Amino Acid Sequence Of Peptide Research Articles

Optimisation and Characterisation of Novel Angiotensin-Converting Enzyme Inhibitory Peptides Prepared by Double Enzymatic Hydrolysis from Agaricus bisporus Scraps.

Food-derived hypotensive peptides have attracted attention in the field of active peptide research in recent years. In this study, based on ACE inhibition rate and using the Box–Behnken central combination design principle to optimise the process of ACE inhibitor peptides prepared by double-enzyme hydrolysis. The amino acid sequences of ACE inhibitor peptides were determined by liquid chromatography mass spectrometry (LC-MS/MS), and their binding to ACE was studied by molecular docking. The optimal processing conditions were 1:1 alkaline protease: compound protease, pH was 8.43, enzymolysis temperature was 44.32 °C, and enzymolysis time was 3.52 h. Under these conditions, the ACE inhibition rate reached 65.12%, and the inhibition rate after separation and purification was 80.68% (IC50 = 0.9 mg/mL). Three novel peptides with ACE inhibitory activity were detected by LC-MS/MS, with sequences LVYP (Leu-Val-Tyr-Pro), VYPW(Val-Tyr-Pro-Trp) and YPWT(Tyr-Pro-Trp-Thr). Molecular docking revealed that the three novel peptides all established hydrogen bonds with the S1(Tyr523, Glu384, Ala354) and S2 (His353) pockets of ACE. Among them, LVYP, VYPW and YPWT, respectively, formed eleven hydrogen bonds, six hydrogen bonds and nine hydrogen bonds with ACE. The study revealed that these peptides have the potential for the development of novel ACE inhibitor drugs and provide a new avenue for high-value utilisation of mushrooms scraps.

Neuroprotective and antioxidant capability of RW20 peptide from histone acetyltransferases caused by oxidative stress-induced neurotoxicity in in vivo zebrafish larval model

Oxidative stress causes disruption of cell macromolecules and resulting neurodegeneration when the antioxidant defense mechanism is inhibited. Oxidative stress is emerging as a key therapeutic target for nerve agent toxicity. Therefore, antioxidant treatment methods are a novel and effective approach to attenuate oxidative stress and neurodegeneration. This study evaluated the neuroprotective and antioxidant ability of a short peptide, RW20 derived from histone acetyltransferases (HATs).Using bioinformatics tools, the short amino acid sequence of RF13 peptide with antioxidant activity was predicted from HAT of Channa striatus transcriptome. The synthesized antioxidant peptide at various concentrations (10–50 μM) was analyzed in in vitro and in vivo antioxidant assay. The neuroprotective effect of RF13 peptide was evaluated in H2O2 stressed zebrafish larvae.The results showed the neuroprotective effect by restoring antioxidant enzymes including SOD, CAT, GPx, GSH, and GST treated with RW20. In addition, the oxidative stress indicator of lipid peroxidation was gradually declined with an increase in the concentration of peptides. It was also shown to attenuate neuronal damage by inhibiting nitric oxide overproduction, thus increasing AChE activity. Further, to validate the neuroprotective effect of RW20, the intracellular ROS level and apoptosis in the zebrafish larvae were analyzed by incubating the larvae along with RW20, which decreased intracellular ROS level and reduced the cell death.Overall, the study demonstrated that RW20 possessed prominent antioxidant activity. Hence, we conclude that RW20 can be developed as a potent therapeutic agent against oxidative stress-induced neurodegenerative diseases.

Recent findings on the cellular and molecular mechanisms of action of novel food-derived antihypertensive peptides

Hypertension impacts negatively on the quality of life of sufferers, and complications associated with uncontrolled hypertension are life-threatening. Hence, many research efforts are exploring the antihypertensive properties of bioactive peptides derived from food proteins using in vitro ACE-inhibitory assay, experimentally-induced and spontaneous hypertensive rats, normotensive and hypertensive human models. In this study, the cellular and molecular mechanisms of blood pressure-lowering properties of novel peptides reported in recent studies (2015-July 30, 2021) were discussed. In addition to common mechanisms such as the inhibition of angiotensin I-converting enzyme (ACE) and renin activities, recently recognized mechanisms through which bioactive peptides exert their antihypertensive properties including the induction of vasodilation via upregulation of cyclo-oxygenase (COX) and prostaglandin receptor and endothelial nitric oxide synthase expression and L-type Ca2+ channel blockade were presented. Similarly, emerging mechanisms of blood pressure-lowering by bioactive peptides such as modulation of inflammation (TNF-α, and other cytokines signaling), oxidative stress (Keap-1/Nrf2/ARE/HO-1 and related signaling pathways), PPAR-γ/caspase3/MAPK signaling pathways and inhibition of lipid accumulation were discussed. The review also highlighted factors that influence the antihypertensive properties of peptides such as method of hydrolysis (type and number of enzymes, and chemical used for hydrolysis, and microbial fermentation), and amino acid sequence and chain length of peptides.

Sesquiterpene Cyclases from the Basidiomycete Cerrena unicolor

Hundreds of terpenoids have been isolated from Basidiomycota, among them are volatile mono- and sesquiterpenes with amazing sensory qualities, representing a promising alternative to essential oils from endangered plant species. Sesquiterpene synthases (STS) appear to be an abundant class of enzymes in these fungi. The basidiomycete Cerrena unicolor, a known sesquiterpene producer, was in silico screened for sesquiterpene cyclases via homology Basic Local Alignment Search Tool searches. Cyclase genes identified were cloned and heterologously expressed in Escherichia coli Bl21 using pCOLD I as the expression vector. Ten cyclases were successfully produced and purified, and their identity was confirmed using amino acid sequencing of tryptic peptides by nano-liquid chromatography-high resolution-electrospray ionization-tandem mass spectrometry. Gas chromatography/mass spectrometry analysis was applied to characterize these cyclases according to the formation of sesquiterpene hydrocarbons and oxidized terpenoids. Bioinformatic characterization and phylogenetic determination allowed for the classification of these diverse fungal enzymes. A representative single and a multi-product STS, respectively, were further analyzed for their dependency from divalent metal cations as a cofactor for the catalytic activity.

NPCdc, a synthetic natriuretic peptide, is a substrate to neprilysin and enhances blood pressure-lowering induced by enalapril in 5/6 nephrectomized rats

NPCdc is a natriuretic peptide synthesized from the amino acid sequence of the Crotalus durissus cascavella snake venom peptide, NP2Casca. NPCdc presents hypotensive and antioxidants effects. This study aimed to investigate in vivo whether angiotensin I-converting enzyme (ACE) inhibition would influence the impact of NPCdc in arterial pressure of rats submitted to 5/6 nephrectomy (Nx). Adult male Wistar rats following a 5/6 Nx were treated with enalapril (NxE group, 10 mg/kg/day, n = 9) or vehicle (Nx group, n = 8) for two weeks. On the 15th day after Nx, rats were anaesthetized and submitted to mean arterial pressure (MAP) determination before and after receiving two intravenous injections of saline (vehicle, n = 9) or NPCdc (0.3 μg/kg dissolved in saline, n = 18) separated by a 20-min interval. The kidneys were submitted to oxidative stress analysis. The basal MAP of the NxE group was nearly 20% lower (P < 0.05) than non-treated rats. NPCdc administration decreased the MAP in both groups; however, in the NxE group, the effects were observed only in the second injection. The peptide also decreased the NADPH oxidase activity in the renal cortex. Additionally, the hydrolysis of NPCdc by recombinant neprilysin (NEP) was monitored by mass spectrometry. NPCdc was cleaved by NEP at different peptides with an inhibition constant (Ki) of 1.5 μM, determined by a competitive assay using the NEP fluorescence resonance energy transfer (FRET) peptide substrate Abz-(d)Arg-Gly-Leu-EDDnp. Docking experiments confirmed the high affinity of NPCdc toward NEP. These findings provide new insights into the antihypertensive and antioxidant mechanism of action of NPCdc. Altogether, the results presented here suggest that NPCdc must be further studied as a potential therapy for cardiorenal syndromes.

Coordination of di-Histidine-containing hexapeptides with cupric ion and its application in electrochemical detection

The lone pair electrons of nitrogen in imidazole ring enable His residue served as an anchor to bind metal ion or as a connector to regulate peptide-metal ions self-assembly. In this paper, a series of hexapeptides with two His residues of different locations in the sequences were synthesized and their coordination performance with Cu2+ were explored. The stoichiometric ratio, binding ability and coordination structures of the peptides with Cu2+ in aqueous solutions were characterized with isothermal titration calorimetry (ITC), 1H nuclear magnetic resonance (1H NMR), ultraviolet visible (UV–Vis) and visible circular dichroism (Vis-CD) spectroscopies. Furthermore, the di-His-containing peptides were immobilized on gold electrodes to detect Cu2+ through the high-efficiency complexation. The results indicated that the binding ability and coordination structures of di-His-containing peptides with Cu2+ were determined by the sequence. GHGGGH showed the highest binding constant with Cu2+ and formed a stable 4 N complex of {NH2, N-, 2Nim}. GHGGGH modified electrodes had the highest sensitivity in the electrochemical detection of Cu2+ in aqueous solutions at neutral pH. This work provides us with a better understanding of the interaction of functional amino acids in peptide sequences with metal ions and offers constructive guidance in designing metal ion binding peptides.

Phage-Phenotype Imaging of Myeloma Plasma Cells by Phage Display

Multiple myeloma (MM) is a malignant disease based on differentiated plasma cells (PCs) in the bone marrow (BM). Flow cytometry and fluorescence microscopy, used to identify a large combination of clusters of differentiation (CDs), are applied for MM immunophenotyping. However, due to the heterogeneous MM immunophenotypes, more antibody panels are necessary for a preliminary diagnosis and for the monitoring of minimal residual disease (MRD). In this study, we evaluated the use of phage clones as probes for the identification of several PCs immunophenotypes from MM patients. First, A 9-mer M13-pVIII phage display library was screened against an MM.1 cells line to identify peptides that selectively recognize MM.1 cells. Then, the most representative phage clones, with amino acid sequences of foreign peptides closer to the consensus, were labelled with isothiocyanate of fluorescein (FITC) and were used to obtain a fluorescent signal on cells in ex-vivo samples by fluorescence microscopy. Selected phage clones were able to discriminate different MM immunophenotypes from patients related to CD45, CD38, CD56, and CD138. Our results highlight the possibility of using a phage-fluorescence probe for the simultaneous examination of the presence/absence of CDs associated with disease usually detected by combination of anti-CD antibodies. The design of a multi-phage imaging panel could represent a highly sensitive approach for the rapid detection of immunophenotype subtypes and the subsequent characterization of patient disease status.

Exploiting of Secondary Raw Materials from Fish Processing Industry as a Source of Bioactive Peptide-Rich Protein Hydrolysates.

Developing peptide-based drugs are very promising to address many of the lifestyle mediated diseases which are prevalent in a major portion of the global population. As an alternative to synthetic peptide-based drugs, derived peptides from natural sources have gained a greater attention in the last two decades. Aquatic organisms including plants, fish and shellfish are known as a rich reservoir of parent protein molecules which can offer novel sequences of amino acids in peptides, having unique bio-functional properties upon hydrolyzing with proteases from different sources. However, rather than exploiting fish and shellfish stocks which are already under pressure due to overexploitation, the processing discards, regarded as secondary raw material, could be a potential choice for peptide based therapeutic development strategies. In this connection, we have attempted to review the scientific reports in this area of research that deal with some of the well-established bioactive properties, such as antihypertensive, anti-oxidative, anti-coagulative, antibacterial and anticarcinogenic properties, with reference to the type of enzymes, substrate used, degree of particular bio-functionality, mechanism, and wherever possible, the active amino acid sequences in peptides. Many of the studies have been conducted on hydrolysate (crude mixture of peptides) enriched with low molecular bioactive peptides. In vitro and in vivo experiments on the potency of bioactive peptides to modulate the human physiological functions beneficially have demonstrated that these peptides can be used in the prevention and treatment of non-communicable lifestyle mediated diseases. The information synthesized under this review could serve as a point of reference to drive further research on and development of functionally active therapeutic natural peptides. Availability of such scientific information is expected to open up new zones of investigation for adding value to underutilized secondary raw materials, which in turn paves the way for sustainability in fish processing. However, there are significant challenges ahead in exploring the fish waste as a source of bioactive peptides, as it demands more studies on mechanisms and structure–function relationship understanding as well as clearance from regulatory and statutory bodies before reaching the end user in the form of supplement or therapeutics.

Properties of peptides released from salmon and carp via simulated human-like gastrointestinal digestion described applying quantitative parameters.

Apart from the classical (experimental) methods, biologically active peptides can be studied via bioinformatics approach, also known as in silico analysis. This study aimed to verify the following research hypothesis: ACE inhibitors and antioxidant peptides can be released from salmon and carp proteins during simulated in silico human-like gastrointestinal digestion. The potential to release biopeptides was evaluated using the BIOPEP-UWM quantitative criteria including the profile of biological activity, frequency of the occurrence (A)/release (AE) of fragments with an ACE inhibitory or antioxidant activity by selected enzymes, and relative frequency of release of bioactive fragments with a given activity by selected enzymes (W). Salmon collagen and myofibrillar proteins of carp turned out to be the best potential source of the searched peptides–ACE inhibitors and antioxidant peptides. Nonetheless, after digestion, the highest numbers of ACE inhibitors and antioxidant peptides were potentially released from the myofibrillar proteins of salmon and carp. Peptide Ranker Score, Pepsite2, and ADMETlab platform were applied to evaluate peptides’ bioactivity potential, their safety and drug-like properties. Among the 63 sequences obtained after the simulated digestion of salmon and carp proteins, 30 were considered potential biopeptides. The amino acid sequences of ACE-inhibiting and antioxidant peptides were predominated by P, G, F, W, R, and L. The predicted high probability of absorption of most analyzed peptides and their low toxicity should be considered as their advantage.

Non-canonical proline-tyrosine interactions with multiple host proteins regulate Ebola virus infection.

The Ebola virus VP30 protein interacts with the viral nucleoprotein and with host protein RBBP6 via PPxPxY motifs that adopt non-canonical orientations, as compared to other proline-rich motifs. An affinity tag-purification mass spectrometry approach identified additional PPxPxY-containing host proteins hnRNP L, hnRNPUL1, and PEG10, as VP30 interactors. hnRNP L and PEG10, like RBBP6, inhibit viral RNA synthesis and EBOV infection, whereas hnRNPUL1 enhances. RBBP6 and hnRNP L modulate VP30 phosphorylation, increase viral transcription, andexert additive effects on viral RNA synthesis. PEG10 has more modest inhibitory effects on EBOV replication. hnRNPUL1 positively affects viral RNA synthesis but in a VP30-independent manner. Binding studies demonstrate variable capacity of the PPxPxY motifs from these proteins to bind VP30, define PxPPPPxY as an optimal binding motif, and identify the fifth proline and the tyrosine as most critical for interaction. Competition binding and hydrogen-deuterium exchange mass spectrometry studies demonstrate that each protein binds a similar interface on VP30. VP30 therefore presents a novel proline recognition domain that is targeted by multiple host proteins to modulate viral transcription.

From Folding to Assembly: Functional Supramolecular Architectures of Peptides Comprised of Non‐Canonical Amino Acids

Front Cover: This review discusses the self-assembly of various noncoded amino acid-containing peptides and their applications in nanobiotechnology. The incorporation of non-coded amino acids in peptide sequences have been shown to form well defined secondary structures, similar to natural proteins, with the ability to self-assemble and generate a wide variety of nanostructures with excellent biocompatibility and biostability. This is reported by Rajkumar Misra, Safra Rudnick-Glick, and Lihi Adler-Abramovich in article number 2100090.

Sequence-Specific Model for Predicting Peptide Collision Cross Section Values in Proteomic Ion Mobility Spectrometry.

The contribution of peptide amino acid sequence to collision cross section values (CCS) has been investigated using a dataset of ∼134 000 peptides of four different charge states (1+ to 4+). The migration data were acquired using a two-dimensional liquid chromatography (LC)/trapped ion mobility spectrometry/quadrupole/time-of-flight mass spectrometry (MS) analysis of HeLa cell digests created using seven different proteases and was converted to CCS values. Following the previously reported modeling approaches using intrinsic size parameters (ISP), we extended this methodology to encode the position of individual residues within a peptide sequence. A generalized prediction model was built by dividing the dataset into eight groups (four charges for both tryptic/nontryptic peptides). Position-dependent ISPs were independently optimized for the eight subsets of peptides, resulting in prediction accuracy of ∼0.981 for the entire population of peptides. We find that ion mobility is strongly affected by the peptide's ability to solvate the positively charged sites. Internal positioning of polar residues and proline leads to decreased CCS values as they improve charge solvation; conversely, this ability decreases with increasing peptide charge due to electrostatic repulsion. Furthermore, higher helical propensity and peptide hydrophobicity result in a preferential formation of extended structures with higher than predicted CCS values. Finally, acidic/basic residues exhibit position-dependent ISP behavior consistent with electrostatic interaction with the peptide macrodipole, which affects the peptide helicity. The MS raw data files have been deposited with the ProteomeXchange Consortium via the jPOST partner repository (http://jpostdb.org) with the dataset identifiers PXD021440/JPST000959, PXD022800/JPST001017, and PXD026087/ JPST001176.

Analysis of peptides in a sheep beta lactoglobulin hydrolysate as a model to evaluate the effect of peptide amino acid sequence on bioactivity

Fungal protease FPII was found to hydrolyse sheep β-lactoglobulin (β-Lg), and the hydrolysate exhibited substantial antioxidant and ACE inhibition bioactivities. From analysis of the peptide sequences in the hydrolysate in relation to bioactivity, synthetic peptides corresponding to four regions of sequence in β-Lg (LAFNPTQLEGQCHV, DTDYKKYLLF, LDAQSAPLRVY and VEELKPTPE) were analysed for bioactivity. Additional synthetic peptides were designed to examine the bioactivity of different parts of the above four sequences, and the effect of amino acid substitutions on bioactivity. The results show that parts of the peptide sequences contribute differently to bioactivity and substitution of amino acids has a substantial effect on antioxidant and ACE inhibition activities.

CATCH Peptides Coassemble into Structurally Heterogeneous β-Sheet Nanofibers with Little Preference to β-Strand Alignment.

Coassembling peptides offer an additional degree of freedom in the design of nanostructured biomaterials when compared to analogous self-assembling peptides. Yet, our understanding of how amino acid sequences encodes coassembled nanofiber structure is limited. Prior work on a charge-complementary pair, CATCH+ and CATCH- peptides, detected like-peptide nearest neighbors (CATCH+:CATCH+ and CATCH-:CATCH-) within coassembled β-sheet nanofibers; these self-associated peptide pairs marked a departure from an "ideal" coassembled structure. In this work, we employ solid-state NMR, isotope-edited FTIR, and coarse-grained molecular dynamics simulations to evaluate the alignment of β-strands within CATCH peptide nanofibers. Both experimental and computational results suggest that CATCH molecules coassemble into structurally heterogeneous nanofibers, which is consistent with our observations in another coassembling system, the King-Webb peptides. Within β-sheet nanofibers, β-strands were found to have nearest neighbors aligned in-register parallel, in-register antiparallel, and out-of-register. In comparison to the King-Webb peptides, CATCH nanofibers exhibit a greater degree of structural heterogeneity. By comparing the amino acid sequences of CATCH and King-Webb peptides, we can begin to unravel sequence-to-structure relationships, which may encode more precise coassembled β-sheet nanostructures.

Enhancement of direct electron transfer in graphene bioelectrodes containing novel cytochrome c553 variants with optimized heme orientation

The highly efficient bioelectrodes based on single layer graphene (SLG) functionalized with pyrene self-assembled monolayer and novel cytochromec553(cytc553)peptide linker variants were rationally designed to optimize the direct electron transfer (DET) between SLG and the heme group of cyt. Through a combination of photoelectrochemical and quantum mechanical (QM/MM) approaches we show that the specific amino acid sequence of a short peptide genetically inserted between the cytc553holoprotein and thesurface anchoring C-terminal His6-tag plays a crucial role in ensuring the optimal orientation and distance of the heme group with respect to the SLG surface. Consequently, efficient DET occurring between graphene and cyt c553 leads to a 20-fold enhancement of the cathodic photocurrent output compared to the previously reported devices of a similar type. The QM/MM modeling implies that a perpendicular or parallel orientation of the heme group with respect to the SLG surface is detrimental to DET, whereas the tilted orientation favors the cathodic photocurrent generation. Our work confirms the possibility of fine-tuning the electronic communication within complex bio-organic nanoarchitectures and interfaces due to optimization of the tilt angle of the heme group, its distance from the SLG surface and optimal HOMO/LUMO levels of the interacting redox centers.

Isolation of a starfish myorelaxant peptide (SMP) isotype from the pyloric caeca of Patiria pectinifera

Peptides are naturally occurring biological molecules that are found in all living organisms. These biologically active peptides play a key role in various biological processes. The aim of this study is the extraction and the purification of bioactive materials that induce relaxation of an apical muscle from the pyloric caeca of Patiria pectinifera. The acidified pyloric caeca extract was partially separated by the solid phase extraction using a stepwise gradient on Sep-Pak C18 cartridge. Among the fractions, materials eluted with 60% methanol/0.1% trifluoroacetic acid was put a thorough of a series of high performance liquid chromatography (HPLC) steps to isolate a neuropeptide with relaxation activity. The purified compound was eluted at 28% acetonitrile in 0.1% trifluoroacetic acid with retention time of 25.8 min on the CAPCELL-PAK C18 reversed-phase column. To determine the molecular weight and the amino acid sequence of the purified peptide, LC-MS and Edman degradation method were used, respectively. The primary structure of the peptide was determined to be FGMGGAYDPLSAGFTD which corresponded to the amino acid sequence of a starfish myorelaxant peptide (SMP) isotype (SMPb) found in the cDNA sequence encoding SMPa and its isotypes. In this study, a muscle relaxant neuropeptide (SMPb) has been isolated from pyloric caeca of starfish P. pectinifera. This is the first report of SMPb isolation on the protein level from P. pectinifera.

Enzymolysis peptides from Mauremys mutica plastron improve the disorder of neurotransmitter system and facilitate sleep-promoting in the PCPA-induced insomnia mice

Ethnopharmacology relevancyFor many centuries, Mauremys mutica is highly valued as a food homologous Chinese herbal medicine. It has been considered useful to sedate, nourish brain and promote sleep. However, the animal experimental evidence of its sleep-promoting activity is missing in literature. Aim of the studyIn this study, PCPA-induced insomnia model was used to explore the sleep-promoting mechanism of enzymolysis peptides from PMM, and its main composition and chemical structure were analyzed. Materials and methodsExperiments were performed using PCPA-induced insomnia model, all animals were intraperitoneally injected with PCPA (350 mg/kg·d) for two days. The sleep-promoting effect evaluated using measuring content of 5-HT, GABA, DA, IL-1, BDNF and expression of 5-HT1A receptor and GABAA receptor α1-subunit in mice brain. Primary structure of peptides was identified by HPLC-ESI-QqTOF-MS/MS. ResultsCompared with the model group, the content of 5-HT, GABA, IL-1, BDNF in mice brain of PMM peptide groups was increased to varying degrees, the content of DA was decreased, and the gene transcription and protein expression of 5-HT1A receptor and GABAA receptor α1-subunit were almost all returned to normal levels. In addition, the primary structures of most abundant nine typical peptides in PMM peptides were identified. ConclusionsThe results showed that PMM peptides could improve the disorder of neurotransmitter system, restore compensatory over-expression 5-HT1A receptor and GABAA receptor α1-subunit, and have a good sleep-promoting effect. The specific amino acid composition, sequence and glycosylation modification of PMM peptides may be the key reason for their activity, which lays a foundation for the subsequent development of sleep-promoting peptide products.

Molecular characterization of diverse defensins (γ-thionins) from Capsicum annuum flowers and their effects on the insect pest Helicoverpa armigera

Plant defensins (γ-thionins) are antimicrobial peptides (AMPs) constituting the host defense system. They are known to interact with the cell membranes to exhibit antifungal and antibacterial activity. Though defensin genes are inducible under biotic and abiotic stress, their involvement in insect herbivore defense is not well studied. In this work we studied the phylogeny, structural diversity, biochemical and functional properties of defensins from Capsicum annuum L with particular emphasis on their interaction with insect and fungal pathogens. Phylogenetic analysis of defensin peptides from Solanaceae, Pinaceae, Fabaceae, Asteraceae, Brassicaceae and similar peptides from invertebrates divided them into ten groups and Capsicum peptides were represented in most of them. Three thionin (CanThio-1, −2, −3) and two defensin (CanDef-20, −21) genes isolated from the flowers of Capsicum annuum were cloned and recombinant proteins were expressed. The mature peptide amino acid sequences of CanThio and CanDef were 54–75 aa with 21.82 to 73.81% similarity. They contained the conserved features of defensin peptides like the gamma core region and the presence of eight cysteine residues. Predicted structural alignment indicated that CanThio and CanDef had structural diversity and structurally unaligned loop regions.The recombinant CanThio and CanDef inhibited 5% - 65% Aspergillus oryzae amylase activity at 20 μM and ~ 20% of Helicoverpa armigera gut amylase activity at 50 μM peptide concentrations. CanThio-2 displayed bovine trypsin inhibition (~14%) while all the CanThio and CanDef inhibited ~20% of H. armigera gut proteinase activity at 20 μM. H. armigera larvae fed on an artificial diet incorporated with recombinant CanThio-2 and CanDef-20 (125 μg/ml) exhibited a 15% reduction in larval mass and 13% reduction in pupal mass. The CanThio and CanDef peptides also exhibited antifungal properties against pathogenic fungus Fusarium oxysporum at 5 μg/ml, where CanThio-2 and CanDef-20 showed the strongest activity, which may be attributed to the positively charged amino acids in the γ-core region.We conclude that despite having relatively similar conserved three-dimensional structures CanThio and CanDef display functional variability, which can be utilized for enhancing insect and fungal resistance in plants.

Analysis of Amino Acid Sequence of Antimicrobial Peptides in the Fermentation Broth of Paecilomyces hepiali by RP-HPLC-MS

Analysis of Amino Acid Sequence of Antimicrobial Peptides in the Fermentation Broth of Paecilomyces hepiali by RP-HPLC-MS

Probing Structural Information of Gas-Phase Peptides by Near-Edge X-ray Absorption Mass Spectrometry.

Near-edge X-ray absorption mass spectrometry (NEXAMS) is an action-spectroscopy technique of growing interest for investigations into the spatial and electronic structure of biomolecules. It has been used successfully to give insights into different aspects of the photodissociation of peptides and to probe the conformation of proteins. It is a current question whether the fragmentation pathways are sensitive toward effects of conformational isomerism, tautomerism, and intramolecular interactions in gas-phase peptides. To address this issue, we studied the cationic fragments of cryogenically cooled gas-phase leucine enkephalin ([LeuEnk+H]+) and methionine enkephalin ([MetEnk+H]+) produced upon soft X-ray photon absorption at the carbon, nitrogen, and oxygen K-edges. The interpretation of the experimental ion yield spectra was supported by density-functional theory and restricted-open-shell configuration interaction with singles (DFT/ROCIS) calculations. The analysis revealed several effects that could not be rationalized based on the peptide's amino acid sequences alone. Clear differences between the partial ion yields measured for both peptides upon C 1s → π*(C═C) excitations in the aromatic amino acid side chains give evidence for a sulfur-aromatic interaction between the methionine and phenylalanine side chain of [MetEnk+H]+. Furthermore, a peak associated with N 1s → π*(C═N) transitions, linked to a tautomeric keto-to-enol conversion of peptide bonds, was only present in the photon energy resolved ion yield spectra of [MetEnk+H]+.