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1. A new diagonal etectrophoretic technique for determining the order of the lysine-containing tryptic peptides of a protein is described. The protein is converted into its trifluoracetyl derivative, digested enzymatically (or chemically), and the resulting peptides separated by paper electrophoresis. The paper is then treated with ammonia vapour, which re-exposes the ε-amino groups of the lysine residues, and submitted to a second electrophoresis at right angles to the first direction. Peptides containing lysine residues, together with the N-terminal peptide of the protein, are found to lie off a diagonal formed by all other peptides, whence they may be readily purified. A study of these peptides enables the order of the lysine-containing tryptic peptides in the protein to be deduced. 2. The technique has been successfully tested with insulin. 3. When the method was applied to porcine pepsin, the four tryptic peptides isolated were easily ordered and the carboxyl-terminal sequence of the protein shown to be Arg-Gln-Tyr-TyrThr-Val-Phe-Asp-Arg-Ala-Asn-Asn-Lys-Val-Gly-Leu-Ala-Pro-Val-Ala. The thee basic ammo acid residues in the molecule are thus found clustering towards the C-terminus of the polypeptide chain. 4. A common ancestral gene for porcine pepsin and bovine (calf) rennin is suggested by the close homology between the C-terminal sequents of the two proteins. [ABSTRACT FROM AUTHOR]

Summary: Maize grain is deficient in lysine. While the opaque2 mutation increases grain lysine, o2 is a transcription factor that regulates a wide network of genes beyond zeins, which leads to pleiotropic and often negative effects. Additionally, the drastic reduction in 19 kDa and 22 kDa alpha‐zeins causes a floury kernel, unsuitable for agricultural use. Quality protein maize (QPM) overcame the undesirable kernel texture through the introgression of modifying alleles. However, QPM still lacks a functional o2 transcription factor, which has a penalty on non‐lysine amino acids due to the o2 mutation. CRISPR/cas9 gives researchers the ability to directly target genes of interest. In this paper, gene editing was used to specifically target the 19 kDa alpha zein gene family. This allows for proteome rebalancing to occur without an o2 mutation and without a total alpha‐zein knockout. The results showed that editing some, but not all, of the 19 kDa zeins resulted in up to 30% more lysine. An edited line displayed an increase of 30% over the wild type. While not quite the 55% lysine increase displayed by QPM, the line had little collateral impact on other amino acid levels compared to QPM. Additionally, the edited line containing a partially reduced 19 kDa showed an advantage in kernel texture that had a complete 19 kDa knockout. These results serve as proof of concept that editing the 19 kDa alpha‐zein family alone can enhance lysine while retaining vitreous endosperm and a functional O2 transcription factor. [ABSTRACT FROM AUTHOR]

Methods for the site-specific modification of proteins are useful for introducing biological probes into proteins and engineering proteins with novel activities. Herein, we genetically encode a novel noncanonical amino acid (ncAA) that contains an aryl isothiocyanate group which can form stable thiourea crosslinks with amines under mild conditions. We show that this ncAA (pNCSF) allows the selective conjugation of proteins to amine-containing molecular probes through formation of a thiourea bridge. pNCSF was also used to replace a native salt bridge in myoglobin with an intramolecular crosslink to a proximal Lys residue, leading to increased thermal stability. Finally, we show that pNCSF can form stable intermolecular crosslinks between two interacting proteins. [ABSTRACT FROM AUTHOR]

Data analysis utilized four growth experiments with mixed diets limiting in lysine, in threonine, and in methionine respectively. All male juvenile Orechromis niloticus [12 g average body weight instead of average (BW) at start, four repetition tanks per diet, 56 days experimental period] provided the database for application of an exponential N-utilization model. Imposing amino acid efficiency data were utilized for modelling of amino acid requirements depending on the level of daily protein deposition. According to the observed average dietary amino acid efficiency of the amino acids under study, 16.3 g/kg of lysine, 8.3 g/kg of threonine and 7.3 g/kg of methionine were established as required in feed content for 187 mg daily protein deposition (50 g BW, feed intake at 3% of BW). Further modelling by use of graded dietary amino acid efficiency yielded strong evidence for the significance of this dietary factor of influence. Current data analysis has led to conclusion, that the applied non-linear modelling of amino acid requirements is an advantageous approach because of its quantitative reflection of graded dietary amino acid efficiency corresponding to protein deposition data. The procedure has the potential to contribute to alternate approaches for improved reliability of recommended quantitative amino acid supply in fish nutrition. [ABSTRACT FROM AUTHOR]

FLOWERING LOCUS C ( FLC) is a major regulator of flowering time in Arabidopsis. Repression of FLC occurs in response to prolonged cold exposure (vernalization) and is associated with an enrichment of the repressive histone modification trimethylated H3 lysine 27 (H3K27me3) and a depletion of the active histone modification H3K4me3 at FLC chromatin. In two cases genes adjacent to FLC are also repressed by vernalization. NEOMYCIN PHOSPHOTRANSFERASE II ( NPTII) adjacent to an FLC transgene is repressed by vernalization, and this is associated with an increase in H3K27me3, demonstrating that the epigenetic repression of FLC can confer a repressed epigenetic state to an adjacent transcription unit. The second case involves the two genes adjacent to the endogenous FLC gene, UPSTREAM OF FLC ( UFC) and DOWNSTREAM OF FLC ( DFC). Both genes are repressed by vernalization ( Finnegan et al., 2004 ), but they require neither cis-acting nor trans-acting factors derived from the FLC gene nor the VERNALIZATION2 (VRN2) complex which trimethylates H3K27. This demonstrates that there are two different mechanisms of gene repression by vernalization. We further show that repression and H3K27 trimethylation of FLC still occurs in mutants of the VRN2 complex. In contrast, the VRN2 complex is essential for repression and H3K27 trimethylation of the FLC-related MADS AFFECTING FLOWERING ( MAF) genes by vernalization. This suggest that other proteins are able to repress FLC, but not MAF, gene expression. [ABSTRACT FROM AUTHOR]

The legumes Canavalia ensiformis and Mucuna pruriens are underexploited in tropical Mexico. Their seeds have good nutritional potential, but contain antinutritional factors. Physicochemical and antinutritional properties were determined for raw flours (RF) and protein isolates (PI) produced from these legumes. Protein content in the PI was 737 g kg–1 for C. ensiformis and 666 g kg–1 for M. pruriens. Protein isolation improved in vitro digestibility, while maintaining high lysine levels and adequate sulphur amino acids content. Antinutritional factors such as cyanogenic glucosides, cyanide precursors from hydrolysis, tannins and trypsin inhibitors were lower in the PIs than in the RFs. The reduction in canavanine levels, a structural analogue of arginine, in the C. ensiformis PI was noteworthy. These PIs thus have potential applications in the development of new food ingredients in tropical regions using processes that improve nutritional value. [ABSTRACT FROM AUTHOR]

Mixtures of Nα-hippurylarginin, Nα-hippuryllysine, and glyoxal were incubated in the absence and presence of Nα-acetylcysteine in order to assess the individual reactivity of these nucleophilic amino acid residues. The incubations were performed under atmospheric and high hydrostatic pressure (400 MPa), and, at the same time, β-casein was reacted with glyoxal. The results showed that arginine is the main partner for glyoxal in the absence of cysteine, whereas a lysine derivatization was not apparent. In the presence of cysteine, however, arginine was almost completely protected from the reaction, whereas a noticeable formation of lysine derivatives, mainly carboxymethyllysine, was observed. Based on these findings, a reaction mechanism is proposed to explain the influence of cysteine on the reaction. [ABSTRACT FROM AUTHOR]

Glycation is a major cause of spontaneous damage to proteins in physiological systems. This is exacerbated in diabetes as a consequence of the increase in glucose and other saccharides derivatives in plasma and at the sites of vascular complications. Protein damage by the formation of early glycation adducts is limited to lysine side chain and N-terminal amino groups whereas later stage adducts, advanced glycation endproducts (AGEs), modify these and also arginine and cysteine residues. Metabolic dysfunction in vascular cells leads to the increased formation of methylglyoxal which adds disproportionately to the glycation damage in hyperglycaemia. AGE-modified proteins undergo cellular proteolysis leading to the formation and urinary excretion of glycation free adducts. AGEs may potentiate the development of diabetic complications by activation of cell responses by AGE-modified proteins interacting with specific cell surface receptors, activation of cell responses by AGE free adducts, impairment of protein–protein and enzyme–substrate interactions by AGE residue formation, and increasing resistance to proteolysis of extracellular matrix proteins. The formation of AGEs is suppressed by intensive glycaemic control, and may in future be suppressed by thiamine and pyridoxamine supplementation, and several other pharmacological agents. Increasing expression of enzymes of the enzymatic defence against glycation provides a novel and potentially effective future therapeutic strategy to suppress protein glycation. [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.

Folding of DNA into chromatin is mediated by binding to histones such as H4; association of DNA with histones is regulated by covalent histone modifications, e.g. acetylation, methylation, and biotinylation. We sought to identify amino-acid residues that are biotinylated in histone H4, and to determine whether acetylation and methylation of histones affect biotinylation. Synthetic peptides spanning fragments of human histone H4 were biotinylated enzymatically using biotinidase. Peptide-bound biotin was probed with streptavidin–peroxidase. Peptides based on the N-terminal sequence of histone H4 were effectively recognized by biotinidase as substrates for biotinylation; in contrast, peptides based on the C-terminal sequences were not biotinylated. Substitution of K8 or K12 with alanine or arginine decreased biotinylation, suggesting that these lysines are targets for biotinylation; K8 and K12 are also known targets for acetylation. Chemical acetylation or methylation of a given lysine decreased subsequent enzymatic biotinylation of neighboring lysines, consistent with cross-talk among histone modifications. Substitution of a given lysine (positive charge) with glutamate (negative charge) abolished biotinylation of neighboring lysines, providing evidence that the net charge of histones has a role in biotinylation. An antibody was generated that specifically recognized histone H4 biotinylated at K12. This antibody was used to detect biotinylated histone H4 in nuclear extracts from human cells. These studies suggest that K8 and K12 in histone H4 are targets for biotinylation, that acetylation and biotinylation compete for the same binding sites, and that acetylation and methylation of histones affect biotinylation of neighboring lysines. [ABSTRACT FROM AUTHOR]

Four major basic proteins termed C, K, N and Q, which are synthesized very late in grain development, have been isolated from barley (Hordum vulgare L.) mutant Bomi 1508. Immunoelectrophoretic monitoring assured a high degree of purity after a few ion exchange and gel filtration steps. Charge microheterogeneity of two of the four antigenically distinct proteins was observed. Some physico‐chemical properties were determined, including molecular mass (C ∼ 28 000; K ∼ 30 000; N ∼ 11 000; Q ∼ 60000), isoelectric point(s) (C ∼ 9.7; K ∼ 10.1–10.3; N ∼ 9.3; Q ∼ 8.9–9.1 at 25°C), and amino acid composition. In total, the four proteins represent ∼ 5% of the salt‐soluble protein in grains of some cultivated barleys. The most basic protein K is rich in lysine (∼ 7.9 mol %) and may account for ∼1% of the grain lysine content in these barleys. [ABSTRACT FROM AUTHOR]

The lysine reagent pyridoxal 5-phosphate was applied to the ADP/ATP carrier (AAC) in order to elucidate topological and functional properties of the numerous lysines within the primary structure. To establish appropriate labeling conditions, the influence of pyridoxal-P on transport and inhibitor binding to the AAC was examined. The ADP/ATP transport is sensitive to low concentrations of pyridoxal-P with a. Ki = 0.4 mM. Binding of [³H]carboxyatractylate and [³H]bongkrekate is largely inhibited by pyridoxal-P treatment with K1 approximately 1 mM. [³H]Carboxyatractylate is not and [³H]bongkrekate weakly removed by pyridoxal-f, whereas [³H]atractylate is displaced to a large extent. Under optimized conditions of pyridoxal-P concentration, of pH and of time exposure, the AAC was exposed to [³H]pyridoxal-P in mitochondria, in submitochondrial particles and in the detergent-solubilized carrier. The [³H]pyridoxal-P-labeled AAC was isolated from mitochondria and particles. After citraconylation thermolysinolytic peptides were prepared. The pyridoxyl-lysinecontaining peptides were purified and the pyridoxal-P incorporation to specific lysines was determined by sequencing. The pyridoxal-P incorporation into the AAC in various states was evaluated with regard to structural and functional aspects. First, by comparing pyridoxal-P incorporation in mitochondria and sonic particles, the segments of the polypeptide chain exposed to the cytosolic and matrix side of the membrane are detected. Second, the additional lysine incorporation into the isolated as compared to the membrane-bound carrier is attributed to the protein collar facing the phospholipid headgroups. Third, the difference between lysine incorporation into the carboxyatractylate-AAC and bongkrekate-AAC complexes reflect either conformational changes or lysines involved in the translocation channel through the protein. Fourth, the additional lysine labeled in the atractylate-carrier complex as compared to the carboxyatractylate-carrier complex is attributed to a cationic site in the binding center. These results are incorporated into a transmembrane folding model of the carrier. [ABSTRACT FROM AUTHOR]

The vitelline coat lysin of a top shell, Tegula pfeifferi, is a single polypeptide consisting of 118 amino acid residues and having a relative molecular mass of 13800. The complete amino acid sequence of the vitelline coat lysin was determined by the analyses of five peptides obtained by cyanogen bromide degradation and three fragments obtained by Staphylococcus aureus protease digestion of the protein. The sequence showed the presence of microheterogeneities in the vicinity of the C-terminal half of the molecule and the existence of two homologous domain structures.

A method for the large-scale isolation of ribosomal proteins is described avoiding pre-separation of 30-S and 50-S subunits. Five proteins isolated in this way were studied with high-resolution 1H NMR at 500 MHz. These are $21, L18, L25, L30 and L33. The results show that L18, L25 and L30 exhibit tertiary structure in solution and indications for secondary structure in $21 are found. Protein L33 appears to be a random coil. Several resonances in the 1H NMR spectra are assigned to particular protons of amino acid residues, e.g. the aromatic ring protons of tyrosines and histidines, and ε-protons of lysines. [ABSTRACT FROM AUTHOR]

1. The proteins of amoebae and plasmodia of strain CL of Physarum polycephahan have been compared by two-dimensional gel clectrophoresis, Both forms of the organism were labelled by growth on formalin-killed bacteria labelled with [35S]sulphate, [3H]lysine or [14C]lysine. Plasmodia were also labelled from radioactive lysine in the medium. 2. Of 306 relatively abundant proteins examined, 26% were phase-specific, that is they were found only in amoebae or in plasmodia. About a quarter of these apparent differences in gene expression may be due to minor changes in charge and/or size. 3. Amongst the 74% of the proteins present in both amoebae and plasmodia, there are substantial differences in differential rates of synthesis and these have been measured for a representative set of proteins by a double-label procedure. [ABSTRACT FROM AUTHOR]

Two groups of crosslinked polyacrylic gels with immobilized lysine and lysine peptides (Lys)5 and (Lys)5-Pro have been used as models for the chromatographic investigation of lysine-peptide-oligonucleotide interactions. One group carries carboxylic groups in addition to the peptide residues in the gel matrix; the other gel type contains no such carboxylic groups in the gel matrix. Nucleotides of the series (dT)2–5, p(dT)1–4, p(dT)1–4p, (dA)2–5, p(dA)1–5 and p(dA)1–4p were chromatographed on these gels under various conditions in an aqueous buffer. On the gels of the first group the nucleotides were retarded only slighly, the positive charges of the e-amino groups being compensated partially or totally by the negatively charged carboxyl groups of the polymer matrix. On the gels of the second group, however, the oligonucleotides underwent specific interactions. These interactions were based primarily upon electrostatic forces between the positively charged ϵ-amino groups of the immobilized peptides and the negatively charged phosphate groups of the oligonucleotides. Our results indicate that, in addition to the electrostatic interaction, the conformation plays a crucial role. We explain the selectivity of the interaction with a conformation-fit mechanism. The origin of this mechanism, which creates specific interactions from unspecific forces, is discussed. [ABSTRACT FROM AUTHOR]

A method has been developed for specific enzymic cleavage of polypeptides at the N-terminal side of modified cysteine residues. Lysine residues are blocked by trifluoroacetylation and cysteine residues subsequently converted to the 2-aminoethyl derivatives. Digestion of the modified polypeptide with the lysine-specific protease from Armillaria mellea (patented by Walton et al., 1972) occurs only at 2-aminoethylcysteine residues. With the β chain of human haemoglobin, which contains 2 cysteine and 11 lysine residues, cleavage was observed at both modified eysteines but at none of the lysines. In the case of a polypeptide from bee venom which contains 4 half-cystine and 5 lysine residues, cleavage occurred at only 2 of the modified cysteines and also at 2 lysine residues. The pattern of cleavage in the latter case can be interpreted in terms of the amino acid sequence of the polypeptide. [ABSTRACT FROM AUTHOR]

Initial velocity kinetics of the reverse direction showed that the saccharopine dehydrogenasecatalyzed reaction was consistent with a mechanism that the addition and release of reactants to and from the enzyme follow an obligatory order. The product inhibition studies in the forward direction also revealed kinetic patterns consistent with the ordered mechanism where the sequence of addition of substrates being NAD+ and saccharopine, and that of release of products being lysine, α-ketoglutarate and NADH. The various kinetic parameters determined at pH 6.8 and 22° are aisc described. [ABSTRACT FROM AUTHOR]