Residue Polypeptide Research Articles

Autoantibody binding and unique enzyme-substrate intermediate conformation of human transglutaminase 3

Transglutaminase 3 (TG3), the autoantigen of dermatitis herpetiformis (DH), is a calcium dependent enzyme that targets glutamine residues in polypeptides for either transamidation or deamidation modifications. To become catalytically active TG3 requires proteolytic cleavage between the core domain and two C-terminal β-barrels (C1C2). Here, we report four X-ray crystal structures representing inactive and active conformations of human TG3 in complex with a TG3-specific Fab fragment of a DH patient derived antibody. We demonstrate that cleaved TG3, upon binding of a substrate-mimicking inhibitor, undergoes a large conformational change as a β-sheet in the catalytic core domain moves and C1C2 detaches. The unique enzyme-substrate conformation of TG3 without C1C2 is recognized by DH autoantibodies. The findings support a model where B-cell receptors of TG3-specific B cells bind and internalize TG3-gluten enzyme-substrate complexes thereby facilitating gluten-antigen presentation, T-cell help and autoantibody production.

On the plasticity of amyloid formation: The impact of destabilizing small to large substitutions on islet amyloid polypeptide amyloid formation.

Amyloids are partially ordered, proteinaceous, β-sheet rich deposits that have been implicated in a wide range of diseases. An even larger set of proteins that do not normally form amyloid in vivo can be induced to do so in vitro. A growing number of structures of amyloid fibrils have been reported and a common feature is the presence of a tightly packed core region in which adjacent monomers pack together in extremely tight interfaces, often referred to as steric zippers. A second common feature of many amyloid fibrils is their polymorphous nature. We examine the consequences of disrupting the tight packing in amyloid fibrils on the kinetics of their formation using the 37 residue polypeptide hormone islet amyloid polypeptide (IAPP, amylin) as a model system. IAPP forms islet amyloid in vivo and is aggressively amyloidogenic in vitro. Six Cryo-EM structures of IAPP amyloid fibrils are available and in all Gly24 is in the core of the structured region and makes tight contacts with other residues. Calculations using the ff14SBonlysc forcefield in Amber20 show that substitutions with larger amino acids significantly disrupt close packing and are predicted to destabilize the various fibril structures. However, Gly to 2-amino butyric acid (2-carbon side chain) and Gly to Leu substitutions actually enhance the rate of amyloid formation. A Pro substitution slows, but does not prevent amyloid formation.

Revealing the role of tunable amino acid residues in elastin-like polypeptides (ELPs)-mediated biomimetic silicification

Elastin-like polypeptides (ELPs) are attractive materials for the green preparation of silica nanoparticles via biomimetic silicification. However, the critical factors affecting the ELP-mediated silicification remain unclear. Herein, the role of tunable amino acid residues of ELPs in silicification was studied using three ELPs (ELPs[V9F-40], ELPs[KV8F-40], and ELPs[K5V4F-40]) and their fusion proteins (ELPs[V9F-40]-SpyCatcher, ELPs[KV8F-40]-SpyCatcher, and ELPs[K5V4F-40]-SpyCatcher) with different contents of lysine residues. Bioinformatics methods were employed for the first time to reveal the key physicochemical parameters correlated with silicification. The specific activity of ELPs was increased with the promotion of lysine content with a high correlation coefficient (R = 0.899). Furthermore, exogenous acidic protein SpyCatcher would hinder the interactions between the silica precursors and ELPs, leading to the significantly decrease in specific activity. The isoelectric point (pI) of ELPs presented the highest correlation to silicification with a coefficient of 0.963. The charges of the ELPs [K5V4F-40] at different pH were calculated based on the sequence or structure. Interestingly, the excellent correlation between charges based on structure and specific activity was obtained. Collectively, the novel methods developed here may pave a new way for rational design of ELPs or other peptides for efficient and green preparation of silica nanomaterials for biomedicine, biocatalysis, and biosensor.

Tagging Peptides with a Redox Responsive Fluorescent Probe Enabled by Photoredox Difunctionalization of Phenylacetylenes with Sulfinates and Disulfides.

Herein, we describe a photoredox three-component atom-transfer radical addition (ATRA) reaction of aryl alkynes directly with dialkyl disulfides and alkylsulfinates, circumventing the utilization of chemically unstable and synthetically challenging S-alkyl alkylthiosulfonates as viable addition partners. A vast array of (E)-β-alkylsulfonylvinyl alkylsulfides was prepared with great regio- and stereoselectivity. Moreover, this powerful tactic could be employed to tag cysteine residues of complex polypeptides in solution or on resin merging with solid phase peptide synthesis (SPPS) techniques. A sulfonyl-derived redox responsive fluorescent probe could be conveniently introduced on the peptide, which displays green fluorescence in cells while showing blue fluorescence in medium. The photophysical investigations reveal that the red shift of the emission fluorescence is attested to reduction of carbonyl group to the corresponding hydroxyl moiety. Interestingly, the fluorescence change of tagged peptide could be reverted in cells by treatment of H2O2, arising from the reoxidation of hydroxyl group back to ketone by the elevated level of reactive oxygen species (ROS).

Neuropeptide Y Antagonizes Development of Pulmonary Fibrosis through IL-1β Inhibition.

Neuropeptide Y (NPY), a 36 amino acid residue polypeptide distributed throughout the nervous system, acts on various immune cells in many organs, including the respiratory system. However, little is known about its role in the pathogenesis of pulmonary fibrosis. This study was performed to determine the effects of NPY on pulmonary fibrosis. NPY-deficient and wild-type mice were intratracheally administered bleomycin. Inflammatory cells, cytokine concentrations, and morphological morphometry of the lungs were analyzed. Serum NPY concentrations were also measured in patients with idiopathic pulmonary fibrosis and healthy control subjects. NPY-deficient mice exhibited significantly enhanced pulmonary fibrosis and higher IL-1β concentrations in the lungs compared with wild-type mice. Exogenous NPY treatment suppressed the development of bleomycin-induced lung fibrosis and decreased IL-1β concentrations in the lungs. Moreover, IL-1β neutralization in NPY-deficient mice attenuated the fibrotic changes. NPY decreased IL-1β release, and Y1 receptor antagonists inhibited IL-1β release and induced epithelial-mesenchymal transition in human alveolar epithelial cells. Patients with idiopathic pulmonary fibrosis had lower NPY and greater IL-1β concentrations in the serums compared with healthy control subjects. NPY expression was mainly observed around bronchial epithelial cells in human idiopathic pulmonary fibrosis lungs. These data suggest that NPY plays aprotective role against pulmonary fibrosis by suppressing IL-1β release, and manipulating the NPY-Y1 receptor axis could be a potential therapeutic strategy for delaying disease progression.

Exploring Nearest Neighbor Interactions and Their Influence on the Gibbs Energy Landscape of Unfolded Proteins and Peptides.

The Flory isolated pair hypothesis (IPH) is one of the corner stones of the random coil model, which is generally invoked to describe the conformational dynamics of unfolded and intrinsically disordered proteins (IDPs). It stipulates, that individual residues sample the entire sterically allowed space of the Ramachandran plot without exhibiting any correlations with the conformational dynamics of its neighbors. However, multiple lines of computational, bioinformatic and experimental evidence suggest that nearest neighbors have a significant influence on the conformational sampling of amino acid residues. This implies that the conformational entropy of unfolded polypeptides and proteins is much less than one would expect based on the Ramachandran plots of individual residues. A further implication is that the Gibbs energies of residues in unfolded proteins or polypeptides are not additive. This review provides an overview of what is currently known and what has yet to be explored regarding nearest neighbor interactions in unfolded proteins.

Molecular characterization and SNPs association with growth‐related traits of myosin heavy chains from the pearl oyster Pinctada fucata martensii

Myosin heavy chains (MYHs) are important proteins in muscle growth and contraction, which may potentially enhance shellfish growth. In this study, the MYH gene from Pinctada fucata martensii (PmMYH) was studied. Sequence analysis revealed that it has an open reading frame of 5823 bp that encode 1940 polypeptide residues. Analysis of PmMYH showed four conserved domains, namely Myosin_N, MYSc, IQ and Myosin_tail_1. Blast analysis confirmed that PmMYH has a high identity with MYH protein sequences from Crassostrea virginica (87.25%), C. gigas (86.15%), Biomphalaria glabrata (78.8%), Pecten maximus (77.15%), Mizuhopecten yessoensis (75.78%) and Argopecten irradians (77.30%). Expression pattern analysis of PmMYH demonstrated that it is widely expressed in tissues and developmental stages, especially in the adductor muscles and late development stage. Furthermore, PmMYH also expressed significantly higher in individuals selected for fast growth compared with those selected for slow growth, thereby suggesting its role on growth-related traits. Ninety-five single nucleotide polymorphisms (SNPs) were identified in the exon region of PmMYH, and 18 of which were significantly associated with growth (p < 0.05). Of the 18 SNPs, 2 were also verified in two independent populations. Therefore, PmMYH and its SNPs could be used as for the gene-assisted selection of P. f. martensii.

An account on the factors determining the extra stability of the β–hairpin from B1 domain of protein G

The folding–unfolding of a 16 residue polypeptide, a β–hairpin in B1 domain of protein G is investigated here to account for the factors assisting the extra stability of the polypeptide in the presence of an explicit solvent and even when a denaturant like urea is present in the medium. It is observed here that the backbone H–bond network well defines the folded state and is even capable of forming the folded state, but it is not the only criteria for making a stable β–hairpin fold. Factors such as the side chain H–bonds and the alignment of the certain hydrophobic group side chains play a prominent role in preserving the β–hairpin structure and thus providing an extra stability to the hairpin architecture. It is also affirmed that the mentioned hydrophobic groups side chain interactions are very crucial in holding the β–hairpin together and without which the hairpin collapses completely. We also confirm that the denaturant urea acts on the GB1–hairpin backbone H–bonds and in the presence of strong hydrophobic interactions with a consistent side chain H–bonding network, the denaturation being comparatively a slower process with respect to the protein devoid of the side chain interactions. Communicated by Ramaswamy H. Sarma

In a mouse model of INCL reduced S‐palmitoylation of cytosolic thioesterase APT1 contributes to microglia proliferation and neuroinflammation

S-palmitoylation is a reversible posttranslational modification in which a 16-carbon saturated fatty acid (generally palmitate) is attached to specific cysteine residues in polypeptides via thioester linkage. Dynamic S-palmitoylation (palmitoylation-depalmitoylation), like phosphorylation-dephosphorylation, regulates the function of numerous proteins, especially in the brain. While a family of 23 palmitoyl-acyl transferases (PATS), commonly known as ZDHHCs, catalyze S-palmitoylation of proteins, the thioesterases, localized either in the cytoplasm (eg, APT1) or in the lysosome (eg, PPT1) mediate depalmitoylation. Previously, we reported that APT1 requires dynamic S-palmitoylation for shuttling between the cytosol and the plasma membrane. APT1 depalmitoylated H-Ras to regulate its signaling pathway that stimulates cell proliferation. Although we demonstrated that APT1 catalyzed its own depalmitoylation, the ZDHHC(s) that S-palmitoylated APT1 had remained unidentified. We report here that ZDHHC5 and ZDHHC23 catalyze APT1 S-palmitoylation. Intriguingly, lysosomal Ppt1-deficiency in Cln1-/- mouse, a reliable animal model of INCL, markedly reduced ZDHHC5 and ZDHHC23 levels. Remarkably, in the brain of these mice decreased ZDHHC5 and ZDHHC23 levels suppressed membrane-bound APT1, thereby, increasing plasma membrane-localized H-Ras, which activated its signaling pathway stimulating microglia proliferation. Increased inflammatory cytokines produced by microglia together with increased complement C1q level contributed to the transformation of astrocytes to neurotoxic A1 phenotype. Importantly, neuroinflammation was ameliorated by treatment of Cln1-/- mice with a PPT1-mimetic small molecule, N-tert(Butyl)hydroxylamine (NtBuHA). Our results revealed a novel pathway to neuropathology in an INCL mouse model and uncovered a previously unrecognized mechanism of the neuroprotective actions of NtBuHA and its potential as a drug target.

Maximizing Blad-containing oligomer fungicidal activity in sweet cultivars of Lupinus albus seeds

During seed germination and plantlet growth, an important aspect of Lupinus β-conglutin proteolysis is the accumulation of blad in the cotyledons. Blad, a 20.4 kDa, 173 residue polypeptide which inhibits fungal growth, is the main subunit of BCO (Blad-containing oligomer), a Lupinus bioactive polypeptide oligomer, which underwent a successful translational research during the last thirty years. The development of this recent broad-spectrum biological fungicide for plant disease control made evident the advantage of being non-toxic to the environment, plants, humans and other animals, an interesting characteristic given the increasing consumer’s concern about food safety. The industrial-scale production of the edible fungicide BCO for agricultural purposes involves germinating and growing for ca. 8 days massive amounts of Lupinus plantlets. Therefore, it becomes economically relevant to maximize/increment the amount of extractable BCO. Although BCO represents an alternative pathway for lupin production in Europe, there is no previous evidence on lupin cultivars concerning BCO activity or its cotyledonary concentration, and therefore on the most promising cultivars for BCO extraction and fungicide production. In this work, the amount of BCO and its level of fungicide activity was evaluated in seven sweet cultivars of L. albus (cvs. Amiga, Energy, Estoril, Ludic, Misak, Multitalia and Rumbo), along 20 days after the onset of germination. In addition, four distinct lots of cv. Energy and six lots of cv Misak, with different harvest years (1997, 2010, 2013 and 2014) and/or different sites of production in Portugal (Alto Alentejo, Baixo Alentejo and Beira Litoral) were also analysed. Quantitative assays demonstrated maximal accumulation of BCO in the cotyledons of 4-days-old L. albus plantlets, apart from significant differences between seeds’ harvest year or production site. The assays also showed that cv. Energy had significant higher accumulation of BCO (31.67 μg mg−1 wet weight) than Rumbo (25.67 μg mg−1) and Misak (22.12 μg mg−1), respectively second and third cultivars with highest accumulation of the oligomer. Additionally, very significant differences were also observed among seeds’ harvest year or production site. These observations reduced dramatically the original purpose of this work on the study of BCO variations at the level of sweet L. albus cultivars, since the ‘noise’ detected in BCO levels caused by the year and location of their production was far greater than its variation among cultivars. The observation that storing for several years the seeds at -20 °C did not seem to decrease BCO activity and suggests that the variation in cotyledonary BCO accumulation is essentially controlled by the prevailing edaphoclimatic conditions during seed formation. Furthermore, the activity tests exhibited significant antifungal activity between 3 and 5 days-old plantlets, with absence of antifungal activity on 8 days-old plantlets or older. Overall this study is of considerable importance to maximize BCO extraction from the cotyledons of sweet L. albus cultivars.

Multivalent Elastin-Like Glycopolypeptides: Subtle Chemical Structure Modifications with High Impact on Lectin Binding Affinity.

A library of synthetic elastin-like glycopolypeptides were synthesized and screened by microscale thermophoresis to identify key structural parameters affecting lectin binding efficacy. While polypeptide backbone size and glycovalency were found to have little influence, the presence of a linker at the anomeric position of galactose and the absence of positive charge on the polypeptide residue holding the sugar unit were found to be critical for the binding to RCA120.

Serine-Selective Bioconjugation.

This Communication reports the first general method for rapid, chemoselective, and modular functionalization of serine residues in native polypeptides, which uses a reagent platform based on the P(V) oxidation state. This redox-economical approach can be used to append nearly any kind of cargo onto serine, generating a stable, benign, and hydrophilic phosphorothioate linkage. The method tolerates all other known nucleophilic functional groups of naturally occurring proteinogenic amino acids. A variety of applications can be envisaged by this expansion of the toolbox of site-selective bioconjugation methods.

Diurnal expression of circadian clock genes period 1 and period 3 in Pelteobagrus vachellii

Circadian clock genes are crucial for generating and sustaining most rhythmic daily functions in the animal kingdom, which entrain the rhythms of biochemical, physiological, and behavioural processes. To better understand the molecular oscillations of the circadian rhythms in darkbarbel catfish (Pelteobagrus vachellii), we isolated and characterized two circadian clock genes in P. vachellii, period 1 (per1), and period 3 (per3). The circadian clock gene per1 was found to encode a 1 428-amino acid polypeptide, including PER-ARNT-SIM (PAS) dimerisation domains, a PAS-associated C-terminal motif (PAC), a short mutable domain (S/M), and a nuclear export signal (NES). The 4 902-bp per3 cDNA includes an open reading frame encoding a 1 292-amino acid residue polypeptide with a PER-ARNT-SIM (PAS) domain, cytoplasmic localisation domain (CLD), interaction site (TIS), and a nuclear localisation signal (NLS). The per1 and per3 gene was constitutively expressed in all examined tissues. Moreover, per1 expression within a light/dark cycles showed rhythmic expression in the diencephalon, brain, liver and intestine, with the acrophase at 15:15, 12:52, 7:51, and 12:55, respectively. Daily expression of per3 was rhythmic in the diencephalon, brain, liver and intestine, with the acrophase at 8:15, 9:54, 10:39, and 10:25 h, respectively. These findings expand our understanding of circadian mechanism at the molecular level in this species.

Characterisation of a novel cold-adapted calcium-activated transglutaminase: implications for medicine and food processing.

Transglutaminases are a family of enzymes that catalyse the cross‐linking of proteins by forming covalent bonds between lysine and glutamine residues in various polypeptides. Cross‐linking reactions are involved in blood clots, skin formation, embryogenesis and apoptosis. Clinically, these enzymes appear to be implicated in neurodegenerative diseases, tumours and coeliac diseases. Transglutaminases have great potential for use in the food industry because of their ability to cross‐link proteins that are not normally linked. Here, a gene coding for transglutaminase from Atlantic cod was cloned into a bacterial expression vector and used to transform protein expression in a strain of Escherichia coli. The successful expression of recombinant transglutaminase protein from Atlantic cod (AcTG‐1) as a soluble protein upon induction at low temperature was confirmed by sodium dodecyl sulfate/polyacrylamide gel electrophoresis, immunoblotting and mass spectrometry analysis. Biochemical characterisation demonstrated that the transglutaminase was active between 0 and 65 °C, but was completely inactivated after 20‐min incubation at 70 °C. Interestingly, the enzyme displayed cold‐adapted features, such as temperature instability combined with high catalytic efficiency at low temperatures (8–16 °C). In addition, the enzyme had optimal activity at 50 °C, a new feature for a cold‐adapted enzyme. AcTG‐1 was active in the pH range from 6 to 9, with an optimum at pH 8, and required 5 mm calcium for maximum activity. Potential calcium‐binding sites in the enzyme were predictable, making the enzyme an appropriate model for studying structure–function relationships in the calcium‐dependent transglutaminase family. In vitro gel analysis revealed that transglutaminase cross‐linked casein, collagen and gelatin. The binding of fish fillets in the presence of recombinant AcTG‐1 provided further macroscopic proof for the potential application of AcTG‐1 as a biological cross‐linker in the food industry. Once binding occurred, fish fillets withstood further processing such as frying, boiling, freeze‐thawing and chilling. The low‐temperature activity and new enzymatic properties of AcTG‐1 appear to offer advantages over commercially available enzymatic glues in the food industry.

Differential effects of serine side chain interactions in amyloid formation by islet amyloid polypeptide.

Islet amyloid polypeptide (IAPP), a 37 residue polypeptide, is the main protein component of islet amyloid deposits produced in the pancreas in Type 2 diabetes. Human IAPP contains five serine residues at positions 19, 20, 28, 29, and 34. Models of the IAPP amyloid fibril indicate a structure composed of two closely aligned columns of IAPP monomers with each monomer contributing to two intermolecular β-strands. Ser 19 and Ser 20 are in the partially ordered β-turn region, which links the two strands, whereas Ser 28, Ser 29, and Ser 34 are in the core region of the amyloid fibril. Ser 29 is involved in contacts between the two columns of monomers and is the part of the steric zipper interface. We undertook a study of individual serine substitutions with the hydrophobic isostere 2-aminobutyric acid (2-Abu) to examine the site-specific role of serine side chains in IAPP amyloid formation. All five variants formed amyloid. The Ser 19 to 2-Abu mutant accelerates amyloid formation by a factor of 3 to 4, while the Ser 29 to 2-Abu mutation modestly slows the rate of amyloid formation. 2-Abu replacements at the other sites had even smaller effects. The data demonstrate that the cross-column interactions made by residue 29 are not essential for amyloid formation and also show that cross-strand networks of hydrogen-bonded Ser side chains, so called Ser-ladders, are not required for IAPP amyloid formation. The effect of the Ser 19 to 2-Abu mutant suggests that residues in this region are important for amyloid formation by IAPP.

In silico analysis of κ-theraphotoxin-Cg2a from Chilobrachys guangxiensis

κ-theraphotoxin-Cg2a is a 29- residue polypeptide extracted from the venomous glands of the Chinese earth tiger tarantula Chilobrachys guangxiensis. Plethoras of cancers are being associated with irregular functions of potassium ion channels. An extensive understanding of the toxin’s interaction with the voltage-gated potassium channels is of utmost necessity for it to be screened as a potential pharmacological molecule which may perhaps serve as toxin-based therapy to manage various cancer channelopathies. Physicochemical properties were studied, the evolutionary analysis was done to visualize the conserved domain among different toxins of tarantula family, docking studies between κ-theraphotoxin-Cg2a and a voltage-gated potassium ion channel was done by ClusPro 2.0. The presence of signal peptide was observed using PSIPRED. Cysteine – disulfide bonds present in the amino acid sequence was predicted by DiANNA server. Multiple sequence alignment illustrated conserved residues with other families of tarantula’s toxin. The docking of κ-theraphotoxin-Cg2a with the voltage-gated potassium channel was found to be interactive. The presence of cysteine –disulfide bonds were observed potentially playing a crucial role in the docking process. The interaction between the receptor and the ligand was found to be interactive which could turn out to help develop strategies to assist in creating potential pharmacological drug-based therapies.

HSA-interaction studies of uranyl complexes of alkyl substituted isothiosemicarbazone

Interaction between Human Serum Albumine (HSA) and six related uranyl complexes of alkyl substituted (methyl, ethyl, allyl, butyl, pentyl and benzyl) isothiosemicarbazone with general formula [UO2L(sovent)] are examined experimentally and theoretically. In order to determine the effects of changing the length of alkyl groups and their consequent differences in hydrophobicity in HSA interaction, UV–Vis, fluorescence and circular dichroism spectroscopies are utilized as probes. The results show that the complexes quench the fluorescence emission of HSA significantly and induce unfolding of the polypeptide of HSA through converting part of the α-helical content to β-structures and random coil. Docking results obtained from Autodock Vina software indicates that the complexes enter the cleft between three domains of HSA during their interaction. Experimental and theoretical data show that hydrophobic forces play an important role in the interaction of the complexes with HSA. It also appears that some hydrogen bonds between some residues of polypeptide with the complexes could be formed. Finally, it becomes clarified that changing alkyl groups does not represent significant alteration in the interaction of uranyl complexes with HSA and the other structural parameters of complexes have more decisive role in HSA-interaction.

B52 promotes alternative splicing of Dscam in Chinese mitten crab, Eriocheir sinensis

B52 is a member of the classical serine/arginine (SR)-rich proteins, which are phylogenetically conserved and play significant roles in mRNA maturation, including alternative splicing. In the present study, the docking site, selector sequences and locus control region of the Chinese mitten crab (Eriocheir sinensis) Down syndrome cell adhesion molecule (EsDscam) were identified. Alternative splicing of Dscam is essential to generate different isoforms. We also isolated and characterised the B52 gene from E. sinensis (EsB52). The 876 bp open reading frame of EsB52 encodes a 291 amino acid residue polypeptide, and EsB52 has two RNA recognition motifs (RRMs) at the N-terminus and an arginine/serine-rich domain at the C-terminus. Each RRM contains two degenerate short submotifs, RNP-1 and RNP2. Analysis of tissue distribution revealed that EsB52 mRNA expression was widespread in all tested tissues, and especially high in brain and hemocytes. In hemocytes, EsB52 was upregulated significantly after stimulation with pathogen-associated molecular patterns and bacteria. Furthermore, EsB52 RNAi decreased the number of Ig7 inclusion in mRNA rather than Ig2 or Ig3. Taken together, these findings suggest that EsB52 acts as an alternative splicing activator of EsDscam.

Single-Molecule Discrimination of Labeled DNAs and Polypeptides Using Photoluminescent-Free TiO2 Nanopores.

Multicolor fluorescence substantially expands the sensing capabilities of nanopores by complementing or substituting the resistive pulsing signals. However, to date single-fluorophore detection in multiple color channels has proven to be challenging primarily due to high photoluminescence (PL) emanating from the silicon nitride (SiN x) membrane. We hypothesize that the high bandgap of titanium oxide (TiO2) would eliminate the PL background when used as a substrate for a nanopore, and hence enable individual fluorophore sensing during the fast passage of biomolecules through the pore. Herein, we introduce a method for fabricating locally supported, free-standing, TiO2 membranes, in which solid-state nanopores can be readily drilled. These devices produce essentially no PL in the blue-to-red visible spectral range, even when excited by multiple lasers simultaneously. At the same time, the TiO2 nanopores exhibit low electrical noise comparable with standard SiN x devices. Importantly, the optical signal-to-background ratio (SBR) in single-molecule sensing is improved by an order of magnitude, enabling the differentiation among labeled DNA molecules of similar length based solely on their labeling scheme. Finally, the increased SBR of the TiO2 devices allows detection of single fluorophores conjugated to the lysine or cysteine residues of short polypeptides, thus introducing the possibility for optical based peptide/protein discrimination in nanopores.

Characterization and functional analysis of serpin-28 gene from silkworm, Bombyx mori

Serine protease inhibitors (Serpins) are a broadly distributed superfamily of proteins with a SERPIN domain and participate in several immune responses. In this study, a serpin-28 gene was identified in B. mori and its role in immune regulation was investigated. This gene has an open reading frame of 1065 bp that encodes a 354-amino acid residue polypeptide containing one SERPIN domain with a predicted molecular weight of 40.3 kDa. Recombinant Bmserpin-28 protein was expressed in Escherichia coli and used to raise rabbit anti-Bmserpin-28 polyclonal antibodies. Quantitative real-time PCR analysis revealed that Bmserpin-28 was expressed in all examined tissues, with maximum expression in the fat body and silk gland. Expression pattern of different developmental stages showed that the highest expression level was in the pupae, while the lowest expression level was recorded at the egg stage. After challenge with four different microorganisms (Escherichia coli, Beauveria bassiana, Micrococcus luteus and B. mori nuclear polyhedrosis virus), the expression pattern of Bmserpin-28 was investigated in fat body and haemocyte samples. A substantial upregulation of Bmserpin-28 expression level was recorded following pathogen challenge in both the tested tissues. Furthermore, RNA interference of Bmserpin-28 resulted in significant upregulation of antimicrobial peptide genes. In summary, our results indicated that Bmserpin-28 may be involved in the innate immunity of B. mori.