Primary Structure Of Protein Research Articles

Topography prediction of helical transmembrane proteins by a new modification of the sliding window method.

Protein functions are specified by its three-dimensional structure, which is usually obtained by X-ray crystallography. Due to difficulty of handling membrane proteins experimentally to date the structure has only been determined for a very limited part of membrane proteins (<4%). Nevertheless, investigation of structure and functions of membrane proteins is important for medicine and pharmacology and, therefore, is of significant interest. Methods of computer modeling based on the data on the primary protein structure or the symbolic amino acid sequence have become an actual alternative to the experimental method of X-ray crystallography for investigating the structure of membrane proteins. Here we presented the results of the study of 35 transmembrane proteins, mainly GPCRs, using the novel method of cascade averaging of hydrophobicity function within the limits of a sliding window. The proposed method allowed revealing 139 transmembrane domains out of 140 (or 99.3%) identified by other methods. Also 236 transmembrane domain boundary positions out of 280 (or 84%) were predicted correctly by the proposed method with deviation from the predictions made by other methods that does not exceed the detection error of this method.

NMR of Conditional Peripheral Membrane Proteins

Conditional membrane proteins associate with membranes in response to binding specific ligands. We report the application of NMR techniques to gain insight into the structure, dynamics, and protein-membrane interactions of two fundamentally different conditional membrane modules: C1 and C2 domains, both from the Protein Kinase Cα (PKCα), that are covalently linked to one another in the primary structure of the parent protein. Using NMR relaxation dispersion techniques, paramagnetic relaxation enhancement (PRE) experiments, and NMR-detected ligand binding studies, we demonstrate the role of conformational plasticity and initial membrane pre-association in modulating the affinity of the C1 domain to its natural cofactor, diacylglycerol. We show that the C2 domain employs a drastically different mechanism of membrane insertion that involves modulation of its electrostatic potential by divalent metal ions. The dynamics of loop and N- and C-terminal regions of C2 changes as a function of metal ligation state, suggesting a possible mechanism for propagating the information about the metal-binding event to other PKCα domains. In aggregate, our studies provide a view of conditional membrane domains as highly dynamic entities, in which conformational plasticity and synergistic action of cytosolic and membrane-embedded ligands define their nuanced signaling response. The functional and structural interplay between C1 and C2 domains will also be discussed in the context of the only two existing multi-domain structures of Protein Kinase C, one of which came from our laboratory.This work is supported by the NSF CAREER (CHE-2791151435) award to TII.

Boost Protein Expression through Co-Expression of LEA-Like Peptide in Escherichia coli

The boost protein expression has been done successfully by simple co-expression with a late embryogenesis abundant (LEA)-like peptide in Escherichia coli. Frequently, overexpression of a recombinant protein fails to provide an adequate yield. In the study, we developed a simple and efficient system for overexpressing transgenic proteins in bacteria by co-expression with an LEA-like peptide. The design of this peptide was based on part of the primary structure of an LEA protein that is known hydrophilic protein to suppress aggregation of other protein molecules. In our system, the expression of the target protein was increased remarkably by co-expression with an LEA-like peptide consisting of only 11 amino acid residues. This could provide a practical method for producing recombinant proteins efficiently.

A two-stage SVM method to predict membrane protein types by incorporating amino acid classifications and physicochemical properties into a general form of Chou's PseAAC

Membrane proteins play important roles in many biochemical processes and are also attractive targets of drug discovery for various diseases. The elucidation of membrane protein types provides clues for understanding the structure and function of proteins. Recently we developed a novel system for predicting protein subnuclear localizations. In this paper, we propose a simplified version of our system for predicting membrane protein types directly from primary protein structures, which incorporates amino acid classifications and physicochemical properties into a general form of pseudo-amino acid composition. In this simplified system, we will design a two-stage multi-class support vector machine combined with a two-step optimal feature selection process, which proves very effective in our experiments. The performance of the present method is evaluated on two benchmark datasets consisting of five types of membrane proteins. The overall accuracies of prediction for five types are 93.25% and 96.61% via the jackknife test and independent dataset test, respectively. These results indicate that our method is effective and valuable for predicting membrane protein types. A web server for the proposed method is available at http://www.juemengt.com/jcc/memty_page.php

Aging as Accelerated Accumulation of Somatic Variants: Whole-Genome Sequencing of Centenarian and Middle-Aged Monozygotic Twin Pairs

It has been postulated that aging is the consequence of an accelerated accumulation of somatic DNA mutations and that subsequent errors in the primary structure of proteins ultimately reach levels sufficient to affect organismal functions. The technical limitations of detecting somatic changes and the lack of insight about the minimum level of erroneous proteins to cause an error catastrophe hampered any firm conclusions on these theories. In this study, we sequenced the whole genome of DNA in whole blood of two pairs of monozygotic (MZ) twins, 40 and 100 years old, by two independent next-generation sequencing (NGS) platforms (Illumina and Complete Genomics). Potentially discordant single-base substitutions supported by both platforms were validated extensively by Sanger, Roche 454, and Ion Torrent sequencing. We demonstrate that the genomes of the two twin pairs are germ-line identical between co-twins, and that the genomes of the 100-year-old MZ twins are discerned by eight confirmed somatic single-base substitutions, five of which are within introns. Putative somatic variation between the 40-year-old twins was not confirmed in the validation phase. We conclude from this systematic effort that by using two independent NGS platforms, somatic single nucleotide substitutions can be detected, and that a century of life did not result in a large number of detectable somatic mutations in blood. The low number of somatic variants observed by using two NGS platforms might provide a framework for detecting disease-related somatic variants in phenotypically discordant MZ twins.

Epigenetic Dominance of Prion Conformers

Although they share certain biological properties with nucleic acid based infectious agents, prions, the causative agents of invariably fatal, transmissible neurodegenerative disorders such as bovine spongiform encephalopathy, sheep scrapie, and human Creutzfeldt Jakob disease, propagate by conformational templating of host encoded proteins. Once thought to be unique to these diseases, this mechanism is now recognized as a ubiquitous means of information transfer in biological systems, including other protein misfolding disorders such as those causing Alzheimer's and Parkinson's diseases. To address the poorly understood mechanism by which host prion protein (PrP) primary structures interact with distinct prion conformations to influence pathogenesis, we produced transgenic (Tg) mice expressing different sheep scrapie susceptibility alleles, varying only at a single amino acid at PrP residue 136. Tg mice expressing ovine PrP with alanine (A) at (OvPrP-A136) infected with SSBP/1 scrapie prions propagated a relatively stable (S) prion conformation, which accumulated as punctate aggregates in the brain, and produced prolonged incubation times. In contrast, Tg mice expressing OvPrP with valine (V) at 136 (OvPrP-V136) infected with the same prions developed disease rapidly, and the converted prion was comprised of an unstable (U), diffusely distributed conformer. Infected Tg mice co-expressing both alleles manifested properties consistent with the U conformer, suggesting a dominant effect resulting from exclusive conversion of OvPrP-V136 but not OvPrP-A136. Surprisingly, however, studies with monoclonal antibody (mAb) PRC5, which discriminates OvPrP-A136 from OvPrP-V136, revealed substantial conversion of OvPrP-A136. Moreover, the resulting OvPrP-A136 prion acquired the characteristics of the U conformer. These results, substantiated by in vitro analyses, indicated that co-expression of OvPrP-V136 altered the conversion potential of OvPrP-A136 from the S to the otherwise unfavorable U conformer. This epigenetic mechanism thus expands the range of selectable conformations that can be adopted by PrP, and therefore the variety of options for strain propagation.

The network of stabilizing contacts in proteins studied by coevolutionary data

The primary structure of proteins, that is their sequence, represents one of the most abundant sets of experimental data concerning biomolecules. The study of correlations in families of co-evolving proteins by means of an inverse Ising-model approach allows to obtain information on their native conformation. Following up on a recent development along this line, we optimize the algorithm to calculate effective energies between the residues, validating the approach both back-calculating interaction energies in a model system, and predicting the free energies associated to mutations in real systems. Making use of these effective energies, we study the network of interactions which stabilizes the native conformation of some well-studied proteins, showing that it displays different properties than the associated contact network.

Characterization of Ile‐His‐Arg‐Phe, a novel rice‐derived vasorelaxing peptide with hypotensive and anorexigenic activities

Recently, we found that dipeptide Arg-Phe (RF) had cholecystokinin (CCK)-dependent vasorelaxing activity. The RF sequence is often observed in the primary structure of natural food proteins. In the current study, we investigated enzymatic conditions for the release of RF-related peptides from rice glutelin, a major storage protein, using gastrointestinal proteases. RF-related peptides were then characterized. It was found that RF and Ile-His-Arg-Phe (IHRF) were released in the chymotrypsin digest of the partial structure of rice glutelin. We then focused on previously unidentified IHRF, corresponding to rice glutelin(155-158). IHRF had vasorelaxing activity in the mesenteric artery of spontaneous hypertensive rats (SHRs). Orally administered IHRF lowered systolic blood pressure in SHRs. The antihypertensive activity of IHRF was more potent and long-lasting than that of RF. IHRF-induced vasorelaxing activity was not blocked by inhibitors of nitric oxide synthase and cyclooxygenase, but by an antagonist for CCK₁ receptor. IHRF also had CCK-like suppressive activities in food intake and gastrointestinal transit. IHRF increased intracellular Ca²⁺ flux and CCK release in the enteroendocrine cell STC-1. IHRF, a novel CCK-dependent vasorelaxing peptide, decreases both blood pressure and food intake in rodents.

Students' understanding of primary and secondary protein structure: Drawing secondary protein structure reveals student understanding better than simple recognition of structures

The interdisciplinary nature of biochemistry courses requires students to use both chemistry and biology knowledge to understand biochemical concepts. Research that has focused on external representations in biochemistry has uncovered student difficulties in comprehending and interpreting external representations in addition to a fragmented understanding of fundamental biochemistry concepts. This project focuses on students' understanding of primary and secondary protein structure and drawings (representations) of hydrogen-bonding in alpha helices and beta sheets. Analysis demonstrated that students can recognize and identify primary protein structure concepts when given a polypeptide. However, when asked to draw alpha helices and beta sheets and explain the role of hydrogen bonding their drawings students exhibited a fragmented understanding that lacked coherence. Faculty are encouraged to have students draw molecular level representations to make their mental models more explicit, complete, and coherent. This is in contrast to recognition and identification tasks, which do not adequately probe mental models and molecular level understanding.

Theoretical models for electrochemical impedance spectroscopy and local ζ-potential of unfolded proteins in nanopores

Single solid-state nanopores find increasing use for electrical detection and/or manipulation of macromolecules. These applications exploit the changes in signals due to the geometry and electrical properties of the molecular species found within the nanopore. The sensitivity and resolution of such measurements are also influenced by the geometric and electrical properties of the nanopore. This paper continues the development of an analytical theory to predict the electrochemical impedance spectra of nanopores by including the influence of the presence of an unfolded protein using the variable topology finite Warburg impedance model previously published by the authors. The local excluded volume of, and charges present on, the segment of protein sampled by the nanopore are shown to influence the shape and peak frequency of the electrochemical impedance spectrum. An analytical theory is used to relate the capacitive response of the electrical double layer at the surface of the protein to both the charge density at the protein surface and the more commonly measured zeta potential. Illustrative examples show how the theory predicts that the varying sequential regions of surface charge density and excluded volume dictated by the protein primary structure may allow for an impedance-based approach to identifying unfolded proteins.

Molecular characterization of caprine KRTAP13-3 in Liaoning cashmere goat in China

Keratin-associated proteins are a main structural component of hair and wool fibers and may have effects on their characteristics. There are four KAP13 gene members that have been reported in humans, and the KAP13-3 gene has been identified in sheep and cattle, but it has not yet been identified in goats. This led us to search for the caprine KAP13-3 gene. In this study, the caprine KAP13-3 gene was polymerase chain reaction (PCR) amplified, and PCR single-stranded conformational polymorphism (PCR-SSCP) analysis was used to detect genetic variation in 214 Liaoning cashmere goats from China. PCR amplicons of 559 bp were obtained. Sequencing of the amplicons confirmed eight different sequences, and they were named alleles A–H. All of these sequences showed a high sequence homology to known KRTAP13-3 sequences from sheep and cattle. Nine nucleotide substitutions were identified within the KRTAP13-3 coding region, and all of these were nonsynonymous. All sequences showed an open reading frame of 471 nucleotides encoding 156 amino acids. This polypeptide contained high levels of serine (23.08–23.72 mol%), cysteine (10.26–11.54 mol%), and threonine (5.66 mol%). The polypeptide had high calculated pI values (9.02–9.59). The variation reported in the caprine KAP13-3 gene might affect gene expression, primary protein structure, and the character of the resultant cashmere fibers.

The Devil and Holy Water: Protein and Carbon Nanotube Hybrids

Integrating carbon nanotubes (CNTs) with biological systems to form hybrid functional assemblies is an innovative research area with great promise for medical, nanotechnology, and materials science applications. The specifics of molecular recognition and catalytic activity of proteins combined with the mechanical and electronic properties of CNTs provides opportunities for physicists, chemists, biologists, and materials scientists to understand and develop new nanomachines, sensors, or any of a number of other molecular assemblies. Researchers know relatively little about the structure, function, and spatial orientation of proteins noncovalently adsorbed on CNTs, yet because the interaction of CNTs with proteins depends strongly on the tridimensional structure of the proteins, many of these questions can be answered in simple terms. In this Account, we describe recent research investigating the properties of CNT/protein hybrids. Proteins act to solvate CNTs and may sort them according to diameter or chirality. In turn, CNTs can support and immobilize enzymes, creating functional materials. Additional applications include proteins that assemble ordered hierarchical objects containing CNTs, and CNTs that act as protein carriers for vaccines, for example. Protein/CNT hybrids can form bioscaffolds and can serve as therapeutic and imaging materials. Proteins can detect CNTs or coat them to make them biocompatible. One of the more challenging applications for protein/CNT hybrids is to make CNT substrates for cell growth and neural interfacing applications. The challenge arises from the structures' interactions with living cells, which poses questions surrounding the (nano)toxicology of CNTs and whether and how CNTs can detect biological processes or sense them as they occur. The surface chemistry of CNTs and proteins, including interactions such as π-π stacking interactions, hydrophobic interactions, surfactant-like interactions, and charge-π interactions, governs the wealth of structures, processes, and functions that appear when such different types of molecules interact. Each residue stars in one of two main roles, and understanding which residues are best suited for which type of interaction can lead to the design of new hybrids. Nonlocally, the peptide or protein primary, secondary, and tertiary structures govern the binding of proteins by CNTs. The conjugation of proteins with CNTs presents some serious difficulties both experimentally and culturally (such as bridging the "jargon barrier" across disciplines). The intersection of these fields lies between communities characterized by distinctly different approaches and methodologies. However, the examples of this Account illustrate that when this barrier is overcome, the exploitation of hybrid CNT-protein systems offers great potential.

Combined N-Glycome and N-Glycoproteome Analysis of the Lotus japonicus Seed Globulin Fraction Shows Conservation of Protein Structure and Glycosylation in Legumes

Legume food allergy, such as allergy toward peanuts and soybeans, is a health issue predicted to worsen as dietary advice recommends higher intake of legume-based foods. Lotus japonicus (Lotus) is an established legume plant model system for studies of symbiotic and pathogenic microbial interactions and, due to its well characterized genotype/phenotype and easily manipulated genome, may also be suitable for studies of legume food allergy. Here we present a comprehensive study of the Lotus N-glycoproteome. The global and site-specific N-glycan structures of Lotus seed globulins were analyzed using mass spectrometry-based glycomics and glycoproteomics techniques. In total, 19 N-glycan structures comprising high mannose (∼20%), pauci-mannosidic (∼40%), and complex forms (∼40%) were determined. The pauci-mannosidic and complex N-glycans contained high amounts of the typical plant determinants β-1,2-xylose and α-1,3-fucose. Two abundant Lotus seed N-glycoproteins were site-specifically profiled; a predicted lectin containing two fully occupied N-glycosylation sites carried predominantly pauci-mannosidic structures in different distributions. In contrast, Lotus convicilin storage protein 2 (LCP2) carried exclusively high mannose N-glycans similar to its homologue, Ara h 1, which is the major allergen in peanut. In silico investigation confirmed that peanut Ara h 1 and Lotus LCP2 are highly similar at the primary and higher protein structure levels. Hence, we suggest that Lotus has the potential to serve as a model system for studying the role of seed proteins and their glycosylation in food allergy.

Sonolysis of High Macroviscosity Systems: Hen Albumen Hydrogel

To understand molecular effects of ultrasound on protein gels (cross-linked, hydrated macromolecular systems of immeasurably high macroviscosity, but low microviscosity), the thick fraction of hen albumen was sonicated. The immeasurably high viscosity of the intact thick fraction decreased to 2.5-4.0 mPa·s (depending on the sample) after a 12 min sonication (0.14 mM of radicals were produced and 19 J g(-1) of thermal energy absorbed) indicating that the 3D protein network was degraded. SDS-PAGE analysis indicated the breaking of intermolecular S-S bridges holding together the protein network rather than the primary structure of constituent proteins. Despite the relatively large concentration of OH radical produced in the sonication time range applied, no protein cross-linking was observed which can be attributed to the high degree of protein glycosylation and protein immobility. Differential scanning calorimetry (DSC) showed that both the amount of bound water and the enthalpy of denaturation of the constituent proteins are not affected by sonication, which is consistent with the SDS-PAGE results. A small increase in sample turbidity can be attributed to the small extent of thermal denaturation occurring in the vicinity of cavitation sites.

Determination of accurate protein monoisotopic mass with the most abundant mass measurable using high-resolution mass spectrometry

While recent developments in mass spectrometry enable direct evaluation of monoisotopic masses (Mmi) of smaller compounds, protein Mmi is mostly determined based on its relationship to average mass (Mav). Here, we propose an alternative approach to determining protein Mmi based on its correlation with the most abundant mass (Mma) measurable using high-resolution mass spectrometry. To test this supposition, we first empirically calculated Mmi and Mma of 6158 Escherichia coli proteins, which helped serendipitously uncover a linear correlation between these two protein masses. With the relationship characterized, liquid chromatography–mass spectrometry was employed to measure Mma of protein samples in its ion cluster with the highest signal in the mass spectrum. Generally, our method produces a short series of likely Mmi in 1-Da steps, and the probability of each likely Mmi is assigned statistically. It is remarkable that the mass error of this Mmi is as miniscule as a few parts per million, indicating that our method is capable of determining protein Mmi with high accuracy. Benefitting from the outstanding performance of modern mass spectrometry, our approach is a significant improvement over others and should be of great utility in the rapid assessment of protein primary structures.

Development and characterization of a novel mAb against bilitranslocase - a new biomarker of renal carcinoma

BackgroundBilitranslocase (TC 2.A.65.1.1) is a bilirubin-specific membrane transporter, found on absorptive (stomach and intestine) and excretory (kidney and liver) epithelia and in vascular endothelium. Polyclonal antibodies have been raised in rabbits in the past, using a synthetic peptide corresponding to AA65-77 of rat liver bilitranslocase, as an antigen. Affinity-purified antibodies from immune sera have been found to inhibit various membrane transport functions, including the bilirubin uptake into human hepatocytes and the uptake of some flavonoids into human vascular endothelial cells. It was described by means of immunohistochemistry using polyclonal antibodies that bilitranslocase expression is severely down-regulated in clear cell renal carcinoma. The aim of our work was development and characterization of high-affinity, specific mAbs against bilitranslocase, which can be used as a potential diagnostic tool in renal cell carcinoma as well as in a wide variety of biological assays on different human tissues.Materials and methodsMice were immunized with a multi-antigen peptide corresponding to segment 65–75 of predicted primary structure of the bilitranslocase protein. By a sequence of cloning, immune- and functional tests, we aimed at obtaining a specific monoclonal antibody which recognizes a 37 kDa membrane protein, and influences the transport activity of bilitranslocase.ResultsOn the basis of previous results, specific IgM monoclonal antibodies were produced in BALB/c mice, in order to further improve and extend the immunological approach to the study of bilitranslocase in renal cancer cells as well as to develop its potential diagnostics use.ConclusionsIn this article we show an immunological approach, based on newly developed monoclonal antibodies, to a detailed biochemical and functional characterization of a protein whose gene and protein structure is still unknown. We were able to demonstrate our novel mAb as a tumor marker candidate of renal cell carcinoma, which may prove useful in the diagnostic procedures.

Physicochemical and Functional Comparability Between the Proposed Biosimilar Rituximab GP2013 and Originator Rituximab

BackgroundRegulatory approval for a biosimilar product is provided on the basis of its comparability to an originator product. A thorough physicochemical and functional comparability exercise is a key element in demonstrating biosimilarity. Here we report the characterization of a proposed biosimilar rituximab (GP2013) and originator rituximab.ObjectiveTo compare GP2013 with originator rituximab using an extensive array of routine analytical and extended characterization methods.MethodsPrimary and higher order protein structures were analyzed using a variety of methods that included high-performance liquid chromatography electrospray ionization mass spectrometry (HPLC-ESI-MS), peptide mapping with UV and MS detection, circular dichroism (CD), Fourier transform infrared (FTIR) spectroscopy, hydrogen deuterium exchange (HDX) MS, 1D 1H nuclear magnetic resonance (NMR) spectroscopy, X-ray crystallography and differential scanning calorimetry (DSC). Charge and amino acid modifications were assessed using cation exchange chromatography (CEX) and peptide mapping using reversed-phase (RP) HPLC. Boronate affinity chromatography was used to determine the relative amount of glycation. Glycans were identified and quantified after 2-aminobenzamide (2-AB) labeling and separation using normal phase HPLC with fluorescence and MS detection, respectively. Glycan site occupancy was determined using reducing capillary electrophoresis with sodium dodecyl sulfate (CE-SDS). Size heterogeneity was determined using reducing and non-reducing CE-SDS, size exclusion chromatography (SEC) and asymmetric flow field flow fractionation (AF4). Biological characterization included a series of bioassays (in vitro target binding, antibody-dependent cell-mediated cytotoxicity [ADCC], complement-dependent cytotoxicity [CDC] and apoptosis) and surface plasmon resonance (SPR) Fc receptor binding assays.ResultsIntact mass analysis of GP2013 and the heavy and light chains using RP HPLC–ESI–MS revealed the expected molecular mass of rituximab. The amino acid sequence was shown to be identical between GP2013 and the originator rituximab. Further sequence confirmation using RP-HPLC-UV/MS peptide mapping showed non-distinguishable chromatograms for Lys-C digested GP2013 and originator rituximab. The higher order structure of GP2013 was shown to be indistinguishable from originator rituximab using a large panel of redundant and orthogonal methods. GP2013 and originator rituximab were comparable with regard to charge variants, specific amino acid modifications and the glycan pattern. GP2013 was also shown to have similar purity, aggregate and particle levels when compared with the originator. Functionally, and by using a comprehensive set of bioassays and binding assays covering a broad range of rituximab’s functional activities, GP2013 could not be distinguished from originator rituximab.ConclusionGP2013 was shown to be physicochemically highly similar to originator rituximab at the level of primary and higher order structure, post-translational modifications and size variants. An extensive functional characterization package indicated that GP2013 has the same biological properties as originator rituximab.

A novel web server predicts amino acid residue protection against hydrogen–deuterium exchange

To clarify the relationship between structural elements and polypeptide chain mobility, a set of statistical analyses of structures is necessary. Because at present proteins with determined spatial structures are much less numerous than those with amino acid sequence known, it is important to be able to predict the extent of proton protection from hydrogen-deuterium (HD) exchange basing solely on the protein primary structure. Here we present a novel web server aimed to predict the degree of amino acid residue protection against HD exchange solely from the primary structure of the protein chain under study. On the basis of the amino acid sequence, the presented server offers the following three possibilities (predictors) for user's choice. First, prediction of the number of contacts occurring in this protein, which is shown to be helpful in estimating the number of protons protected against HD exchange (sensitivity 0.71). Second, probability of H-bonding in this protein, which is useful for finding the number of unprotected protons (specificity 0.71). The last is the use of an artificial predictor. Also, we report on mass spectrometry analysis of HD exchange that has been first applied to free amino acids. Its results showed a good agreement with theoretical data (number of protons) for 10 globular proteins (correlation coefficient 0.73). We pioneered in compiling two datasets of experimental HD exchange data for 35 proteins. The H-Protection server is available for users at http://bioinfo.protres.ru/ogp/ Supplementary data are available at Bioinformatics online.

Statistical analysis of pentose phosphate pathway genes from eubacteria and eukarya reveals translational selection as a major force in shaping codon usage pattern.

Comparative analysis of metabolic pathways among widely diverse species provides an excellent opportunity to extract information about the functional relation of organisms and pentose phosphate pathway exemplifies one such pathway. A comparative codon usage analysis of the pentose phosphate pathway genes of a diverse group of organisms representing different niches and the related factors affecting codon usage with special reference to the major forces influencing codon usage patterns was carried out. It was observed that organism specific codon usage bias percolates into vital metabolic pathway genes irrespective of their near universality. A clear distinction in the codon usage pattern of gram positive and gram negative bacteria, which is a major classification criterion for bacteria, in terms of pentose phosphate pathway was an important observation of this study. The codon utilization scheme in all the organisms indicates the presence of translation selection as a major force in shaping codon usage. Another key observation was the segregation of the H. sapiens genes as a separate cluster by correspondence analysis, which is primarily attributed to the different codon usage pattern in this genus along with its longer gene lengths. We have also analyzed the amino acid distribution comparison of transketolase protein primary structures among all the organisms and found that there is a certain degree of predictability in the composition profile except in A. fumigatus and H. sapiens, where few exceptions are prominent. In A. fumigatus, a human pathogen responsible for invasive aspergillosis, a significantly different codon usage pattern, which finally translated into its amino acid composition model portraying a unique profile in a key pentose phosphate pathway enzyme transketolase was observed.

Characterization of Tyr‐Leu‐Gly, a novel anxiolytic‐like peptide released from bovine α S ‐casein

We found previously that dipeptide YL exhibits orally active anxiolytic activity comparable to diazepam. The YL sequence is often observed in the primary structure of natural food proteins. In the present study, we investigated whether YL and YL analogues are released from bovine αS-casein by gastrointestinal proteases. YLG, corresponding to αS1-casein (aa 91-93), was more effectively released from αS-casein than YL by pepsin-pancreatin digestion, mimicking gastrointestinal enzymatic conditions. Using the synthetic model peptide, we determined that trypsin cleaved the N terminus of YLG, and elastase and carboxypeptidase contributed to cleave the C-terminus. YLG exhibited orally active anxiolytic-like activity in the elevated plus maze and open-field tests in mice. The anxiolytic-like activity of YLG was inhibited by WAY100135, SCH23390 or bicuculline, antagonists of serotonin 5-HT1A, dopamine D1, and GABA(A) receptors, respectively; however, YLG had no affinity for these receptors. The pepsin-pancreatin digest of αS-Casein also exhibited anxiolytic-like activity. Meanwhile, anxiolytic-like activity of α-casozepine, an αS1-casein-derived decapeptide with YL sequence in the N terminus, was blocked by WAY100135, SCH23390, or bicuculline, equally to YLG and YL; however, it was not detected in the pepsin-pancreatic digest. Taken together, we found that YLG is released after pepsin-pancreatic digestion of αS-casein and exhibits potent anxiolytic-like activity via activation of serotonin, dopamine, and the GABA receptor system.