Bioengineered Chimeric Spider Silk‐Uranium Binding Proteins

Heavy metals constitute a source of environmental pollution. Here, novel functional hybrid biomaterials for specific interactions with heavy metals are designed by bioengineering consensus sequence repeats from spider silk of Nephila clavipes with repeats of a uranium peptide recognition motif from a mutated 33‐residue of calmodulin protein from Paramecium tetraurelia. The self‐assembly features of the silk to control nanoscale organic/inorganic material interfaces provides new biomaterials for uranium recovery. With subsequent enzymatic digestion of the silk to concentrate the sequestered metals, options can be envisaged to use these new chimeric protein systems in environmental engineering, including to remediate environments contaminated by uranium.

     

Oxopiperazine capping: Formation of oxopiperazine-containing peptoids via C-terminal cyclization

An unexpected side reaction was observed in peptoids containing a C-terminal carboxamide with a 2-aminoethyl side chain. The reaction proceeded via cyclization and release of NH₃, resulting in C-terminal oxopiperazine formation, analogous to pyroglutamate formation from N-terminal glutamine in peptides. Reaction conditions that promote oxopiperazine formation were developed. In particular, the addition of organic bases accelerated the cyclization, thus providing a simple strategy for the post-synthetic C-terminal capping of peptoids.     

Spidroin-Inspired,High-Strength,Loofah-Shaped Protein Fiber for Capturing Uranium from Seawater

The unique three-dimensional structure of spidrion determines the outstanding mechanical properties of the spider silk fiber. Inspired by the similarity of the three-dimensional structure of superb-uranyl binding protein (SUP) to that of spidroin, a dual-SUP (DSUP) chimeric protein fiber with high tensile strength is designed. The DSUP hydrogel fiber exhibits a loofah-shape structure by the cross-interaction of the protein nanofiber. Full exposure of abundant functional uranyl-binding sites in the stretchable loofah-shape hydrogel protein fiber give the DSUP fiber a groundbreaking uranium extraction capacity of 17.45 mg g⁻¹ with an ultrashort saturation time of 3 days in natural seawater. This work reports the design of an adsorbent with ultrahigh uranium extraction capacity and explores a strategy for fabricating artificial high-strength functional non-spidroin protein fiber.     

Synthesis of a secretoglobin 3A2 type C (98–139) fragment by Dawson’s native chemical ligation

A secretoglobin 3A2 type C (98–139) peptide was synthesized by native chemical ligation between ¹¹⁵Ile and ¹¹⁶Cys residues using Dawson’s linker. The peptide-N-acyl-benzimidazolinone-glycine amide, a C-terminal thioesters precursor, was provided from 3-amino-4-(methylamino)benzoic acid. In addition, an N-terminal cysteine fragment, the (116–139) peptide, was prepared by ordinary Fmoc-solid phase peptide synthesis. Native chemical ligation of the (98–115) fragment with the Dawson’s linker and the (116–139) peptide smoothly proceeded to give SCGB3A2 type C (98–139) peptide.     

Silk-based nanocomplexes with tumor-homing peptides for tumor-specific gene delivery

Nanoscale complexes of recombinant silk molecules containing THPs with DNA are designed as less cytotoxic and highly target‐specific gene carriers. Genetically engineered silk proteins containing poly(L ‐lysine) domains to interact with pDNA and the THP to bind to specific tumorigenic cells for target‐specific pDNA delivery are prepared, followed by in vitro transfection into MDA‐MB‐435 melanoma cells, highly metastatic human breast tumor MDA‐MB‐231 cells, and non‐tumorigenic MCF‐10A breast epithelial cells. The silk/poly(L ‐lysine) block copolymer containing Lyp1 (ML‐Lyp1) shows significant differences from silk/poly(L ‐lysine) block copolymer containing F3 (ML‐F3) in cytotoxicity to MCF10A cells. ML‐F3 is the most promising candidate for target delivery into tumorigenic cells.

     

Spidroin‐Inspired,High‐Strength,Loofah‐Shaped Protein Fiber for Capturing Uranium from Seawater

The unique three‐dimensional structure of spidrion determines the outstanding mechanical properties of the spider silk fiber. Inspired by the similarity of the three‐dimensional structure of superb‐uranyl binding protein (SUP) to that of spidroin, a dual‐SUP (DSUP) chimeric protein fiber with high tensile strength is designed. The DSUP hydrogel fiber exhibits a loofah‐shape structure by the cross‐interaction of the protein nanofiber. Full exposure of abundant functional uranyl‐binding sites in the stretchable loofah‐shape hydrogel protein fiber give the DSUP fiber a groundbreaking uranium extraction capacity of 17.45 mg g^?1 with an ultrashort saturation time of 3 days in natural seawater. This work reports the design of an adsorbent with ultrahigh uranium extraction capacity and explores a strategy for fabricating artificial high‐strength functional non‐spidroin protein fiber.     

Aptamer-mediated turn-on fluorescence assay for prion protein based on guanine quenched fluophor

An aptamer-participated haprin structure was designed by employing cellular prion protein (PrP^C) as a model protein, and thus an aptamer-mediated turn-on fluorescence assay for proteins was developed in this contribution. The designed aptamer-participated haprin structure consists of three segments. Namely, an aptamer sequence located in the loop, three guanine bases at 3′-terminal, and a fluophor modified at 5′-terminal. It was found that the guanine bases at the 3′-terminal could quench the fluorescence of the fluophor such as tetramethyl-6-carboxyrhodamine (TAMRA) at the 5′-terminal about 76.6% via electron transfer if the guanine bases are close enough to the fluophor, and the quenched fluorescence could get restored when the target protein is present since the interaction, which could be confirmed by measuring fluorescence lifetime, between TAMRA-aptamer and the target protein forces the guanines away from TAMRA so that TAMRA-modified aptamer changes into turn-on state. A linear relationship was then constructed between the turn-on fluorescence intensity and the concentration of PrP^C in the range from 1.1 to 44.7 μg/mL with a limit of detection of 0.3 μg/mL (3σ).     

铁和锰对桑蚕丝蛋白构象转变的影响

采用电感耦合等离子体质谱和原子吸收光谱对桑蚕丝腺体和蚕茧丝中Fe和Mn的含量进行表征, 同时采用拉曼光谱研究Fe(III)和Mn(II)对高浓度再生桑蚕丝蛋白水溶液中丝蛋白构象的影响. 研究结果表明, 桑蚕丝纤维中Fe的含量高于其在丝腺体中的含量, 而Mn的含量则相反. 在丝腺体中, Fe的含量从丝腺体的后部到前部逐渐增加; 但Mn含量的变化趋势与Fe不同, 它在丝腺体中部的含量最低. Fe(III)能够诱导丝蛋白的构象由无规线团和/或螺旋结构向β-折叠转变, 但是Mn(II)对丝蛋白的构象没有明显的影响.     

蜘蛛与蚕的人工强制纺丝研究进展

为制备高性能人造蜘蛛丝和蚕丝,人们在蜘蛛丝蛋白的基因重组、再生蜘蛛丝蛋白或蚕丝蛋白的仿生纺丝,以及蜘蛛与蚕的人工强制纺丝方面开展了大量工作.研究了人工强制纺丝,比较了蜘蛛牵引丝和蚕丝在不同纺丝条件下的力学性能.结果表明:在一定范围内增加纺丝速率有利于提高蜘蛛丝和蚕丝的力学性能,此外,环境温度、纺丝状态等条件对丝的性能也...     

A comparison of the peptide fragmentation obtained from a reflector matrix-assisted laser desorption-ionization time-of-flight and a tandem four sector mass spectrometer

The types, extent, and overall distribution of peptide fragmentation produced by matrix-assisted laser desorption-ionization-postsource decay (MALDI-PSD) on a reflector time-of-flight mass spectrometer were compared with those obtained from high and low energy collision-induced dissociation (CID) on a four-sector mass spectrometer and from liquid secondary ion mass spectrometry (LSIMS) ion source fragmentation and LSIMS metastable ion (MI) decomposition on a two-sector mass spectrometer. The model peptides studied had sequences and compositions that yielded predominantly either N- or C-terminal fragmentation from CID. For des-Arg¹ and des-Arg⁹ bradykinin (i.e., H-PPGFSPFR-OH and H-RP-PGFSPF-OH, respectively), the types of fragment ions and the extent to which each type is formed in both MALDI-PSD and low energy CID spectra are remarkably similar. This observation suggests that both methods deposit comparable internal energies (IE) into [M + H]⁺ precursor ions. The distribution of N-terminal, C-terminal, immonium, and internal fragmentation from MALDI-PSD spectra of des-Arg¹ and des-Arg⁹ bradykinin did not change dramatically with respect to the terminal arginine position, contrary to those from LSIMS MI decomposition, high and low energy CID spectra. This observation in combination with the prominent immonium, internal, and minus 17 fragment ion types in PSD indicates that the imparted IE from MALDI and the 14 µs of flight time may promote steady-state decomposition kinetics. Fragmentation distributions of MALDI-PSD spectra are also similar to those in LSIMS spectra. This implies that the distribution of protonation sites in [M + H]⁺ is comparable for both techniques.     

Changes in Amino Acid Profiles and Bioactive Compounds of Thai Silk Cocoons as Affected by Water Extraction

Silk proteins have many advantageous components including proteins and pigments. The proteins—sericin and fibroin—have been widely studied for medical applications due to their good physiochemical properties and biological activities. Various strains of cocoon display different compositions such as amino-acid profiles and levels of antioxidant activity. Therefore, the objectives of this study were to find a suitable silk protein extraction method to obtain products with chemical and biological properties suitable as functional foods in two strains of Bombyx mori silk cocoon (Nangsew strains; yellow cocoon) and Samia ricini silk cocoon (Eri strains; white cocoon) extracted by water at 100 °C for 2, 4, 6 and 8 h. The results showed that Nangsew strains extracted for 6 h contained the highest amounts of protein, amino acids, total phenolics (TPC) and total flavonoids (TFC), plus DPPH radical-scavenging activity, ABTS radical scavenging capacity, and ferric reducing antioxidant power (FRAP), anti-glycation, α-amylase and α-glucosidase inhibition. The longer extraction time produced higher concentrations of amino acids, contributing to sweet and umami tastes in both silk strains. It seemed that the bitterness decreased as the extraction time increased, resulting in improvements in the sweetness and umami of silk-protein extracts.     

Asymmetric epoxidation of electron-poor olefins-V : Influence on stereoselectivity of the structure of poly-α-aminoacids used as catalysts

New poly-α-aminoacids modified at the C orN-terminal groups are synthetised and employed in the asymmetric epoxidation of chalcone. Their influence on the stereoselectivity of the reaction is studied.     

Effects of side-chains ofN-acetylamino acids on the structures of their triphenyltin(IV) complexes

Triphenyltin(IV) complexes ofN-acetylglycine,N-acetyl-L-leucine,N-acetyl-L-asparagine andN-acetyl-L-tyrosine were prepared by two methods and characterized by means of different spectroscopic methods (FTIR, multinuclear,¹H,¹³C and¹¹⁹Sn NMR and¹¹⁹Sn Mössbauer). The spectroscopic data indicated that theN-acetylglycine complex adopts a trigonal-bipyramidal structure in which the monodentate carboxylate and the amide-C=O group are bound to the same organotin(IV) moiety. The other three complexes are linear oligomers in which the planar Ph₃Sn(IV) is coordinated axially by a monodentate carboxylate and an amide-C=O from two different ligands. At theC-terminal end of the oligomer chain there is a tetracoordinated tin(IV) with a monodentate carboxylate as donor group.     

Synthetic adhesive attachment discs inspired by spider's pyriform silk architecture

Spiders attach their major ampullate silk dragline fibers to surfaces using attachment discs spun from pyriform silk fibers. These attachment discs allow spiders to move safely from place to place while secured to a dragline and to attach their webs to a variety of surfaces. Here, we demonstrate a simple electrospinning process to mimic the “staple‐pin” architecture used by spiders for these attachment discs. Experimental and theoretical evidence are provided to confirm the advantages of thousands of micron‐size “staple‐pins” and their low peeling angles to enhance the adhesive forces required to peel the natural and synthetic attachment discs. These results provide a unique architectural design for fabricating new adhesives that uses very little material for various biomedical and material science applications. © 2014 Wiley Periodicals, Inc. J Polym Sci Part B: Polym. Phys. 2014 , 52, 553–560     

Selective determination of N-terminal tyrosine containing peptides by a novel fluorescence reaction with borate,hydroxylamine and cobalt(II)

A fluorimetric method is proposed for determining N-terminal tyrosine-containing peptides, of which some peptides such as enkephalins and kyotorphin are of physiological importance. An intense fluorescence is produced when the peptide is heated at 100°C for 3 min in a weakly alkaline medium containing borate, hydroxylamine and cobalt(II). The fluorescent species is stabilized with β-mercaptoethanol, with excitation and emission maxima at 335 and 430 nm, respectively. The method is highly selective for N-terminal tyrosine-containing peptides, with a detection limit of 43–69 pmol ml^?1.     

Tunable Anticancer Activity of Furoylthiourea-Based RuII–Arene Complexes and Their Mechanism of Action

Fourteen new Ru^II–arene (p-cymene/benzene) complexes ( C1 – C14 ) have been synthesized by varying the N-terminal substituent in the furoylthiourea ligand and satisfactorily characterized by using analytical and spectroscopic techniques. Electrostatic potential maps predicted that the electronic effect of the substituents was mostly localized, with some influence seen on the labile chloride ligands. The structure–activity relationships of the Ru–p-cymene and Ru–benzene complexes showed opposite trends. All the complexes were found to be highly toxic towards IMR-32 cancer cells, with C5 (Ru–p-cymene complex containing C₆H₂(CH₃)₃ as N-terminal substituent) and C13 (Ru–benzene complex containing C₆H₄(CF₃) as N-terminal substituent) showing the highest activity among each set of complexes, and hence they were chosen for further study. These complexes showed different behavior in aqueous solutions, and were also found to catalytically oxidize glutathione. They also promoted cell death by apoptosis and cell cycle arrest. Furthermore, the complexes showed good binding ability with the receptors Pim-1 kinase and vascular endothelial growth factor receptor 2, commonly overexpressed in cancer cells.     

Novel enantiopure non-C2-symmetric NCN-pincer palladium complexes with l-proline chiral auxiliaries: mer η3-N,C,N versus square planar η4-N,C,N,O coordination

New chiral NCN-pincer palladium complexes containing proline ester moieties as chiral auxiliaries have been synthesized. The parent ligands 2,6-bis{[(S)-2-(methoxycarbonyl)-1-pyrrolidinyl]methyl}-1-bromobenzene L^Me and 2,6-bis{[(S)-2-(benzoxycarbonyl)-1-pyrrolidinyl]methyl}-1-bromobenzene L^Bn were prepared in a single synthetic step and were obtained enantiomerically pure. Neutral arylpalladium bromide complexes 1a and 1b, formed upon treatment of the respective ligands L^Me and L^Bn with [Pd₂(dba)₃]·CHCl₃, were isolated as mixtures of three stereoisomers (S_NS_NS_CS_C, R_NS_NS_CS_C and R_NR_NS_CS_C). The ratio of stereoisomers is approximately 1:1:0.6 in the case of methyl ester derivative 1a, whereas the bulkier benzyl ester derivative 1b predominantly forms the (S_NS_NS_CS_C)-stereoisomer. Upon abstraction of the bromide ion from unresolved mixtures of 1a and 1b, cationic complexes 2 and 3, respectively, form as single diastereoisomers in which one of the ester prolinate carbonyl groups is coordinated to palladium according to X-ray crystal structure determination. This coordination of a carbonyl group to the metal has a substantial influence on the stereochemistry and results in the formation of a single diastereoisomer, having the (R_NR_NS_CS_C)-configuration, regardless of the stereochemistry or ratio of stereoisomers of the starting bromide compound. The structures of compounds 2 and 3 were somewhat unexpected since formation of the corresponding cationic [Pd(NCN)(OH₂)]⁺ complexes was anticipated. In preliminary tests of these cationic complexes as catalysts in the enantioselective aldol condensation of benzaldehyde with methyl isocyanoacetate, modest selectivities were observed.     

Novel fragmentation of N-diisopropyloxyphosphoryl dipeptides and tripeptides by fast atom bombardment mass spectrometry

Positive ion fast atom bombardment mass spectrometry of N-diisopropyloxyphosphoryl dipeptides and tripeptides showed a novel cleavage pattern in that only the N-phosphoryl fragment ions gave intense peaks while the C-terminal series of ions was suppressed. The base peak was the N-phosphoryl imino ion responding to the N-terminal residue. These advantages are superior to those of other types of N-protecting groups.     

Reinforcing Silk Scaffolds with Silk Particles

Silk fibroin is a useful protein polymer for biomaterials and tissue engineering. In this work, porogen leached scaffolds prepared from aqueous and HFIP silk solutions were reinforced through the addition of silk particles. This led to about 40 times increase in the specific compressive modulus and the yield strength of HFIP‐based scaffolds. This increase in mechanical properties resulted from the high interfacial cohesion between the silk matrix and the reinforcing silk particles, due to partial solubility of the silk particles in HFIP. The porosity of scaffolds was reduced from ≈90% (control) to ≈75% for the HFIP systems containing 200% particle reinforcement, while maintaining pore interconnectivity. The presence of the particles slowed the enzymatic degradation of silk scaffolds.

     

In Vivo Biological Responses to Silk Proteins Functionalized with Bone Sialoprotein

Recombinant 6mer + BSP protein, combining six repeats of the consensus sequence for Nephila clavipes dragline (6mer) and bone sialoprotein sequence (BSP), shows good support for cell viability and induces the nucleation of hydroxyapatite and tricalcium phosphate during osteoblast in vitro culture. The present study is conducted to characterize this bioengineered protein‐based biomaterial further for in vivo behavior related to biocompatibility. 6mer + BSP protein films are implanted in subcutaneous pouches in the back of mice and responses are evaluated by flow cytometry and histology. The results show no major differences between the inflammatory responses induced by 6mer + BSP films and the responses observed for the controls. Thus, this new chimeric protein could represent an alternative for bone regeneration applications.