Design and facile production of recombinant resilin-like polypeptides: gene construction and a rapid protein purification method

Resilin is an elastic protein found in specialized regions of the cuticle of insects, which displays unique resilience and fatigue lifetime properties. As is the case with many elastomeric proteins, including elastin, gliadin and spider silks, resilin contains distinct repetitive domains that appear to confer elastic properties to the protein. Recent work within our laboratory has demonstrated that cloning and expression of exon 1 of the Drosophila melanogaster CG15920 gene, encoding a putative resilin-like protein, results in a recombinant protein that can be photochemically crosslinked to form a highly resilient, elastic biomaterial (Rec1 resilin). The current study describes a recursive cloning strategy for generating synthetic genes encoding multiple copies of consensus polypeptides, based on the repetitive domains within resilin-like genes from D. melanogaster and Anopheles gambiae. A simple non-chromatographic purification method that can be applied to these synthetic proteins and Rec1 is also reported. These methods for the design and purification of resilin-like periodic polypeptides will facilitate the future investigation of structural and functional properties of resilin, and the development of novel highly resilient biomaterials.     

Biomimetic protein‐based elastomeric hydrogels for biomedical applications

In recent years, protein‐based elastomeric hydrogels have gained increased research interest in biomedical applications for their remarkable self‐assembly behaviour, tunable 3D porous structure, high resilience (elasticity), fatigue lifetime (durability), water uptake, excellent biocompatibility and biological activity. The proteins and polypeptides can be derived naturally (animal or insect sources) or by recombinant (bacterial expression) routes and can be crosslinked via physical or chemical approaches to obtain elastomeric hydrogels. Here we review and present the recent accomplishments in the synthesis, fabrication and biomedical applications of protein‐based elastomeric hydrogels such as elastin, resilin, flagelliform spider silk and their derivatives. © 2013 Society of Chemical Industry     

Structure‐Specific Recognition of Friedreich's Ataxia (GAA)n Repeats by Benzoquinoquinoxaline Derivatives

Expansion of GAA triplet repeats in intron 1 of the FXN gene reduces frataxin expression and causes Friedreich's ataxia. (GAA)_n repeats form non‐B‐DNA structures, including triple helix H‐DNA and higher‐order structures (sticky DNA). In the proposed mechanisms of frataxin gene silencing, central unanswered questions involve the characterization of non‐B‐DNA structure(s) that are strongly suggested to play a role in frataxin expression. Here we examined (GAA)_n binding by triplex‐stabilizing benzoquinoquinoxaline (BQQ) and the corresponding triplex‐DNA‐cleaving BQQ‐1,10‐phenanthroline (BQQ‐OP) compounds. We also examined the ability of these compounds to act as structural probes for H‐DNA formation within higher‐order structures at pathological frataxin sequences in plasmids. DNA‐complex‐formation analyses with a gel‐mobility‐shift assay and sequence‐specific probing of H‐DNA‐forming (GAA)_n sequences by single‐strand oligonucleotides and triplex‐directed cleavage demonstrated that a parallel pyrimidine (rather than purine) triplex is the more stable motif formed at (GAA)_n repeats under physiologically relevant conditions.     

Design of Cyclic Peptides Featuring Proline Predominantly in the cis Conformation under Physiological Conditions

Turns are secondary‐structure elements that are omnipresent in natively folded polypeptide chains. A large variety of four‐residue β‐turns exist, which differ mainly in the backbone dihedral angle values of the two central residues i+1 and i+2. The βVI‐type turns are of particular biological interest because the i+2 residue is always a proline in the cis conformation and might thus serve as target of peptidyl prolyl cis/trans isomerases (PPIases). We have designed cyclic hexapeptides containing two proline residues that predominantly adopt the cis conformation in aqueous solution. NMR data and MD calculations indicated that the cyclic peptide sequences c‐(‐D Xaa‐Ser‐Pro‐D Xaa‐Lys‐Pro‐) result in highly symmetric backbone structures when both prolines are in the cis conformation and the D ‐amino acids are either alanine or phenylalanine residues. Replacement of the serine residue either by phosphoserine or by tyrosine compromises this symmetry, but further increases the cis conformation content of both prolines. As a result, we obtained a cyclic hexapeptide that exists almost exclusively as the cis‐Pro/cis‐Pro conformer but shows no cis/trans interconversion even in the presence of the PPIase Pin1, apparently due to an energetically quite favorable but highly restricted conformational space.     

Molecular recognition: polymers comprising isomeric meta‐/para‐isopropylphenol and N‐isopropylacrylamide and their supramolecular complexes with methylated β‐cyclodextrin

We describe the molecular recognition of polymeric attached isomers meta‐isopropylphenol and para‐isopropylphenol, respectively, in a copolymer of N‐isopropylacrylamide (NIPAAm) by use of randomly methylated β‐cyclodextrin (RAMEB‐CD). The acrylic monomers 4‐ and 3‐isopropylphenylacrylate were synthesized and radically copolymerized with NIPAAm yielding the corresponding polymers. The supramolecular structures resulting from complexation with RAMEB‐CD were characterized using dynamic light scattering, turbidity, NMR spectroscopy and isothermal titration. We found differences in binding constant, mean coil size and cloud point as a result of different complexations of the mentioned polymer‐bound isomers with RAMEB‐CD. Copyright © 2012 Society of Chemical Industry     

Connecting Active‐Site Loop Conformations and Catalysis in Triosephosphate Isomerase: Insights from a Rare Variation at Residue 96 in the Plasmodial Enzyme

Despite extensive research into triosephosphate isomerases (TIMs), there exists a gap in understanding of the remarkable conjunction between catalytic loop‐6 (residues 166–176) movement and the conformational flip of Glu165 (catalytic base) upon substrate binding that primes the active site for efficient catalysis. The overwhelming occurrence of serine at position 96 (98 % of the 6277 unique TIM sequences), spatially proximal to E165 and the loop‐6 residues, raises questions about its role in catalysis. Notably, Plasmodium falciparum TIM has an extremely rare residue—phenylalanine—at this position whereas, curiously, the mutant F96S was catalytically defective. We have obtained insights into the influence of residue 96 on the loop‐6 conformational flip and E165 positioning by combining kinetic and structural studies on the PfTIM F96 mutants F96Y, F96A, F96S/S73A, and F96S/L167V with sequence conservation analysis and comparative analysis of the available apo and holo structures of the enzyme from diverse organisms.     

Novel building blocks for oligonuclear copper complexes derived from β‐ketoenamines of histidine

Condensation products of L‐histidine with the 3‐oxoenolethers diethyl‐ethoxymethylene‐malonate ( 1 ) and ethyl‐ethoxymethylene‐cyanoacetate ( 2 ) react with copper(II) as di‐anionic ligands to give neutral 1:1 complexes Cu‐ His1 and Cu‐ His2 . Both complexes crystallize as oligonuclear units, even from strongly donating solvents like N‐methylimidazole (Meim) (Cu‐ His1 ) and pyridine (Cu‐ His2 ). X‐ray structure analyses show supramolecular structures, formed of two (Cu‐ His1 ) or four (Cu‐ His2 ) formula units of the complex, which arrange to macrocycles by means of intermolecular coordination of the imidazole‐N. Strong H‐bridges result in a face‐to‐face orientation of the hydrophilic sites of two great rings. ESI‐MS investigations in pyridine solution give evidence for the existence of dimeric, tetrameric and – in case of Cu‐ His2 – trimeric units, besides the monomeric adducts with one pyridine. In contrast to the dimeric or tetrameric (“cubane‐like”) copper(II) complexes of amino alcohols and their β‐ketoenamines, the complexes Cu‐ His1 and Cu‐ His2 show no significant spin coupling from room temperature down to 4 K. The complexes Cu‐ His1 and Cu‐ His2 give no electrochemically reversible Cu^II/I reduction in pyridine. However, the isolation of a stable diamagnetic copper(I) complex of the methylester derivative, Cu^I‐ HisMe1 , supports the assumption, that similar histidine‐derived copper complexes should display reversible redox behaviour and catalytic activity in reactions with O₂.     

Exclusively Heteronuclear 13C‐Detected Amino‐Acid‐Selective NMR Experiments for the Study of Intrinsically Disordered Proteins (IDPs)

Carbon‐13 direct‐detection NMR methods have proved to be very useful for the characterization of intrinsically disordered proteins (IDPs). Here we present a suite of experiments in which amino‐acid‐selective editing blocks are encoded in CACON‐ and CANCO‐type sequences to give ¹³C‐detected spectra containing correlations arising from a particular type or group of amino acid(s). These two general types of experiments provide the complementary intra‐ and inter‐residue correlations necessary for sequence‐specific assignment of backbone resonance frequencies. We demonstrate the capabilities of these experiments on two IDPs: fully reduced Cox17 and WIP^C. The proposed approach constitutes an independent strategy to simplify crowded spectra as well as to perform sequence‐specific assignment, thereby demonstrating its potential to study IDPs.     

Evaluation of β‐Amino Acid Replacements in Protein Loops: Effects on Conformational Stability and Structure

β‐Amino acids have a backbone that is expanded by one carbon atom relative to α‐amino acids, and β residues have been investigated as subunits in protein‐like molecules that adopt discrete and predictable conformations. Two classes of β residue have been widely explored in the context of generating α‐helix‐like conformations: β³‐amino acids, which are homologous to α‐amino acids and bear a side chain on the backbone carbon adjacent to nitrogen, and residues constrained by a five‐membered ring, such the one derived from trans‐2‐aminocyclopentanecarboxylic acid (ACPC). Substitution of α residues with their β³ homologues within an α‐helix‐forming sequence generally causes a decrease in conformational stability. Use of a ring‐constrained β residue, however, can offset the destabilizing effect of α→β substitution. Here we extend the study of α→β substitutions, involving both β³ and ACPC residues, to short loops within a small tertiary motif. We start from previously reported variants of the Pin1 WW domain that contain a two‐, three‐, or four‐residue β‐hairpin loop, and we evaluate α→β replacements at each loop position for each variant. By referral to the ?,ψ angles of the native structure, one can choose a stereochemically appropriate ACPC residue. Use of such logically chosen ACPC residues enhances conformational stability in several cases. Crystal structures of three β‐containing Pin1 WW domain variants show that a native‐like tertiary structure is maintained in each case.     

Defining the Potential of Aglycone Modifications for Affinity/Selectivity Enhancement against Medically Relevant Lectins: Synthesis,Activity Screening,and HSQC‐Based NMR Analysis

The emerging significance of lectins for pathophysiological processes provides incentive for the design of potent inhibitors. To this end, systematic assessment of contributions to affinity and selectivity by distinct types of synthetic tailoring of glycosides is a salient step, here taken for the aglyconic modifications of two disaccharide core structures. Firstly we report the synthesis of seven N‐linked‐lactosides and of eight O‐linked N‐acetyllactosamines, each substituted with a 1,2,3‐triazole unit, prepared by copper‐catalyzed azide–alkyne cycloaddition (CuAAC). The totally regioselective β‐D ‐(1→4) galactosylation of a 6‐O‐TBDPSi‐protected N‐acetylglucosamine acceptor provided efficient access to the N‐acetyllactosamine precursor. The resulting compounds were then systematically tested for lectin reactivity in two binding assays of increasing biorelevance (inhibition of lectin binding to a surface‐presented glycoprotein and to cell surfaces). As well as a plant toxin, we also screened the relative inhibitory potential with adhesion/growth‐regulatory galectins (total of eight proteins). This type of modification yielded up to 2.5‐fold enhancement for prototype proteins, with further increases for galectins‐3 and ‐4. Moreover, the availability of ¹⁵N‐labeled proteins and full assignments enabled ¹H,¹⁵N HSQC‐based measurements for hu‐ man galectins‐1, ‐3, and ‐7 against p‐nitrophenyl lactopyranoside, a frequently tested standard inhibitor containing an aromatic aglycone. The measurements confirmed the highest affinity against galectin‐3 and detected chemical shift differences in its hydrophobic core upon ligand binding, besides common alterations around the canonical contact site for the lactoside residue. What can be accomplished in terms of affinity/selectivity by this type of core extension having been determined, the applied combined strategy should be instrumental for proceeding with defining structure–activity correlations at other bioinspired sites in glycans and beyond the tested lectin types.     

Quasi‐alternating polyesteramides from ε‐caprolactone and α‐amino acids

Glycine‐ɛ‐caprolactone‐based and α‐alanine‐ɛ‐caprolactone‐based polyesteramides with a strong tendency to form alternating sequences (degree of randomness = 1.64 and 1.31) were synthesized by melt polycondensation of intermediate hydroxy‐ and ethyl ester‐terminated amides. These intermediates were synthesized by the reaction of equimolar amounts of ɛ‐caprolactone and glycine or L‐α‐alanine ethyl esters in mild conditions. The structure and microstructure of these polyesteramides are discussed on the basis of an in‐depth nuclear magnetic resonance study. Both polyesteramides are semi‐crystalline, but the glycine‐based one presents the highest melting enthalpy. This polyesteramide also exhibits higher Young's modulus and stress at break than its α‐ and β‐alanine counterparts. © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016 , 133, 44220.     

Bioengineering functional copolymers: V. Synthesis,LCST, and thermal behavior of poly(N‐isopropyl acrylamide‐co‐p‐vinylphenylboronic acid)

New boron‐containing stimuli‐responsive (pH‐ and temperature‐sensitive) copolymers were synthesized and characterized. Structure and composition of copolymers were determined by FTIR and ¹H‐NMR spectroscopy, and elemental analysis and titration (N and B contents for NIPA and VPBA unit, respectively). By DSC and XRD measurements, it is established that the synthesized copolymers have a semicrystalline structure due to formation of intra‐ and/or intermolecular H‐bonded supramolecular architecture. The copolymer composition–structure–property relationship indicates semicrystalline structure of copolymers with different compositions, degrees of crystallinity, and thermal and stimuli‐responsive behaviors depends on the content of boron‐containing monomer linkage. Results of DSC, DTA, and TGA analyses indicated that copolymers have T_g and T_m and high thermal stability. These water‐soluble and temperature‐ and pH‐sensitive amphiphilic copolymers can be used as polymeric carries for delivery of biological entities for diverse biomedical use, including boron neutron capture therapy. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 95: 573–582, 2005     

Influence of NaCl on Granulometric Characteristics and Polymorphism in Batch‐Cooling Crystallization of Glycine

The effect of NaCl added in different quantities on thermodynamic properties, granulometric characteristics, and structure of glycine in a crystallization process was investigated. Solubilities of α‐ and γ‐polymorphs in the presence of varying amounts of NaCl were analyzed. In order to examine the impact of the additive on granulometric properties of glycine, crystal morphology was examined by observing crystals under a scanning electron microscope. Crystal size distribution was determined by sieve analysis. By X‐ray diffraction analysis, the critical concentration of NaCl at which the structure of glycine changed, could be defined. The purity of obtained polymorphs was confirmed by Fourier transform infrared spectroscopy.     

Biotransformation of (+)‐ and (−)‐bornyl acetate using the plant parasitic fungus Glomerella cingulata as a biocatalyst

The microbial transformations of (+)‐ and (?)‐bornyl acetate were investigated using the plant parasitic fungus, Glomerella cingulata. As a result, (+)‐ and (?)‐bornyl acetate were converted to (+)‐ and (?)‐5‐exo‐hydroxybornyl acetate, (+)‐ and (?)‐5‐oxobornyl acetate and (+)‐ and (?)‐borneol respectively. The structures of the metabolic products were determined by spectroscopic data. © 2001 Society of Chemical Industry     

Fully bio‐based poly(ɛ‐capolactone)/poly(lactide) alternating multiblock supramolecular polymers: Synthesis,crystallization behavior,and properties

Supramolecular poly(?‐capolactone)/poly(lactide) alternating multiblock copolymers were prepared by UPy‐functionalized poly(lactide)‐b‐ poly(?‐capolactone)‐b‐ poly(lactide) copolymers. The prepared supramolecular polymers (SMPs) exhibit the characteristic properties of thermoplastic elastomers. The stereo multiblock SMPs (sc‐SMPs) were formed by blending UPy‐functionalized poly(l ‐lactide)‐b‐ PCL‐b‐ poly(l ‐lactide) (l ‐SMPs) and UPy‐functionalized poly(d ‐lactide)‐b‐ PCL‐b‐ poly(d ‐lactide) (d ‐SMPs) due to stereocomplexation of the PLLA and PDLA blocks. Sc‐SMPs with low content of d ‐SMPs (≤20%) are transparent, elastic solids, while those having high d ‐SMPs content are opaque, brittle solids. The effects of l ‐SMPs/d ‐SMPs mixing ratios on thermal, crystallization behaviors, crystal structure, mechanical and hydrophilic properties of sc‐SMPs were deeply investigated. The incorporation of UPy groups depresses the crystallization of polymer, and the stereocomplex formation accelerates the crystallization rate. The used initiator functionalized polyhedral oligomeric silsesquioxanes causes a different effect on the crystallization of PLA and PCL blocks. The tensile strength and elongation at break of l _d /d _d ‐SMPs (d represents the initiator diethylene glycol) are significantly larger than that of l _p /d _p ‐SMPs (p represents the initiator polyhedral oligomeric silsesquioxanes), and their heat resistance and hydrophilicity can be also modulated by the l ‐SMPs/d ‐SMPs mixing ratios and the different initiators. © 2017 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2017 , 134 , 45575.     

Improving Binding Affinity and Stability of Peptide Ligands by Substituting Glycines with D‐Amino Acids

Improving the binding affinity and/or stability of peptide ligands often requires testing of large numbers of variants to identify beneficial mutations. Herein we propose a type of mutation that promises a high success rate. In a bicyclic peptide inhibitor of the cancer‐related protease urokinase‐type plasminogen activator (uPA), we observed a glycine residue that has a positive ? dihedral angle when bound to the target. We hypothesized that replacing it with a D ‐amino acid, which favors positive ? angles, could enhance the binding affinity and/or proteolytic resistance. Mutation of this specific glycine to D ‐serine in the bicyclic peptide indeed improved inhibitory activity (1.75‐fold) and stability (fourfold). X‐ray‐structure analysis of the inhibitors in complex with uPA showed that the peptide backbone conformation was conserved. Analysis of known cyclic peptide ligands showed that glycine is one of the most frequent amino acids, and that glycines with positive ? angles are found in many protein‐bound peptides. These results suggest that the glycine‐to‐D ‐amino acid mutagenesis strategy could be broadly applied.     

Light‐induced cooperative electron–proton transfer in hydrogen‐bonded networks of N,N‐diaryl substituted 1,4‐bisimines and meso‐1,2‐diaryl‐1,2‐ethanediols

The 1:1 cocrystallization of 1,4‐diaryl‐1,4‐bisimines (Ar–CHN–CH₂‐)₂ 4 – 11 and substituted meso‐1,2‐diaryl‐1,2‐ethanediols 1 – 3 leads to supramolecular structures in which the diol is hydrogen bonded by one of its hydroxy groups to an imine nitrogen atom of a 1,4‐bisimine. The second functionality in each molecule leads to the generation of ladderlike polymeric structures where each molecule of the diol is linked to two molecules of the 1,4‐bisimine and vice versa. If the diol carries electron donor groups in the aromatic residue and the 1,4‐bisimine correspondingly acceptor groups, then charge transfer interactions are observed. The excited CT complex which corresponds to a radical ion pair is stabilized by migration of a proton of a hydroxy group to the nitrogen atom of an imino group. This is supported by the appearance of a N–H vibration in the IR spectra. The reorganization is also accompanied by changes in the UV/Vis spectra and by the generation of paramagnetism in the crystalline material. The results represent a type of photochromism which has its origin in a light‐induced cooperative electron–proton transfer. The photochromism is thermally reversible.     

Acetylcholine Promotes Binding of α‐Conotoxin MII at α3β2 Nicotinic Acetylcholine Receptors

α‐Conotoxin MII (α‐CTxMII) is a 16‐residue peptide with the sequence GCCSNPVCHLEHSNLC, containing Cys2–Cys8 and Cys3–Cys16 disulfide bonds. This peptide, isolated from the venom of the marine cone snail Conus magus, is a potent and selective antagonist of neuronal nicotinic acetylcholine receptors (nAChRs). To evaluate the impact of channel–ligand interactions on ligand‐binding affinity, homology models of the heteropentameric α₃β₂‐nAChR were constructed. The models were created in MODELLER with the aid of experimentally characterized structures of the Torpedo marmorata‐nAChR (Tm‐nAChR, PDB ID: 2BG9) and the Aplysia californica‐acetylcholine binding protein (Ac‐AChBP, PDB ID: 2BR8) as templates for the α₃‐ and β₂‐subunit isoforms derived from rat neuronal nAChR primary amino acid sequences. Molecular docking calculations were performed with AutoDock to evaluate interactions of the heteropentameric nAChR homology models with the ligands acetylcholine (ACh) and α‐CTxMII. The nAChR homology models described here bind ACh with binding energies commensurate with those of previously reported systems, and identify critical interactions that facilitate both ACh and α‐CTxMII ligand binding. The docking calculations revealed an increased binding affinity of the α₃β₂‐nAChR for α‐CTxMII with ACh bound to the receptor, and this was confirmed through two‐electrode voltage clamp experiments on oocytes from Xenopus laevis. These findings provide insights into the inhibition and mechanism of electrostatically driven antagonist properties of the α‐CTxMIIs on nAChRs.     

C3‐Symmetrical π‐Scaffolds: Useful Building Blocks to Construct Helical Supramolecular Polymers

In this review, we highlight some relevant examples of C₃‐symmetrical molecules that have been reported to form supramolecular polymers and helical aggregates. In particular, the number and type of non‐covalent forces are key to bias the supramolecular polymerization leading from a simple isodesmic or cooperative mechanism to a more complex self‐assembly process, i. e. pathway complexity. Furthermore, the attachment of stereogenic centres at the peripheral side chains of the C₃‐systems provokes efficient transfer and amplification of chirality phenomena when directional and specific non‐covalent interactions operate. Interestingly, the incorporation of hydrophilic side chains induces the formation of organized aggregates in aqueous media with potential biomedical applications. Overall, the examples shown in this review on C₃‐symmetrical scaffolds illustrate the relevance of this molecular shape in the development of functional supramolecular structures.