported to be glycomimetic sequences, mainly on the basis of their inhibitory effect on the Gal‐1–asialofetuin (ASF) interaction. However, the results regarding the efficacy of the Tyr‐Xxx‐Tyr motif as a glycomimetic inhibitor are still controversial. The present STD and trNOE NMR experiments reveal that the Tyr‐Xxx‐Tyr peptides studied do not bind to Gal‐1, whereas their binding to ASF is clearly detected. ¹⁵N,¹H HSQC titrations with ¹⁵N‐labeled Gal‐1 confirm the absence of any peptide–Gal‐1 interaction. These data indicate that the Tyr‐Xxx‐Tyr peptides tested in this work are not glycomimetics as they interact with ASF via an unrevealed molecular linkage.     

Structural Studies on the Reaction of Isopenicillin N Synthase with a Sterically Demanding Depsipeptide Substrate Analogue

Isopenicillin N synthase (IPNS) is a nonheme iron(II)‐dependent oxidase that catalyses the central step in penicillin biosynthesis, conversion of the tripeptide δ‐L ‐α‐aminoadipoyl‐L ‐cysteinyl‐D ‐valine (ACV) to isopenicillin N (IPN). This report describes mechanistic studies using the analogue δ‐(L ‐α‐aminoadipoyl)‐(3S‐methyl)‐L ‐cysteine D ‐α‐hydroxyisovaleryl ester (A^SmCOV), designed to intercept the catalytic cycle at an early stage. A^SmCOV incorporates two modifications from the natural substrate: the second and third residues are joined by an ester, so this analogue lacks the key amide of ACV and cannot form a β‐lactam; and the cysteinyl residue is substituted at its β‐carbon, bearing a (3S)‐methyl group. It was anticipated that this methyl group will impinge directly on the site in which the co‐substrate dioxygen binds. The novel depsipeptide A^SmCOV was prepared in 13 steps and crystallised with IPNS anaerobically. The 1.65 Å structure of the IPNS–Fe^II–A^SmCOV complex reveals that the additional β‐methyl group is not oriented directly into the oxygen binding site, but does increase steric demand in the active site and increases disorder in the position of the isovaleryl side chain. Crystals of IPNS–Fe^II–A^SmCOV were incubated with high‐pressure oxygen gas, driving substrate turnover to a single product, an ene‐thiol/C‐hydroxylated depsipeptide. A mechanism is proposed for the reaction of A^SmCOV with IPNS, linking this result to previous crystallographic studies with related depsipeptides and solution‐phase experiments with cysteine‐methylated tripeptides. This result demonstrates that a (3S)‐methyl group at the substrate cysteinyl β‐carbon is not in itself a block to IPNS activity as previously proposed, and sheds further light on the steric complexities of IPNS catalysis.     

Synthesis and characterization of novel polyimides containing triazoles units in the main chain by click chemistry

In this study, some polyimides containing triazoles units in the main chain was prepared from the polymerization of dialkynes including imide linkages and diazides in the presence of Cu (I) catalyst in yield of 76.2–87.6%, with inherent viscosity of 0.37–0.53 dL g^?1. The obtained polymers are soluble in polar aprotic solvents such as N,N‐dimethyformamide (DMF), N,N‐dimethyacetamide (DMAc), dimethyl sulfoxide (DMSO), and N‐methyl‐2‐pyrrolidone (NMP). These polymers were characterized using FT‐IR, ¹H‐NMR, and elemental analysis techniques. Their thermal stability was evaluated with thermogravimetric (TGA) analysis and differential scanning calorimetry (DSC) techniques under a nitrogen atmosphere which is indicative of their good thermal stability. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2012     

The Role of Al in Cross‐Linking of Alkali‐Activated Slag Cements

The structural development of a calcium (sodium) aluminosilicate hydrate (C–(N‐)A–S–H) gel system, obtained through the reaction of sodium metasilicate and ground granulated blast furnace slag, is assessed by high‐resolution ²⁹Si and ²⁷Al MAS NMR spectroscopy during the first 2 yr after mixing. The cements formed primarily consist of C–(N‐)A–S–H gels, with hydrotalcite and disordered alkali aluminosilicate gels also identified in the solid product assemblages. Deconvolution of the ²⁷Al MAS NMR spectra enables the identification of three distinct tetrahedral Al sites, consistent with the ²⁹Si MAS NMR data, where Q³(1Al), Q⁴(3Al), and Q⁴(4Al) silicate sites are identified. These results suggest significant levels of cross‐linking in the C–(N‐)A–S–H gel and the presence of an additional highly polymerized aluminosilicate product. The mean chain length, extent of cross‐linking, and Al/Si ratio of the C–(N‐)A–S–H gel decrease slightly over time. The de‐cross‐linking effect is explained by the key role of Al in mixed cross‐linked/non‐cross‐linked C–(N‐)A–S–H gels, because the cross‐linked components have much lower Al‐binding capacities than the noncross‐linked components. These results show that the aluminosilicate chain lengths and chemical compositions of the fundamental structural components in C–(N‐)A–S–H gels vary in a way that is not immediately evident from the overall bulk chemistry.     

Stereochemical assignments of the nuclear magnetic resonance spectra of isobornyl acrylate/methacrylonitrile copolymers

Isobornyl acrylate (B)/methacrylonitrile (N) copolymers with different compositions were synthesized by the free‐radical bulk polymerization with azobisisobutyronitrile as the initiator under a nitrogen atmosphere at 70°C. The copolymer compositions were calculated from quantitative ¹³C(¹H)NMR spectra. The reactivity ratios of the comonomers in the B/N copolymers determined from the linear Kelen–Tudos method and nonlinear error‐in‐variable method were r_B = 0.66 ± 0.11 and r_N = 1.54 ± 0.22 and r_B = 0.74 and r_N = 1.65, respectively. The complete spectral assignments of the ¹H‐NMR and ¹³C(¹H)‐NMR spectra were carried out with the help of distortionless enhancement by polarization transfer, two‐dimensional (2D) heteronuclear single quantum coherence, and 2D total correlation spectroscopy. The nitrile carbon of the N unit and the methine and OCH carbons of the B unit were assigned to triad compositional sequences, whereas the β‐methylene carbons of the B and N units were assigned to the tetrad compositional and configurational sequences. The α‐methyl carbon of the N unit was also assigned to the triad level of configurational and compositional sequences. Similarly, the nitrile and quaternary carbon resonances with the methine, methylene, and methyl protons were studied in detail with 2D heteronuclear multiple‐bond correlation spectra. © 2012 Wiley Periodicals, Inc. J Appl Polym Sci, 2012     

Control of Electrical Resistivity in Silicon Carbide Ceramics Sintered with Aluminum Nitride and Yttria

The electrical properties of β‐SiC ceramics were found to be adjustable through appropriate AlN–Y₂O₃ codoping. Polycrystalline β‐SiC specimens were obtained by hot pressing silicon carbide (SiC) powder mixtures containing AlN and Y₂O₃ as sintering additives in a nitrogen atmosphere. The electrical resistivity of the SiC specimens, which exhibited n‐type character, increased with AlN doping and decreased with Y₂O₃ doping. The increase in resistivity is attributed to Al‐derived acceptors trapping carriers excited from the N‐derived donors. The results suggest that the electrical resistivity of the β‐SiC ceramics may be varied in the 10⁴–10^?3 Ω·cm range by manipulating the compensation of the two impurity states. The photoluminescence (PL) spectrum of the specimens was found to evolve with the addition of dopants. The presence of N‐donor and Al‐acceptor states within the band gap of 3C–SiC could be identified by analyzing the PL data.     

Extraction and characterization of lignins from maize stem and sugarcane bagasse

Lignins were isolated from maize stem and sugarcane bagasse by using mild dioxane or acidic dioxane solution. The result of nitrobenzene oxidation of the isolated lignins shows that there is a high proportion of p‐hydroxyphenyl alcohol in the lignins of maize stem and sugarcane bagasse. The lignins isolated from maize stem and sugarcane bagasse have relatively same value of the weight‐average (M _w = 3405–3868 g mol⁻¹) and number‐average (M _n = 1411–1612 g mol⁻¹) molecular weights, and polydispersity (M _w/M _n = 2.24–2.51). Acidic dioxane treatment did attack the β‐aryl ether structures in lignins, in particular for β‐aryl syringyl ethers, and broke the ester bonds between arabinose and ferulic acid that etherified to lignins, and it also cleaved lots of bonds in hemicellulosic polymer. The proportion of β‐O‐4 (threo) guaiacyl units is higher than that of β‐O‐4 (erthreo) guaiacyl units. The phenyl glycoside and benzyl ether linkages between lignin and hemicelluloses are also demonstrated in NMR analysis. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011