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Not all proteins form well defined three-dimensional structures in their native states. Some amino-acid sequences appear to strongly favour the disordered state, whereas some can apparently transition between disordered and ordered states under the influence of changes in the biological environment, thereby playing an important role in processes such as signalling. Although important biologically, for the structural biologist disordered regions of proteins can be disastrous even preventing successful structure determination. The accurate prediction of disorder is therefore important, not least for directing the design of expression constructs so as to maximize the chances of successful structure determination. Such design criteria have become integral to the construct-design strategies of laboratories within the Structural Proteomics In Europe (SPINE) consortium. This paper assesses the current state of the art in disorder prediction in terms of prediction reliability and considers how best to use these methods to guide construct design. Finally, it presents a brief discussion as to how methods of prediction might be improved in the future. [ABSTRACT FROM AUTHOR]

In this paper we report the synthesis of new azabicycloalkane scaffolds, which could be exploited to obtain cRGD-based bioconjugates that may find promising application for targeted drug delivery, theranostic, and general cancer-cell labeling. By exploiting a Hosomi-Sakurai intramolecular allylation reaction we efficiently converted a silylated aldehyde precursor into 7,5-fused lactam scaffolds endowed with an exocyclic double bond. The presence of the vinyl function should make it possible to conjugate bioactive compounds to selectively carry them to tumor sites. The optimized synthetic sequence allows the gram-scale preparation of the target scaffolds in a few steps and good 39% overall yield from readily accessible materials. The high reactivity of the exocyclic olefin moiety was ascertained by performing a Heck coupling reaction with 1-bromo-4-nitrobenzene, which gave the corresponding functionalized derivatives in good (80-91%) yields. [ABSTRACT FROM AUTHOR]

A 64-kDa membrane-bound glycoprotein (gp64) of the cellular slime mold Polysphondylium pallidum, is a putative cell/cell adhesion protein identified by adhesion-blocking antibody fragments (Fab). gp64 is expressed on the cell surface of growth-phase cells and seems to mediate cell/cell adhesion. This paper describes an improved purification method based on the lipophilic nature of this protein. A critical step in the purification method is to collect an insoluble top layer appearing during ammonium sulfate precipitation. The sequence of cDNA encoding gp64 and its deduced amino acid sequence have been determined previously. Based on cDNA sequence data, the structure of gp64 protein was analyzed: almost all amino acid compositions and partial amino acid sequences of lysyl-endopeptidase-digested peptides of gp64 were determined by protein analysis; all six asparagine- linked glycosylation sites (Asn-Xaa-Ser/Thr) in fact contain carbohydrates, and all 36 cysteine residues were involved in forming disulfide bridges. From these data, gp64 seems to be a unique protein among cell/cell adhesion proteins. [ABSTRACT FROM AUTHOR]

The complete primary structure of the constant part of the μ-chain-disease protein, BOT, was established. It includes the whole CH2, CH3 and CH4 domains. Two amino acid changes were found, at positions 309 (Ser ↵ Gly) and 333 (Val ↵ Gly) (GAL numbering). In two additional monoclonal n chains (SCO and CO), the same positions showed an amino acid variability. From these data it may be concluded that four types of μ chains exist in the human: (1) GAL type with Ser-309 and Val-333; (2) OU type with Gly-309 and Val-333; (3) SCO type with Ser-309 and Gly-333; (4) BOT/CO type with Gly-309 and Gly-333. The meaning of this molecular polymorphism is discussed. [ABSTRACT FROM AUTHOR]

Recent advances in optical bioimaging have prompted the need for minimal chemical reporters that can retain the molecular recognition properties and activity profiles of biomolecules. As a result, several methodologies to reduce the size of fluorescent and Raman labels to a few atoms (e.g. single aryl fluorophores, Raman‐active triple bonds and isotopes) and embed them into building blocks (e.g. amino acids, nucleobases, sugars) to construct native‐like supramolecular structures have been described. The integration of small optical reporters into biomolecules has also led to smart molecular entities that were previously inaccessible in an expedite manner. In this article, we review recent chemical approaches to synthesize miniaturized optical tags as well as some of their multiple applications in biological imaging. [ABSTRACT FROM AUTHOR]

Abstract: So far, various approaches for phylogenetic analysis have been developed. Almost all of them put stress on analyzing nucleic acid sequences or protein primary structures. In this paper, we take the physicochemical properties of amino acids into account and introduce the hydropathy profile of amino acids into phylogenetic analysis. We find that this introduction is effectual and our method may be used to complement phylogenetic analysis. [Copyright &y& Elsevier]

Prebiotic peptide synthesis and the origin of the genetic code are central issues concerning the origin of life. The question of how they are possibly correlated on the primordial Earth remains perplexing, although numerous experiments have been carried out to explain the prebiotic chemistry of peptide synthesis and the genetic code origin. The purpose of this article is to review the chemical reactions occurred during the synthesis of peptides and the origin of the genetic code in the early Earth aqueous environment. Meanwhile, we attempt to review their relationship as well. At last, from our perspective, the chiral properties of biomolecules should be taken into account in the prebiotic chemical scenarios, which may contribute to some breakthroughs in the further research of this field. [ABSTRACT FROM AUTHOR]

Owing to the involvement in various biological processes, developing sensors for the detection of amino acids is an important area of research. Pillar[n]arenes are elegant macrocyclic systems that have been used as potential host systems for various applications including the sensing of ions and molecules. Since its inclusion in 2008, pillar[n]arenes and its derivatives have received much attention among the researchers, and these molecular systems are used as excellent host systems for the formation of inclusion complexes with various guest molecules which have relevance both in chemistry and biology. Herein, we highlight the recent developments on the sensing of amino acids by pillar[n]arene derivatives. The majority of the sensors we discussed here detect amino acids by fluorescence techniques and their sensing was further supported by various other spectroscopic methods. In some cases sensing was achieved visually by the change in colour of the host system upon interaction with the amino acids. [ABSTRACT FROM AUTHOR]

The Thr-102 mutant of yeast iso-l-cytochrome c is a useful system for the study of structure-function relationships in this important class of electron transfer proteins, but little is known about its structure. Furthermore, few assignments of individual amino acid residues in yeast iso-l-cytochrome c have been made by proton NM R. Here we report assignments for nearly half of the amino acids in the reduced Thr-102 mutant of yeast iso1-cytochrome c. We also report assignments for the oxidized Thr-102 mutant. While the crystal structure of the reduced iso-l-cytochrome c (N. B. not the Thr-102 mutant) has been reported, there is currently little structural information concerning its solution structure and none concerning the oxidized protein. There is also no information concerning the structure of either oxidation state of the Thr-102 mutant. Comparison of the chemical shift and NOE data for the reduced Thr-102 mutant and comparison of paramagnetic shifts for analogous residues between this mutant and horse-heart and tuna cytochromes c reveal that both the basic fold of Thr-102 yeast isoI-cytochrome c and the region around the site of the mutation are the same as those found in the latter two proteins. It is concluded that the results from structure function studies using the Thr-102 mutant will be applicable to eukaryotic cytochrome c in general. This knowledge allows us to proceed to a description of some mutants of yeast iso-l-cytochrome c in the next paper. [ABSTRACT FROM AUTHOR]

This paper experimentally verifies the idea presented earlier that the contact of nonpolar clusters located on the surface of protein molecules with water destabilizes proteins. It is demonstrated that protein stabilization can be achieved by artificial hydrophilization of the surface area of protein globules by chemical modification. Two experimental systems are studied for the verification of the hydrophilization approach. 1. The surface tyrosine residues of trypsin are transformed to aminotyrosines using a two-step modification procedure: nitration by tetranitromethane followed by reduction with sodium dithionite. The modified enzyme is much more stable against irreversible thermoinactivation: the stabilizing effect increases with the number of aminotyrosine residues in trypsin and the modified enzyme can become even 100 times more stable than the native one. 2. α-Chymotrypsin is covalently modified by treatment with anhydrides or chloroanhydrides of aromatic carboxytic acids. As a result, different numbers of additional carboxylic groups (up to five depending on the structure of the modifying reagent) are introduced into each Lys residue modified. Acylation of all available amino groups of α-chymotrypsin by cyclic anhydrides of pyromellitic and mellitic acids results in a substantial hydrophilization of the protein as estimated by partitioning in an aqueous Ficoll-400/Dextran-70 biphasic system. These modified enzyme preparations are extremely stable against irreversible thermal inactivation at elevated temperatures (65-98 °C); their thermostability is practically equal to the stability of proteolytic enzymes from extremely thermophilic bacteria, the most stable proteinases known to date. [ABSTRACT FROM AUTHOR]

Red kangaroo (Macropus rufus) ribonuclease was isolated from pancreatic tissue by affinity chromatography. The amino acid sequence was determined by automatic sequencing of overlapping large fragments and by analysis of shorter peptides obtained by digestion with a number of proteolytic enzymes. The polypeptide chain consists of 122 amino acid residues. Compared to other ribonucleases, the N-terminal residue and residue 114 are deleted. In other pancreatic ribonucleases position 114 is occupied by a cis proline residue in an external loop at the surface of the molecule. Other remarkable substitutions are the presence of a tyrosine residue at position 123 instead of a serine which forms a hydrogen bond with the pyrimidine ring of a nucleotide substrate, and a number of hydrophobic- hydrophilic interchanges in the sequence 51–55, which forms part of an α-helix in bovine ribo-nuclease and exhibits few substitutions in the placental mammals. Kangaroo ribonuclease contains no carbohydrate, although the enzyme possesses a recognition site for carbohydrate attachment in the sequence Asn-Val-Thr (62–64). The enzyme differs at about 35–40% of the positions from all other mammalian pancreatic ribonucleases sequenced to date, which is in agreement with the early divergence between the marsupials and the placental mammals. From fragmentary data a tentative sequence of red-necked wallaby (Macropus rufogriseus) pancreatic ribonuclease has been derived. Eight differences with the kangaroo sequence were found. [ABSTRACT FROM AUTHOR]

1. Different types of bacteriorhodopsin chromophores and their reactions are described. This includes intermediates of the reconstitution reaction of the purple complex, intermediates of the photochemical cycle and a photochemically active 500-nm chromophore. In contrast to the native chromophore (purple complex) some of these species are reducible by borohydride yielding stable retinyl proteins. 2. Hydrolysis and/or extraction of the retinyl proteins reveals that in the corresponding non reduced parent chromophores the retinal is bound either noncovalently or covalently to lysyl residues of the polypeptide chain. 3. Retinol, retinyl lysine and their retro isomers isolated from the various reduced chromophores are identified by thin-layer chromatography and mass spectrometry. 4. The absorption spectra of the retinyl proteins and binding studies with retinol and retinal indicate that the cyclohexene ring and the side chain of the retinyl moiety are forced into a coplanar conformation by interaction with the protein. The three peaked absorption band of the reduced chromophores is due to this planarized conformation and not to a retro configuration of the retinyl moiety. 5. Isomerization to retro compounds can be achieved by HCl as is known to be the case for retinyl compounds in solution. In addition photochemical isomerization is observed in the case of planarized retinyl moieties. Thus the native protein structure is responsible not only for a specific conformation of the retinyl moiety but also for its specific reactivity. [ABSTRACT FROM AUTHOR]

The primary structure of the most acidic (pI = 4.50) of the two major parvalbumins from frog (Rana esculenta) has been determined from a study of its trypsic peptides and of overlapping peptides generated by limited trypsic digestion, chymotrypsic digestion and N-bromosuccinimide cleavage of the protein. The amino acid sequence so obtained is considered in comparison with those know for other parvalbumins and for rabbit troponin-C. [ABSTRACT FROM AUTHOR]

Structural analysis of modern pharmaceutical practices allows for the identification of two rapidly growing trends: the introduction of tailor‐made amino acids and the exploitation of fluorinated motifs. Curiously, the former represents one of the most ubiquitous classes of naturally occurring compounds, whereas the latter is the most xenobiotic and comprised virtually entirely of man‐made derivatives. Herein, 39 selected compounds, featuring both of these traits in the same molecule, are profiled. The total synthesis, source of the corresponding amino acids and fluorinated residues, and medicinal chemistry aspects and biological properties of the molecules are discussed. [ABSTRACT FROM AUTHOR]

Novel emitters that do not contain traditional chromophores but only electron‐rich moieties (e. g. amine, C=O, −OH, ether, and imide), which are classified as nonconventional luminophores, have been more frequently reported. Although more and more examples have been demonstrated, their emission mechanism remains unclear. The clustering‐triggered emission (CTE) mechanism has previously been proposed to rationalize the luminescence of unconventional chromophores. Moreover, great attention has been paid to the distinctive inherent luminescence from nonaromatic biomolecules such as cellulose, starch, sugars, and nonaromatic amino acids and proteins. In this Review, we summarize these unconventional biomolecular luminophores and apply the CTE mechanism to rationalize such a phenomenon. This Review may shed new light on the understanding of intrinsic emission of nonaromatic biomolecules and decipher the intrinsic fluorescence from cells and tissues. [ABSTRACT FROM AUTHOR]

In this study, a set of hybrid images was designed as a new and up‐to‐date unexamined type of external visual representations in the chemistry classroom. One challenging teaching theme was chosen ("Amino acids and proteins"), and the hybrid images were used among novices—primary school students. For the purpose of the study, two groups were formed: experimental and control. The experimental group was taught using the hybrid images, while the control group was subjected to a traditional approach. After conducting the experiment, no statistically significant difference in the average performances between the two groups was found. However, the experimental group outperformed the control group in several test items. Additionally, misconceptions appeared within the C group. The results showed that several hybrid images with a potentially good methodological design might help students to develop conceptual understanding of some important concepts of amino acids and proteins (e.g., formation of peptide bonds). © 2019 International Union of Biochemistry and Molecular Biology, 47(6):644–655, 2019. [ABSTRACT FROM AUTHOR]

Abstract: BACKGROUND: Levodopa is a precursor of several neurotransmitters, such as dopamine, and is used in the treatment of Parkinson's disease. In this work, an alternative strategy was studied to separate levodopa from similar biomolecules using aqueous two‐phase systems (ATPS). RESULTS: Ternary ATPS composed of polyethylene glycol (PEG) 400 or ionic liquids (ILs), citrate buffer (K3C6H5O7/C6H8O7) at pH 7.0 and water, and quaternary ATPS composed of PEG 400, K3C6H5O7/C6H8O7 at pH 7.0, water and the same ILs at 5 wt%, were studied. The respective liquid–liquid phase diagrams were determined at 298 K to appraise the mixture compositions required to form two‐phase systems, followed by studies of the partition of levodopa and structurally similar biomolecules (dopamine, L‐phenylalanine, and L‐tyrosine). Their partition coefficients and extraction efficiencies have been determined, and the selectivity of the ATPS to separate levodopa from the remaining biomolecules evaluated. CONCLUSION: The results obtained indicated that PEG‐based ATPS were the most effective to separate levodopa from L‐phenylalanine while the separation from the other biomolecules was better using IL‐based ATPS, in particular those based on [P4444]Cl and [N4444]Cl, with extraction efficiencies of levodopa to the salt‐rich phase ranging between 62.7 and 74.0%, and of the remaining biomolecules to polymer/IL‐rich phase up to 91.5%. © 2017 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

ABSTRACT A series of pH/redox dual stimuli-responsive poly(2-methacryloyloxyethyl phosphorylcholine)25- block-poly( l-histidine) n (p[MPC])25- b-p[His]n, n = 20, 35, 50, and 75) copolymers consisting of a pH-responsive p(His) n block and a biocompatible phospholipid analog p(MPC) block connected by a redox-responsive disulfide linker have been synthesized. The block copolymers are self-assembled into uniform micelles (∼100 nm) in which doxorubicin (Dox) is efficiently encapsulated. The in vitro release profile shows an enhanced release of Dox at low pH (5.0) in 10 mM glutathione (GSH). The in vitro cell viability assays performed using various cell lines show that the blank hybrid micelles have no acute or intrinsic toxicity. A pH-dependent cytotoxicity is observed with the Dox-loaded micelles, especially at pH 5.0. Moreover, confocal microscopy images and flow cytometry results show the pH-dependent cellular uptake of Dox-loaded micelles. Therefore, the Dox-loaded micelles can be considered a good candidate for cancer therapy. © 2017 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2017, 55, 2061-2070 [ABSTRACT FROM AUTHOR]

Exerting control over the size, morphology, and complexity of metal and semiconductor nanoparticles and nanostructures is a requisite for exploring novel phenomena, and the potential applications of these nanomaterials. Bottom-up colloidal chemistry syntheses can benefit from using biomolecules as active elements to influence the formation of inorganic nanoparticles. In this review, we will discuss how three main biomolecule types, (namely DNA; amino acids, peptides, and proteins; and enzymes), can affect the growth of metal and semiconductor nanoparticles. We will present and discuss the templating and non-templating roles of those biomolecules, featuring key aspects and prospects of biomolecule-assisted metal and semiconductor nanoparticle growth. [ABSTRACT FROM AUTHOR]

Although the formation mechanism of biogenically metallic nanoparticles is broadly associated to enzyme mediation, major attention has been given to the role of proteins and peptides in oxido-reduction of metallic ions leading to these nanostructures. Among the wide range of biomolecules that can act not only as capping agents but also as non-enzymatic agents to form nanoparticles, disulphide bridge-containing peptides and amino acids particularly stand out. The literature proposes that they actively participate in the process of nanoparticles' synthesis, with thiols groups and disulphide bridge moieties as the reaction catalytic sites. Similarly, denaturated enzymes containing exposed S-S or S-H moieties are also able to reduce metallic ions to form nanoparticles. This mini-review is focused on the biogenic synthesis of metallic nanoparticles such as gold, silver, copper, platinum, palladium, lead and selenium, in which proteins, peptides, reductases and even oxido-reductases act as non-enzymatic catalysts of the reduction reaction, opening economically and ecologically favourable perspectives in the nanoparticles synthesis field. [ABSTRACT FROM AUTHOR]

Reactive molecular dynamics simulations are performed to study the interaction of reactive oxygen species, such as OH, HO2 and H2O2, with the endotoxic biomolecule lipid A of the gramnegative bacterium Escherichia coli. It is found that the aforementioned plasma species can destroy the lipid A, which consequently results in reducing its toxic activity. A clear difference is observed in the bond breaking mechanisms upon impact of HO2 radicals and H2O² molecules on one hand and OH radicals on the other hand. Our simulation results are in good agreement with experimental observations. [ABSTRACT FROM AUTHOR]

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RATIONALE The post-translational modification known as glycation affects the physiological properties of peptides and proteins. Glycation is particularly important during hyperglycaemia where α-dicarbonyl compounds are generated. These compounds react with proteins to generate α-dicarbonyl-derived glycation products, which are correlated with diabetic complications such as nephropathy, retinopathy, and neuropathy, among others. One of these α-dicarbonyl compounds is ethanedial, also known as glyoxal. Thereby, glyoxal binding to protein/peptides is studied by electron capture dissociation (ECD) and collisionally activated dissociation (CAD). METHODS Acetylated and non-acetylated undecapeptides containing one lysine and one arginine susceptible of glycation were reacted with glyoxal under pseudo-physiological and MeOH/H2O (50:50) conditions. Two types of glyoxal-derived AGEs were fragmented by ECD and CAD using 12 Tesla Fourier transform ion cyclotron resonance mass spectrometry (FTICRMS). RESULTS Reaction with glyoxal under different reaction conditions showed the addition of C2O and C2H2O2, which corresponded to a net increase on the peptide mass of 39.9949 Da and 58.0055 Da, respectively. The binding site was assigned within an error <1 ppm, using ECD and CAD. The results indicated that both types of glyoxal-derived AGEs are formed at the side chain of arginine located in position 3. CONCLUSIONS Types and binding sites of glyoxal-derived AGEs were investigated in peptides containing one arginine-one lysine using FTICRMS. Two net mass additions to the mass of the peptide were assigned as C2O and C2H2O2, which were located at the arginine side chain. In addition, these mass additions (C2O and C2H2O2) observed in the peptides were unaffected by different reaction conditions. Copyright © 2013 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Light‐responsive vesicles: Derivatives of an α‐amino acid with two azobenzene side chains self‐assemble into vesicular structures that undergo morphological transitions upon light irradiation (see scheme). This is the first example of photoresponsive vesicle‐like supramolecular structures formed exclusively by peptides or derivatized amino acids. The entrapment and subsequent light‐induced release of functionalized gold nanoparticles are also shown. [ABSTRACT FROM AUTHOR]

On the move: Two isostructural, homochiral CuII coordination frameworks based on amino acids (D‐ and L‐PGA) were synthesized (see figure). Dynamic behavior by solid‐state structural transformation in single‐crystal‐to‐single‐crystal fashion was demonstrated. The extent of structural dynamism was shown by guest inclusion studies. Reversible sol–gel formation and anion‐tuning morphology of the compounds is also discussed. [ABSTRACT FROM AUTHOR]

The molecular mechanism by which dual-specificity RasGAPs of the Gap1 subfamily activate the GTP hydrolysis of both Rap and Ras is an unresolved phenomenon. RasGAPs and RapGAPs use different strategies to stimulate the GTPase reaction of their cognate G-proteins. RasGAPs contribute an arginine finger to orient through the Gln61 of Ras the nucleophilic water molecule. RapGAP contributes an asparagine (Asn thumb) into the active site to substitute for the missing Gln61. Here, by using steady-state kinetic assays and time-resolved Fourier-transform infrared spectroscopy (FTIR) experiments with wild type and mutant proteins, we unravel the remarkable mechanism for the specificity switch. The plasticity of GAP1IP4BP and RASAL is mediated by the extra GTPase-activating protein (GAP) domains, which promote a different orientation of Ras and Rap's switch-II and catalytic residues in the active site. Thereby, Gln63 in Rap adopts the catalytic role normally taken by Gln61 of Ras. This re-orientation requires specific interactions between switch-II of Rap and helix-α6 of GAPs. This supports the notion that the specificities of fl proteins versus GAP domains are potentially different. [ABSTRACT FROM AUTHOR]

In Drosophila, the PIWI proteins, Aubergine (Aub), AGO3, and Piwi are expressed in germlines and function in silencing transposons by associating with PIWI-interacting RNAs (piRNAs). Recent studies show that PIWI proteins contain symmetric dimethyl-arginines (sDMAs) and that dPRMT5/Capsuleen/DART5 is the modifying enzyme. Here, we show that Tudor (Tud), one of Tud domain-containing proteins, associates with Aub and AGO3, specifically through their sDMA modifications and that these three proteins form heteromeric complexes. piRNA precursor-like molecules are detected in these complexes. The expression levels of Aub and AGO3, along with their degree of sDMA modification, were not changed by tud mutations. However, the population of transposon-derived piRNAs associated with Aub and AGO3 was altered by tud mutations, whereas the total amounts of small RNAs on Aub and AGO3 was increased. Loss of dprmt5 did not change the stability of Aub, but impaired its association with Tud and lowered piRNA association with Aub. Thus, in germline cells, piRNAs are quality-controlled by dPRMT5 that modifies PIWI proteins, in tight association with Tud. [ABSTRACT FROM AUTHOR]

The recent discovery of new potent therapeutic molecules that do not reach the clinic due to poor delivery and low bioavailability have made of delivery a key stone in therapeutic development. Several technologies have been designed to improve cellular uptake of therapeutic molecules, including cell-penetrating peptides (CPPs). CPPs were first discovered based on the potency of several proteins to enter cells. Numerous CPPs have been described so far, which can be grouped into two major classes, the first requiring chemical linkage with the drug for cellular internalization and the second involving formation of stable, non-covalent complexes with drugs. Nowadays, CPPs constitute very promising tools for non-invasive cellular import of cargo and have been successfully applied for in vitro and in vivo delivery of therapeutic molecules varying from small chemical molecule, nucleic acids, proteins, peptides, liposomes and particles. This review will focus on the structure/function and cellular uptake mechanism of CPPs in the general context of drug delivery. We will also highlight the application of peptide carriers for the delivery of therapeutic molecules and provide an update of their clinical evaluation. [ABSTRACT FROM AUTHOR]

Proteins and insolubles were recovered from whole Antarctic krill via novel isoelectric solubilization/precipitation using different pH treatments. The protein recovery yield was 45% to 50% (dry basis). The recovered proteins had higher ( P < 0.05) content of essential amino acids (EAAs) and non-EAAs as well as higher ( P < 0.05) ratio of total EAA/total AA than whole krill. The EAAs constituted almost 50% of total AAs. The least extreme pH treatments (pHs 3 and 12) yielded highest ( P < 0.05) content of EAAs. The quality of recovered proteins was high based on EAAs meeting FAO/WHO/UNU recommendations for adults and infants. The basic pH yielded proteins with the lowest ( P < 0.05) amount of minerals and the highest ( P < 0.05) amount of Ca, P, and Mg in the insolubles when compared to the acidic treatments. However, both basic and acidic treatments effectively removed minerals from recovered proteins without the removal of the exoskeleton before processing. Therefore, besides high-quality proteins, the insolubles may provide a mineral supplement in the animal diet. [ABSTRACT FROM AUTHOR]

We previously found that selective restriction of amino acids inhibits invasion of two androgen-independent human prostate cancer cell lines, DU145 and PC3. Here we show that the restriction of tyrosine (Tyr) and phenylalanine (Phe), methionine (Met) or glutamine (Gln) modulates the activity of G proteins and affects the balance between two actin-binding proteins, cofilin and profilin, in these two cell lines. Selective amino acid restriction differentially reduces G protein binding to GTP in DU145 cells. Tyr/Phe deprivation reduces the amount of Rho-GTP and Rac1-GTP. Met deprivation reduces the amount of Ras-GTP and Rho-GTP, and Gln deprivation decreases Ras-GTP, Rac-GTP, and Cdc42-GTP. Restriction of these amino acids increases the amount of profilin, cofilin and phosphorylation of cofilin-Ser3. Increased PAK1 expression and phosphorylation of PAK1-Thr423, and Ser199/204 are consistent with the increased phosphorylation of LIMK1-Thr508. In PC3 cells, Tyr/Phe or Gln deprivation reduces the amount of Ras-GTP, and all of the examined amino acid restrictions reduce the amount of profilin. PAK1, LIMK1 and cofilin are not significantly altered. These data reveal that specific amino acid deprivation differentially affects actin dynamics in DU145 and PC3. Modulation on Rho, Rac, PAK1, and LIMK1 likely alter the balance between cofilin and profilin in DU145 cells. In contrast, profilin is inhibited in PC3 cells. These effects modulate directionality and motility to inhibit invasion. J. Cell. Physiol. 217: 184–193, 2008. © 2008 Wiley-Liss, Inc. [ABSTRACT FROM AUTHOR]

The function of the CNS depends on the correct regulation of neurotransmitter receptors by interacting proteins. Here, we screened a retinal cDNA library for proteins interacting with the intracellular C-terminus of the metabotropic glutamate receptor isoform 8a (mGluR8a). The band 4.1B protein binds to the C-termini of mGluR8a and mGluR8b, co-localizes with these glutamate receptors in transfected mammalian cells, facilitates their cell surface expression and inhibits the mGluR8 mediated reduction of intracellular cAMP concentrations. In contrast, no interaction with 4.1B was observed for other mGluRs tested. Amino acids encoded by exons 19 and 20 of 4.1B and a stretch of four basic amino acids present in the mGluR8 C-termini mediate the protein interaction. Besides binding to 4.1B, mGluR8 isoforms interact with 4.1G, 4.1N, and 4.1R. Because band 4.1 transcripts undergo extensive alternative splicing, we analyzed the splicing pattern of interacting regions and detected a 4.1B isoform expressed specifically in the retina. Within this tissue, mGluR8 and 4.1B, 4.1G, 4.1N, and 4.1R show a comparable distribution, being expressed in both synaptic layers and in somata of the ganglion cell layer. In summary, our studies identified band 4.1 proteins as new players for the mGluR8 mediated signal transduction. [ABSTRACT FROM AUTHOR]

Three ammonium transport systems of the Mep/Amt/Rh superfamily contribute to ammonium uptake for use as a nitrogen source in Saccharomyces cerevisiae. A specific sensor role has further been proposed for Mep2 in the stimulation of pseudohyphal development during ammonium limitation. Optimal ammonium transport by the Mep proteins requires the Npr1 kinase, a potential target of the target-of-rapamycin signalling pathway. We show here that the growth impairment of cells lacking Npr1 on many nitrogen sources is shared by cells deprived of the three Mep proteins and is a consequence of deficient ammonium retrieval. Expression of a newly isolated Npr1-independent and hyperactive Mep2 in cells lacking Npr1 and/or the Mep proteins restores growth on low ammonium but also on other nitrogen sources. This hyperactive Mep2 variant efficiently counteracts ammonium excretion. Hence, ammonium uptake activity plays an important role in compensating for leakage of catabolic ammonium. Our data also reveal that the requirement of Npr1 for ammonium-induced pseudohyphal growth is an indirect consequence of its necessity for Mep2-mediated ammonium transport. Finally, we show that Mep2 participates, through ammonium leakage compensation, in pseudohyphal growth induced by amino acid starvation. This argues further in favour of tight coupling of Mep2 transport and sensor functions. [ABSTRACT FROM AUTHOR]

This study investigated the functional impact ofO-linked glycosylation of von Willebrand Factor (VWF) A1 domains on the interaction with platelet receptors. Native or mutant VWF-A1-domains were transiently overexpressed on COS-7 cells as membrane glycosylphosphatidylinositol (GPI)-anchored FLAG-tagged fusion proteins. Cytofluometric analysis assured comparable levels of A1-domain expression among native and mutant homologues as well as for different culture conditions. Expressing native VWF-A1-domains underO-linked glycosylation blocking conditions increased the platelet aggregatory responses observed for fully glycosylated forms. Utilizing a neuronal network for prediction ofO-linked glycosylation of mammalian proteins, threonine (T) and serine (S) residues located in the VWF-A1-loop flanking regions– not in the loop itself– were determined to be glycosylated n-terminal at amino acids T485, S490, T492 and T493 and c-terminal at T705. Simultaneous selective charge-to-alanine mutation of S490, T492 and T493 led to gain in aggregatory responses. When compared with native forms, equivalent alterations of T485 did not dictate functional differences. Any alanine-substitution for T705 revealed a substantial loss in aggregatory effects– possibly as a result of structural desintegration of the VWF-A1-binding site for glycoprotein (GP) Ib. These data suggest specificO-linked glycosylation of the amino-terminal VWF-A1-loop-flanking region to have a negative regulatory impact on the A1-domain affinity of non-activated human VWF for human platelet-GPIb. [ABSTRACT FROM AUTHOR]

The role of vertebrate histone proteins or histone derived peptides as innate immune effectors has only recently been appreciated. In this study, high levels of core histone proteins H2A, H2B, H3 and H4 were found in hemocytes from the Pacific white shrimp,Litopenaeus vannamei. The proteins were identified by in-gel digestion, mass spectrometry analysis, and homology searching. TheL. vannameihistone proteins were found to be highly homologous to histones of other species. Based on this homology, histone H2A was cloned and its N-terminus was found to resemble the known antimicrobial histone peptides buforin I, parasin, and hipposin. Consequently, a 38 amino acid synthetic peptide identical to the N-terminus of shrimp H2A was synthesized and assayed, along with endogenous histones H2A, H2B, and H4, for growth inhibition againstMicrococcus luteus. Histone H2A, purified to homogeneity, completely inhibited growth of the Gram-positive bacterium at 4.5 µmwhile a mixture of histones H2B and H4 was active at 3 µm. In addition, a fraction containing a fragment of histone H1 was also found to be active. The synthetic peptide similar to buforin was active at submicromolar concentrations. These data indicate, for the first time, that shrimp hemocyte histone proteins possess antimicrobial activity and represent a defense mechanism previously unreported in an invertebrate. Histones may be a component of innate immunity more widely conserved, and of earlier origin, than previously thought. [ABSTRACT FROM AUTHOR]

The HbpR protein is theσ54-dependent transcription activator for 2-hydroxybiphenyl degradation inPseudomonas azelaica. The ability of HbpR and XylR, which share 35% amino acid sequence identity, to cross-activate the P hbpC and P u promoters was investigated by determining HbpR- or XylR-mediated luciferase expression and by DNA binding assays. XylR measurably activated the P hbpC promoter in the presence of the effector m-xylene, both inEscherichia coliandPseudomonas putida. HbpR weakly stimulated the P u promoter inE. colibut not inP. azelaica. Poor HbpR-dependent activation from P u was caused by a weak binding to the operator region. To create promoters efficiently activated by both regulators, the HbpR binding sites on P hbpC were gradually changed into the XylR binding sites of P u by site-directed mutagenesis. Inducible luciferase expression from mutated promoters was tested inE. colion a two plasmid system, and from mono copy gene fusions inP. azelaicaandP. putida. Some mutants were efficiently activated by both HbpR and XylR, showing that promoters can be created which are permissive for both regulators. Others achieved a higher XylR-dependent transcription than from P u itself. Mutants were also obtained which displayed a tenfold lower uninduced expression level by HbpR than the wild-type P hbpC, while keeping the same maximal induction level. On the basis of these results, a dual-responsive bioreporter strain ofP. azelaicawas created, containing both XylR and HbpR, and activating luciferase expression from the same single promoter independently withm-xylene and 2-hydroxybiphenyl. [ABSTRACT FROM AUTHOR]

A novel tripeptide motif called a nest has recently been described in proteins with the function of binding anionic, or partially anionic, atoms such as carbonyl 0 atoms. In the present work, a search for nests in small polypeptides stored in the Cambridge Structural Database is reported. 37 unique examples were found: over half form part of hydrogen-bond arrangements resembling those in proteins, such as Schellman/ paperclip loop motifs, various types of β-turn and Asx-turns or Ser/Thr-turns, while a third are in novel situations, some involving binding to anionic groups from other molecules within the crystal complex. An example is the antibiotic vancomycin, which incorporates a prominent nest forming part of a peptide-binding site. This nest binds the carboxylate of the C-terminal D-alanine of the bacterial cell-wall precursor peptide, thereby inhibiting the final step of bacterial cell-wall synthesis. As in proteins, a number of nests occur in short peptides with an alternating glycine/L-amino- acid sequence but, uniquely to non-ribosomally synthesized short peptides, several nests within them are constructed from alternating D- and L-amino acids, and such sequences seem to specially favour nests. [ABSTRACT FROM AUTHOR]

Focuses on the study which shows that lysine acetylation has been shown to occur in many protein targets. Mechanisms used by lysine acetylation; Docking sites for bromodomain proteins; Relationship between acetyllysine and bromodomains.

A candidate human imidazoline-1 receptor, designated imidazoline receptor antisera-selected (IRAS) protein, was cloned based on immunoreactivity with antiserum against a purified imidazoline receptor binding peptide (IRBP antiserum). Human IRAS is 167 kD in size, different from 33- to 85-kD IRBP bands previously linked to the human platelet I1 receptor. To explore the possible relationship between IRAS and these smaller proteins, seven different epitope-specific antisera against IRAS were raised in rabbits for comparison with IRBP antiserum. Focus was on antiserum227-241, corresponding to amino acids #227 to 241 in IRAS, because this antiserum was found uniquely able to immunoprecipitate non-denatured 85-kD and 170-kD forms of IRAS from a human megakaryoblastoma cell line (MEG01), a model of platelet-producing cells. Human platelets lacked the 170-kD form of IRAS, but 33-kD and 85-kD bands were detectable and seemed to be possible fragments of full-length IRAS. The intensity of the 85-kD band detected by antiserum227-241 was significantly correlated (r 5 0.62, P 5 0.04) with the intensity of the 33-kD band across 11 human platelet samples. A positive correlation between the intensities of the 33-kD and 85-kD bands is consistent with both being fragments of IRAS. [ABSTRACT FROM AUTHOR]

The human I1-imidazoline receptor candidate gene, iras, has previously been cloned and mapped to locus 3p21.1-9 (also known as Nischarin; accession #AC006208). By comparison to a database of expressed sequence tags (ESTs), three alternatively spliced transcripts have been deduced. A map of 21 exons was constructed for the medium-length transcript (IRAS-M) containing 5,232 base pairs (bp) and encoding 1,504 amino acids (aas). Introns 13B and 13C are inserted into the two alternative transcripts, forming IRAS-S and IRAS-L mRNA (short and long isoforms). Northern blots confirmed the existence of these mRNA isoforms. In most brain regions the order of mRNA abundance was IRAS-M > IRAS-L > IRAS-S mRNA. Although aas 1 through 510 are theoretically identical, truncated proteins could be derived from IRAS-S (2,678 bp transcript yields 515 aas) and IRAS-L (9,457 bp transcript yields 583 aas). Because exon-16 of the iras gene has been proposed to encode the functional domains of imidazoline and a-5 integrin binding, only IRAS-M is expected to possess I1 receptor properties. Subtype-selective cDNA expression constructs were therefore generated and used to transfect CHO cells. High-affinity I1 binding was endowed by IRAS-M and IRAS-L, but not by IRAS-S transfection. [ABSTRACT FROM AUTHOR]

Presents a study that proposed a laboratory exercise in biochemistry to guide students to the comprehension of basic aspects in determining structure and physicochemical properties of proteins. Amino acid composition of human proteins and silk fibroin; Flow chart for fractionation of plasma proteins by precipitation with ammonium sulfate; Relative dimensions of human proteins.

Directly upstream of the Halobacterium salinarum transducer genes basT and htpIV we identified two open reading frames (orfs) with significant homologies to genes encoding binding proteins for amino acids and compatible solutes, respectively. Behavioral testing of deletion mutants indicates that halobacterial chemotaxis towards branched-chain amino acids as well as compatible osmolytes of the betaine family requires both a binding and a transducer protein. We therefore named the binding/transducer proteins BasB/BasT for branched-chain and sulfur-containing amino acids and CosB/CosT for compatible solutes. Our data support a signaling mechanism with the binding proteins functioning as lipid-anchored receptors interacting with the extracellular domain of their cognate transducers. Inspection of the halobacterial genome suggests that BasB and CosB exclusively mediate chemotaxis responses without any additional role in transport, which is in contrast to bacterial binding proteins, which are always part of ABC transport systems. The CosB/CosT system is the first instance of a chemotaxis signaling pathway for organic osmolytes in the living world and natural abundance 13C-NMR analysis of cytoplasmic extracts suggests that H.salinarum utilizes these solutes for osmotic adaptation. [ABSTRACT FROM AUTHOR]

A Sin3-interacting domain (SID) originally described in Mad proteins is necessary for both transcriptional repression and growth suppression by these transcription factors. We recently reported that a structurally and functionally related Mad1-like SID is also present in five Sp1-like repressor proteins (TIEG1, TIEG2, BTEB1, BTEB3 and BTEB4), demonstrating that SID-mSin3A interactions have a wider functional impact on transcriptional repression. SID-mSin3A interaction is necessary for the anti-proliferative function of Mad, TIEG and BTEB proteins. It remains to be established, however, whether the SID-mSin3A interaction is constitutive or regulated. Here, we describe that the Mad1-like SID domain of the Sp1-like repressor TIEG2 is inhibited by the epidermal growth factor (EGF)-Ras-MEK1-ERK2 signaling pathway, via phosphorylation of four serine/threonine sites adjacent to the SID. This phenomenon disrupts the SID-mSin3A interaction and thereby inhibits TIEG2's repression activity. Thus, these results show for the first time that the repression of a SID-containing protein is regulated by signaling rather than functioning in a constitutive manner, extending our understanding of how the function of SID-containing repressors may be controlled. [ABSTRACT FROM AUTHOR]

11S REGs (PA28s) are multimeric rings that bind proteasomes and stimulate peptide hydrolysis. Whereas REGα activates proteasomal hydrolysis of peptides with hydrophobic, acidic or basic residues in the P1 position, REGγ only activates cleavage after basic residues. We have isolated REGγ mutants capable of activating the hydrolysis of fluorogenic peptides diagnostic for all three active proteasome β subunits. The most robust REGγ specificity mutants involve substitution of Glu or Asp for Lys188. REGγ(K188ELD) variants are virtually identical to REGα in proteasome activation but assemble into less stable hepta- mers/hexamers. Based on the REGα crystal structure, Lys188 of REGγ faces the aqueous channel through the heptamer, raising the possibility that REG channels function as substrate-selective gates. However, covalent modification of proteasome chymotrypsin- like subunits by 125I-YL3-VS demonstrates that REGγ(K188E)'s activation of all three proteasome active sites is not due to relaxed gating. We propose that decreased stability of REGγ(K188E) heptamers allows them to change conformation upon proteasome binding, thus relieving inhibition of the CT and PGPH sites normally imposed by the wild-type REGγ molecule. [ABSTRACT FROM AUTHOR]

The presence of endoproteinases in the intercellular washing fluid of the first wheat (Triticum aestivum) leaf 13 days after sowing was investigated. Two activities were detected after separation of the intercellular fluid proteins by SDS-PAGE using slab gels containing gelatin as substrate. Their sizes were 70 and 100 kDa. Both enzymes hydrolyzed gelatin and casein. They also seem to be involved in the degradation of a 40 kDa protein component of the intercellular fluid. These results indicate that both enzymes are endoproteinases. They were also obtained from leaves previously submitted to intercellular fluid extraction. However, the 100 kDa enzyme yield was low, indicating that it is mainly located in the intercellular washing fluid. Assays performed with either specific substrates or inhibitors indicate that both enzymes are serine proteinases. [ABSTRACT FROM AUTHOR]

Cereal plants were grown for 3 weeks under sterile conditions in solution culture with their roots either unrestricted or growing between glass ballotini. The exudation of both amino acids and carbohydrates was increased when the roots were grown through a solid medium and the total quantity exuded was equivalent to about 9% of the dry matter increment of the roots. [ABSTRACT FROM AUTHOR]

Autumnal changes in total nitrogen, salt‐extractable protein and amino acid concentrations in leaves and adjacent bark of black alder [Ainus glutinosa (L.) Gaertn.], eastern cottonwood (Populus deltoides Bartr. ex Marsh.) and white basswood (Tilia heterophylla Vent.) were determined for trees growing on minespoils and a prairiederived loamy soil in central Illinois. The composition of free amino acids in foliage was also determined at peak concentration for each tree species during late senescence. Total nitrogen concentration in the leaves decreased slowly throughout most of the fall for all species. In the final stages of senescence, total leaf nitrogen concentrations were about halved in eastern cottonwood and white basswood but continued to decrease slowly in black alder. The concentration of salt‐extractable proteins in leaves of all species peaked early in the fall and then declined prior to leaf abscission. This decline coincided with an increase in the concentration of free amino acids in the leaves. The increase stabilized in both eastern poplar and white basswood but continued in black alder. Glutamine in black alder and eastern cottonwood, and asparagine in white basswood were the most abundant free amino acids at the time of peak concentration of total free amino acids in senescent leaves. Bark of trees of all species had higher nitrogen concentrations and higher proportions of salt‐extractable proteins to estimated total proteins after leaf senescence than during the preceding summer. Results indicate that autumnal fluxes in leaf and bark nitrogen fractions of alder can differ substantially from fluxes in other broadleaved winter‐deciduous trees in a way which suggests that alder does not effectively conserve leaf nitrogen through retranslocation to bark tissue. [ABSTRACT FROM AUTHOR]

The transition from the growth to the maturation phase in developing seeds of Agrostemma githago L. was found to coincide with major changes in the rate of protein synthesis, the kinds of proteins synthesized, and the composition of the non‐proteinbound amino acid pool. Coincident changes were observed in viability and the ability to withstand desiccation. Desiccated mature Agrostemma seeds are dormant and need at least three months of after‐ripening. In imbibed dormant and after‐ripened seeds no synthesis of storage proteins was observed with the exception of one particular set of storage proteins. Dormant and after‐ripened seeds synthesized the same kinds of proteins during early imbibition, indicating an almost identical metabolic state which differs considerably from that of developing seeds. [ABSTRACT FROM AUTHOR]

The N-trifluoroacetyl-L-prolyl-leucyl methyl ester (TFA-L- prolyl-leucine) was synthesized to quantitate the amounts of D- and L-leucine in the amino acid pool fraction of tobacco callus tissue. TFA-L-prolyl-D-leucine and TFA-L-prolyl-L-leucine could be separated on gas liquid chromatography, with the former eluting about 45 s before the latter. The chromatographed TFA-L-prolyl-L-leucine was collected from the gas liquid chromatograph and found to contain equal molar amounts of leucine and proline. The identity of the two gas liquid chromatography peaks was confirmed by gas liquid chromatography/mass spectrometry. Although callus tissue incorporated both exogenously added D- and L-leucine into its amino acid pool fraction, the rate of uptake of D-leucine appeared to be substantially less than that of L-leucine. When callus tissue was labeled on ³H-L-leucine medium for 24 h and then transferred to nonradioactive medium for 24 h, about 50% of the tritium remaining in the leucine pool was not available for protein synthesis (nonprecursor pool fraction). However, all of the tritium was associated with L-leucine, and the specific radioactivity of leucine in collected TFA-L-prolyl-leucine was the same as that of leucine in the crude pool fraction. Thus, the nonprecursor pool is L-leucine. [ABSTRACT FROM AUTHOR]

To identify the ssDNA-binding domain of Escherichia coli RecA protein, we examined the ssDNA-binding capabilities of synthetic peptides, the sequences of which were derived from the C- and N-termini and from sequences within loops L1 and L2 of the RecA molecule identified from the crystal structure. Synthetic peptides derived from amino acid residues 185–219 of several bacterial RecA proteins, which include loop L2 of RecA, bound to ssDNA in filter-binding assays, whereas three separate synthetic peptides corresponding to single point mutants of E. coti RecA in this region did not. The binding of RecA to ssDNA examined using a gel-shill assay was inhibited by a synthetic peptide derived from this ssDNA-binding region, but not by synthetic peptides derived from amino acid residues 301–329 of the C-terminus or from N-terminal residues 6–39. A peptide corresponding to amino acid positions 152–169 of the RecA molecule and spanning loop L1 and its flanking regions did not bind ssDNA at peptide concentrations up to 250 μM. We have also defined a synthetic 20-amino-acid peptide that comprises amino acid residues 193–212 and includes loop L2 of RecA as the minimum unit that can bind to ssDNA from this region of RecA. Finally, two maltose-binding protein-RecA fusion proteins were made, one containing amino acid residues 185–224 of RecA and the other the last 51 C-terminal residues of RecA (amino acid residues 303–353). In contrast to the C-terminus-derived fusion protein, the fusion protein containing the putative DNA-binding site demonstrated significant binding to single-stranded oligonucleotides in both filter-binding and gel-shift assays. These findings suggest that a portion of the region extending from amino acid residues 193–212 is either part of or the whole ssDNA-binding domain of the RecA protein. [ABSTRACT FROM AUTHOR]

A purification procedure consisting of DEAE-Sephacel chromatography, heparin-Sepharose CL-6B chromatography and Mono-S chromatography led to the isolation of three proteins endowed with RNase activity from the thermoacidophilic archaebacterium Sulfolobus solfataricus. They were referred to as p1, p2 and p3, according to their elution order from the Mono-S column. Complete amino acid sequence of p2 and partial sequence of p3 displayed high sequence similarity to the 7-kDa DNA-binding proteins previously isolated in Sulfolobus strains [Choli, T., Wittman-Liebold, B. & Reinhardt, R. (1988) J. Biol. Chem. 263, 7087-7093]. The molecular mass of p2, calculated from sequence data, was 7.02 kDa, which compares fairly well with the value of 7.4 kDa determined by SDS/PAGE. Gel filtration of the molecule under native conditions displayed, however, a largely prevailing form with an assessed molecular mass of 13.0 kDa, which points to a dimeric structure. Kinetic characterization of protein p2 showed a broad pH optimum in the range 6.7 - 7.6 using yeast RNA as substrate; also, it was shown that activity was unaffected by EDTA, Mg2+ and phosphate. The enzyme did not accept as substrate any homopolyribonucleotide, which points to a rather narrow substrate specificity. This was also confirmed by incubating p2 with tRNAfMet Met (fMet, N-formylmethionine) from Escherichia coli: the hydrolysis products were thus identified as 3'-phosphooligonucleotides. [ABSTRACT FROM AUTHOR]