A sucrose-binding protein and ß-conglycinins regulate soybean seed protein content and control multiple seed traits.

Expanded agriculture production is required to support the world's population but can impose substantial environmental and climate change costs, particularly with intensifying animal production and protein demand. Shifting from an animal- to a plant-based protein diet has numerous health benefits. Soybean (Glycine max (L.) Merr.) is a major source of protein for human food and animal feed; improved soybean protein content and amino acid composition could provide high-quality soymeal for animal feed, healthier human foods, and a reduced carbon footprint. Nonetheless, during the soybean genome evolution, a balance was established between the amount of seed protein, oil, and carbohydrate content, burdening the development of soybean cultivars with high proteins. We isolated two high-seed protein (HP) soybean mutants, HP1 and HP2, with improved seed amino acid composition and stachyose content, pointing to their involvement in controlling seed rebalancing phenomenon. HP1 encodes ß-conglycinin (GmCG-1) and HP2 encodes Sucrose Binding Protein (GmSBP-1), which are both highly expressed in soybean seeds. Mutations in GmSBP-1, GmCG-1, and the paralog GmCG-2 resulted in increased protein levels, confirming their role as general regulators of seed protein content, amino acid seed composition, and seed vigor. Biodiversity analysis of GmCG and GmSBP across 108 soybean accessions revealed haplotypes correlated with protein and seed carbohydrate content. Furthermore, our data revealed an unprecedented role of GmCG and GmSBP proteins in improving seed vigor, crude protein, and amino acid digestibility. Since GmSBP and GmCG are present in most seed plants analyzed, these genes could be targeted to improve multiple seed traits.

Catalytic resilience of multicomponent aromatic ring-hydroxylating dioxygenases in Pseudomonas for degradation of polycyclic aromatic hydrocarbons.

Hydrophobic organic compounds, either natural or introduced through anthropogenic activities, pose a serious threat to all spheres of life, including humankind. These hydrophobic compounds are recalcitrant and difficult to degrade by the microbial system; however, microbes have also evolved their metabolic and degradative potential. Pseudomonas species have been reported to have a multipotential role in the biodegradation of aromatic hydrocarbons through aromatic ring-hydroxylating dioxygenases (ARHDs). The structural complexity of different hydrophobic substrates and their chemically inert nature demands the explicit role of evolutionary conserved multicomponent enzyme ARHDs. These enzymes catalyze ring activation and subsequent oxidation by adding two molecular oxygen atoms onto the vicinal carbon of the aromatic nucleus. This critical metabolic step in the aerobic mode of degradation of polycyclic aromatic hydrocarbons (PAHs) catalyzed by ARHDs can also be explored through protein molecular docking studies. Protein data analysis enables an understanding of molecular processes and monitoring complex biodegradation reactions. This review summarizes the molecular characterization of five ARHDs from Pseudomonas species already reported for PAH degradation. Homology modeling for the amino acid sequences encoding the catalytic α-subunit of ARHDs and their docking analyses with PAHs suggested that the enzyme active sites show flexibility around the catalytic pocket for binding of low molecular weight (LMW) and high molecular weight (HMW) PAH substrates (naphthalene, phenanthrene, pyrene, benzo[α]pyrene). The alpha subunit harbours variable catalytic pockets and broader channels, allowing relaxed enzyme specificity toward PAHs. ARHD's ability to accommodate different LMW and HMW PAHs demonstrates its 'plasticity', meeting the catabolic demand of the PAH degraders.

Trp2027Cys mutation evolves in Digitaria insularis with cross-resistance to ACCase inhibitors.

Sourgrass (Digitaria insularis) is one of the most problematic weeds in South America because glyphosate resistance is widespread across most crop production regions. Acetyl coenzyme A carboxylase (ACCase)-inhibiting herbicides have been intensively used to manage D. insularis, which substantially increased selection pressure for this class of herbicides. We confirmed resistance to ACCase herbicides in a D. insularis population from Brazil and characterized its molecular basis. Resistant plants showed high level of resistance to haloxyfop (resistance factor, RF = 613-fold), low level of resistance to pinoxaden (RF = 3.6-fold), and no resistance to clethodim. A target-site mutation, Trp2027Cys, was found in the ACCase sequence from resistant plants. A protein homology model shows that the Trp2027Cys mutation is near the herbicide-binding pocket formed between two ACCase chains, and is predicted to obstruct the access of aryloxyphenoxypropionates (FOP) herbicides to the binding site. A qPCR-based single nucleotide polymorphism genotyping method was validated to discriminate susceptible (wild-type Trp2027) and resistant (mutant Cys2027) alleles. All resistant plants were homozygous for the mutation and the assay could be used for early detection of resistance in D. insularis field samples with suspected resistance to ACCase inhibitors.

Homology Modeling of the Human P-glycoprotein (ABCB1) and Insights into Ligand Binding through Molecular Docking Studies.

The ABCB1 transporter also known as P-glycoprotein (P-gp) is a transmembrane protein belonging to the ATP binding cassette super-family of transporters; it is a xenobiotic efflux pump that limits intracellular drug accumulation by pumping the compounds out of cells. P-gp contributes to a decrease of toxicity and possesses broad substrate specificity. It is involved in the failure of numerous anticancer and antiviral chemotherapies due to the multidrug resistance (MDR) phenomenon, where it removes the chemotherapeutics out of the targeted cells. Understanding the details of the ligand-P-gp interaction is therefore crucial for the development of drugs that might overcome the MRD phenomenon and for obtaining a more effective prediction of the toxicity of certain compounds. In this work, an in silico modeling was performed using homology modeling and molecular docking methods with the aim of better understanding the ligand-P-gp interactions. Based on different mouse P-gp structural templates from the PDB repository, a 3D model of the human P-gp (hP-gp) was constructed by means of protein homology modeling. The homology model was then used to perform molecular docking calculations on a set of thirteen compounds, including some well-known compounds that interact with P-gp as substrates, inhibitors, or both. The sum of ranking differences (SRD) was employed for the comparison of the different scoring functions used in the docking calculations. A consensus-ranking scheme was employed for the selection of the top-ranked pose for each docked ligand. The docking results showed that a high number of π interactions, mainly π-sigma, π-alkyl, and π-π type of interactions, together with the simultaneous presence of hydrogen bond interactions contribute to the stability of the ligand-protein complex in the binding site. It was also observed that some interacting residues in hP-gp are the same when compared to those observed in a co-crystallized ligand (PBDE-100) with mouse P-gp (PDB ID: 4XWK). Our in silico approach is consistent with available experimental results regarding P-gp efflux transport assay; therefore it could be useful in the prediction of the role of new compounds in systemic toxicity.

Computational Study of C-X-C Chemokine Receptor (CXCR)3 Binding with Its Natural Agonists Chemokine (C-X-C Motif) Ligand (CXCL)9, 10 and 11 and with Synthetic Antagonists: Insights of Receptor Activation towards Drug Design for Vitiligo.

Vitiligo is a hypopigmentary skin pathology resulting from the death of melanocytes due to the activity of CD8+ cytotoxic lymphocytes and overexpression of chemokines. These include CXCL9, CXCL10, and CXCL11 and its receptor CXCR3, both in peripheral cells of the immune system and in the skin of patients diagnosed with vitiligo. The three-dimensional structure of CXCR3 and CXCL9 has not been reported experimentally; thus, homology modeling and molecular dynamics could be useful for the study of this chemotaxis-promoter axis. In this work, a homology model of CXCR3 and CXCL9 and the structure of the CXCR3/Gαi/0ßγ complex with post-translational modifications of CXCR3 are reported for the study of the interaction of chemokines with CXCR3 through all-atom (AA-MD) and coarse-grained molecular dynamics (CG-MD) simulations. AA-MD and CG-MD simulations showed the first activation step of the CXCR3 receptor with all chemokines and the second activation step in the CXCR3-CXCL10 complex through a decrease in the distance between the chemokine and the transmembrane region of CXCR3 and the separation of the ßγ complex from the α subunit in the G-protein. Additionally, a general protein-ligand interaction model was calculated, based on known antagonists binding to CXCR3. These results contribute to understanding the activation mechanism of CXCR3 and the design of new molecules that inhibit chemokine binding or antagonize the receptor, provoking a decrease of chemotaxis caused by the CXCR3/chemokines axis.

Identification, classification and transcriptional profiles of dirigent domain-containing proteins in sugarcane.

Dirigent (DIR) proteins, encoded by DIR genes, are referred to as "dirigent" because they direct the outcome of the coupling of the monolignol coniferyl alcohol into (+) or (-) pinoresinol, the first intermediates in the enantiocomplementary pathways for lignan biosynthesis. DIR domain-containing or DIR-like proteins are, thus, termed for not having a clear characterization. A transcriptome- and genome-wide survey of DIR domain-containing proteins in sugarcane was carried out, in addition to phylogenetic, physicochemical and transcriptional analyses. A total of 120 non-redundant sequences containing the DIR domain were identified and classified into 64 groups according to phylogenetic and sequence alignment analyses. In silico analysis of transcript abundance showed that these sequences are expressed at low levels in leaves and genes in the same phylogenetic clade have similar expression patterns. Expression analysis of ShDIR1-like transcripts in the culm internodes of sugarcane demonstrates their abundance in mature internodes, their induction by nitrogen fertilization and their predominant expression in cells that have a lignified secondary cell wall, such as vascular bundles of young internodes and parenchymal cells of the pith of mature internodes. Due to the lack of information about the functional role of DIR in plants, a possible relationship is discussed between the ShDIR1-like transcriptional profile and cell wall development in parenchyma cells of sugarcane culm, which typically accumulates large amounts of sucrose. The number of genes encoding the DIR domain-containing proteins in sugarcane is intriguing and is an indication per se that these proteins may have an important metabolic role and thus deserve to be better studied.

CASP11 refinement experiments with ROSETTA.

We report new Rosetta-based approaches to tackling the major issues that confound protein structure refinement, and the testing of these approaches in the CASP11 experiment. Automated refinement protocols were developed that integrate a range of sampling methods using parallel computation and multiobjective optimization. In CASP11, we used a more aggressive large-scale structure rebuilding approach for poor starting models, and a less aggressive local rebuilding plus core refinement approach for starting models likely to be closer to the native structure. The more incorrectly modeled a structure was predicted to be, the more it was allowed to vary during refinement. The CASP11 experiment revealed strengths and weaknesses of the approaches: the high-resolution strategy incorporating local rebuilding with core refinement consistently improved starting structures, while the low-resolution strategy incorporating the reconstruction of large parts of the structures improved starting models in some cases but often considerably worsened them, largely because of model selection issues. Overall, the results suggest the high-resolution refinement protocol is a promising method orthogonal to other approaches, while the low-resolution refinement method clearly requires further development. Proteins 2016; 84(Suppl 1):314-322. © 2015 Wiley Periodicals, Inc.

Integration of food raw materials, food microbiology, and food additives: systematic research and comprehensive insights into sweet sorghum juice, Clostridium tyrobutyricum TGL-A236 and bio-butyric acid.

Introduction: Sweet sorghum juice is a typical production feedstock for natural, eco-friendly sweeteners and beverages. Clostridium tyrobutyricum is one of the widely used microorganisms in the food industry, and its principal product, bio-butyric acid is an important food additive. There are no published reports of Clostridium tyrobutyricum producing butyric acid using SSJ as the sole substrate without adding exogenous substances, which could reach a food-additive grade. This study focuses on tailoring a cost-effective, safe, and sustainable process and strategy for their production and application. Methods: This study modeled the enzymolysis of non-reducing sugars via the first/second-order kinetics and added food-grade diatomite to the hydrolysate. Qualitative and quantitative analysis were performed using high-performance liquid chromatography, gas chromatography-mass spectrometer, full-scale laser diffraction method, ultra-performance liquid chromatography-tandem mass spectrometry, the cell double-staining assay, transmission electron microscopy, and Oxford nanopore technology sequencing. Quantitative real-time polymerase chain reaction, pathway and process enrichment analysis, and homology modeling were conducted for mutant genes. Results: The treated sweet sorghum juice showed promising results, containing 70.60 g/L glucose and 63.09 g/L fructose, with a sucrose hydrolysis rate of 98.29% and a minimal sucrose loss rate of 0.87%. Furthermore, 99.62% of the colloidal particles and 82.13% of the starch particles were removed, and the concentrations of hazardous substances were effectively reduced. A food microorganism Clostridium tyrobutyricum TGL-A236 with deep utilization value was developed, which showed superior performance by converting 30.65% glucose and 37.22% fructose to 24.1364 g/L bio-butyric acid in a treated sweet sorghum juice (1:1 dilution) fermentation broth. This titer was 2.12 times higher than that of the original strain, with a butyric acid selectivity of 86.36%. Finally, the Genome atlas view, Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG), and evolutionary genealogy of genes: Non-supervised Orthologous (eggNOG) functional annotations, three-dimensional structure and protein cavity prediction of five non-synonymous variant genes were obtained. Conclusion: This study not only includes a systematic process flow and in-depth elucidation of relevant mechanisms but also provides a new strategy for green processing of food raw materials, improving food microbial performance, and ensuring the safe production of food additives.

Ligand-based virtual screening, molecular dynamics, and biological evaluation of repurposed drugs as inhibitors of Trypanosoma cruzi proteasome.

Chagas disease is a well-known Neglected Tropical Disease, mostly endemic in continental Latin America, but that has spread to North America and Europe. Unfortunately, current treatments against such disease are ineffective and produce known and undesirable side effects. To find novel effective drug candidates to treat Chagas disease, we uniquely explore the Trypanosoma cruzi proteasome as a recent biological target and, also, apply drug repurposing through different computational methodologies. For this, we initially applied protein homology modeling to build a robust model of proteasome ß4/ß5 subunits, since there is no crystallographic structure of this target. Then, we used it on a drug repurposing via a virtual screening campaign starting with more than 8,000 drugs and including the methodologies: ligand-based similarity, toxicity predictions, and molecular docking. Three drugs were selected concerning their favorable interactions at the protein binding site and subsequently submitted to molecular dynamics simulations, which allowed us to elucidate their behavior and compare such theoretical results with experimental ones, obtained in biological assays also described in this paper.Communicated by Ramaswamy H. Sarma.

Computational biology insights into genotype-clinical phenotype-protein phenotype relationships between novel SLC26A2 variants identified in inherited skeletal dysplasias.

BACKGROUND: Pathogenic variants in the transmembrane sulfate transporter protein SLC26A2 are associated with different phenotypes of inherited chondrodysplasias. As limited data is published from India, in this study we sought to elucidate the molecular basis of inherited chondrodysplasias in an Indian cohort. METHODS: Molecular screening of 32 fetuses with antenatally diagnosed lethal skeletal dysplasia was performed by next generation sequencing and Sanger sequencing. The genotype-protein phenotype characterization was done using computational biology techniques like homology modelling, stability and pathogenicity predictions. RESULTS: We identified five rare autosomal recessive SLC26A2 [NM_000112.4] variants, including three homozygous c.796dupA(p.Thr266Asnfs*12), c.1724delA(p.Lys575Serfs*10), and c.1375_1377dup(p.Val459dup) and two heterozygous variants (c.532C > T(p.Arg178*)) and (c.1382C > T(p.Ala461Val)) in compound heterozygous form in a total of four foetuses. Genotype-protein phenotype annotations highlighted that the clinically severe achondrogenesis 1B causative c.796dupA(p.Thr266Asnfs*12) and c.1724delA(p.Lys575Serfs*10)variants impact SLC26A2 protein structure by deletion of the protein core and transmembrane STAS domains, respectively. In clinically moderate atelosteogenesis type 2 phenotype, the c.1382C > T(p.Ala461Val) variant is predicted to distort alpha helix conformation and alter the bonding properties and free energy dynamics of transmembrane domains and the c.532C > T(p.Arg178*) variant results in loss of both core transmembrane and STAS domains of the SLC26A2 protein. The c.1375_1377dup(p.Val459dup) variant identified in clinically milder atelosteogenesis type II-diastrophic dysplasia spectrum lethal phenotype is predicted to decrease the Qualitative Model Energy Analysis (QMean), which affects major geometrical aspects of the SLC26A2 protein structure. CONCLUSION: We expand the spectrum of SLC26A2 related lethal chondrodysplasia and report three novel variants correlating clinical severity and protein phenotype within the lethal spectrum of this rare dysplasia. We demonstrate the relevance of structural characterization to aid novel variant reclassification to provide better prenatal management and reproductive options to families with lethal antenatal skeletal disorder.

Novel STAG3 variant associated with primary ovarian insufficiency and non-obstructive azoospermia in an Iranian consanguineous family.

Non-obstructive azoospermia (NOA) and primary ovarian insufficiency (POI) present the most severe forms of male and female infertility. In the last decade, the increasing use of whole exome sequencing (WES) in genomics studies of these conditions has led to the introduction of a number of novel genes and variants especially in meiotic genes with restricted expression to gonads. In this study, exome sequencing of a consanguineous Iranian family with one POI and two NOA cases in three siblings showed that all three patients were double homozygous for a novel in-frame deletion and a novel missense variant in STAG3 (NM_001282717.1:c.1942G > A: p.Ala648Thr; NM_001282717.1:c.1951_1953del: p. Leu652del). Both variants occur within a short proximity of each other affecting the relatively conserved armadillo-type fold superfamily feature. STAG3 is a specific meiotic cohesin complex component that interacts with the α-kleisin subunit through this feature. Protein homology modeling indicated that the in-frame deletion destabilizes kleisin biding by STAG3. Although the missense variant did not seem to affect the binding significantly, protein homology modeling suggests that it further destabilizes kleisin binding when in double homozygous state with the deletion. Our findings are in line with several other studies having associated deleterious variants affecting this region with male and female infertility in humans and mouse models. This is the first report associating an in-frame STAG3 variant with NOA and POI in a single family. SUMMARY SENTENCE: A patient with primary ovarian failure and her two brothers with non-obstructive azoospermia were double homozygous for a novel in-frame deletion and a novel missense variant in STAG3 that potentially disrupt the protein's meiotic functions.

The Se S/N interactions as a possible mechanism of δ-aminolevulinic acid dehydratase enzyme inhibition by organoselenium compounds: A computational study.

Organoselenium compounds present many pharmacological properties and are promising drugs. However, toxicological effects associated with inhibition of thiol-containing enzymes, such as the δ-aminolevulinic acid dehydratase (δ-AlaD), have been described. The molecular mechanism(s) by which they inhibit thiol-containing enzymes at the atomic level, is still not well known. The use of computational methods to understand the physical-chemical properties and biological activity of chemicals is essential to the rational design of new drugs. In this work, we propose an in silico study to understand the δ-AlaD inhibition mechanism by diphenyl diselenide (DPDS) and its putative metabolite, phenylseleninic acid (PSA), using δ-AlaD enzymes from Homo sapiens (Hsδ-AlaD), Drosophila melanogaster (Dmδ-AlaD) and Cucumis sativus (Csδ-AlaD). Protein modeling homology, molecular docking, and DFT calculations are combined in this study. According to the molecular docking, DPDS and PSA might bind in the Hsδ-AlaD and Dmδ-AlaD active sites interacting with the cysteine residues by Se…S interactions. On the other hand, the DPDS does not access the active site of the Csδ-AlaD (a non-thiol protein), while the PSA interacts with the amino acids residues from the active site, such as the Lys291. These interactions might lead to the formation of a covalent bond, and consequently, to the enzyme inhibition. In fact, DFT calculations (mPW1PW91/def2TZVP) demonstrated that the selenylamide bond formation is energetically favored. The in silico data showed here are in accordance with previous experimental studies, and help us to understand the reactivity and biological activity of organoselenium compounds.

Harnessing a P450 fatty acid decarboxylase from Macrococcus caseolyticus for microbial biosynthesis of odd chain terminal alkenes.

Alkenes are industrially important platform chemicals with broad applications. In this study, we report a direct microbial biosynthesis of terminal alkenes from fermentable sugars by harnessing a P450 fatty acid (FA) decarboxylase from Macrococcus caseolyticus (OleTMC). We first characterized OleTMC and demonstrated its in vitro H2O2-independent activities towards linear C10:0-C18:0 FAs, with higher activity for C16:0-C18:0 FAs. Next, we engineered a de novo alkene biosynthesis pathway, consisting of OleTMC and an engineered E. coli thioesterase (TesA) with compatible substrate specificities, and introduced this pathway into E. coli for terminal alkene biosynthesis from glucose. The recombinant E. coli EcNN101 produced a total of 17.78 ±â€¯0.63 mg/L odd-chain terminal alkenes, comprising of 0.9% ±â€¯0.5% C11 alkene, 12.7% ±â€¯2.2% C13 alkene, 82.7% ±â€¯1.7% C15 alkene, and 3.7% ±â€¯0.8% C17 alkene, and a yield of 0.87 ±â€¯0.03 (mg/g) on glucose. To improve alkene production, we identified and overcame the electron transfer limitation in OleTMC, by introducing a two-component redox system, consisting of a putidaredoxin reductase (CamA) and a putidaredoxin (CamB) from Pseudomonas putida, into EcNN101, and demonstrated the alkene production increased ~2.8 fold. Finally, to better understand the substrate specificities of OleTMC observed, we employed in silico protein modeling to illuminate the functional role of FA binding pocket.

Enhancing Structure Prediction and Design of Soluble and Membrane Proteins with Explicit Solvent-Protein Interactions.

Solvent molecules interact intimately with proteins and can profoundly regulate their structure and function. However, accurately and efficiently modeling protein solvation effects at the molecular level has been challenging. Here, we present a method that improves the atomic-level modeling of soluble and membrane protein structures and binding by efficiently predicting de novo protein-solvent molecule interactions. The method predicted with unprecedented accuracy buried water molecule positions, solvated protein conformations, and challenging mutational effects on protein binding. When applied to homology modeling, solvent-bound membrane protein structures, pockets, and cavities were recapitulated with near-atomic precision even from distant homologs. Blindly refined atomic-level structures of evolutionary distant G protein-coupled receptors imply strikingly different functional roles of buried solvent between receptor classes. The method should prove useful for refining low-resolution protein structures, accurately modeling drug-binding sites in structurally uncharacterized receptors, and designing solvent-mediated protein catalysis, recognition, ligand binding, and membrane protein signaling.

Molecular and Computational Studies on Apoptotic Pathway Regulator, Bcl-2 Gene from Breast Cancer Cell Line MCF-7.

Cancer is a dreadful disease constituting abnormal growth and proliferation of malignant cells in the body. Next to lung cancer, breast cancer is the most common form of cancer affecting women. The apoptotic pathway regulators, B cell lymphoma family of protein, play a key role in various malignancies defining cancer and their constitutive expression plays an integral role in breast cancer chemotherapy. The research work discusses the identification and molecular cloning of a B cell lymphoma like gene from human breast cancer cell line. The open reading frame of the gene consisted of 965 nucleotides, encoding a protein of 380 amino acids with a predicted molecular weight of 42.5 kilodalton. The predicted physiochemical properties of the gene were as follows: Isoelectric point - 9.49, molecular formula - C1893H3004N534O548S16, total number of negatively charged residues, (Aspartate+Glutamate) - 26, total number of positively charged residues, (Arginine+Lysine)-39, instability index-42.08 (unstable protein) and grand average of hydropathicity is -0.202. Additionally, phobius prediction suggested non-cytoplasmic localization of the putative protein. The presence of secondary structure in the protein was determined by Memsat program. A 3 dimensional protein homology model was generated using threading based method of protein modeling for structural and functional annotation of the putative protein. Future prospects accounts for the biochemical characterization of the enzyme including in vitro assays on breast cancer cell line would establish the functional characteristics of the protein and its physiological mechanisms in breast cancer development and its therapeutic-target role in future.

Structural characterization and IgE epitope analysis of arginine kinase from Scylla paramamosain.

Arginine kinase (AK) is reported to be the pan-allergen of shellfish. However, there is limited information on its IgE epitopes and structural characteristics. In this study, AK from Scylla paramamosain was purified and characterized. The purified AK is a glycoprotein with the molecular weight of 40 kDa and it demonstrates cross-reactivity with the related allergens present in other shellfish. The cDNA of S. paramamosain AK was cloned, which encodes 357 amino acid residues. Nine linear epitopes and seven conformational epitopes were predicted following bioinformatics analysis. In addition, the entire recombinant AK (rAK) and three partial recombinant AKs (rAK1, rAK2, and rAK3) were successfully expressed in Escherichia coli BL21 (DE3). The proteins of rAK1, rAK2 and rAK have strong IgE reactivity with the pooled sera from crab allergic patients, while rAK3 has significantly weaker IgE reactivity, which indicates that the IgE epitopes of AK are mainly distributed in the regions of rAK1 and rAK2. Furthermore, three experimental linear epitopes (epitope 1: AA 127-141, epitope 2: AA 141-155, and epitope 3: AA 211-225) were discovered in the region of rAK1 and rAK2 using synthetized overlapping peptides. The experimental linear epitopes were mapped onto the protein homology model of AK. Meanwhile, in the IgE-binding assays of the sera from nine crab allergic patients, only three sera reacted with the denatured, linear AK as shown by Western-blotting, eight sera reacted with the native, folded AK by both dot-blotting and ELISA, which indicates that the conformational IgE epitopes of S. paramamosain AK may be more predominant.