Recognition of Higher Order Patterns in Proteins: Immunologic Kernels

By applying analysis of the principal components of amino acid physical properties we predicted cathepsin cleavage sites, MHC binding affinity, and probability of B-cell epitope binding of peptides in tetanus toxin and in ten diverse additional proteins. Cross-correlation of these metrics, for peptides of all possible amino acid index positions, each evaluated in the context of a ±25 amino acid flanking region, indicated that there is a strongly repetitive pattern of short peptides of approximately thirty amino acids each bounded by cathepsin cleavage sites and each comprising B-cell linear epitopes, MHC–I and MHC-II binding peptides. Such “immunologic kernel” peptides comprise all signals necessary for adaptive immunologic cognition, response and recall. The patterns described indicate a higher order spatial integration that forms a symbolic logic coordinating the adaptive immune system.     

Word Decoding of Protein Amino Acid Sequences with Availability Analysis: A Linguistic Approach

The amino acid sequences of proteins determine their three-dimensional structures and functions. However, how sequence information is related to structures and functions is still enigmatic. In this study, we show that at least a part of the sequence information can be extracted by treating amino acid sequences of proteins as a collection of English words, based on a working hypothesis that amino acid sequences of proteins are composed of short constituent amino acid sequences (SCSs) or “words”. We first confirmed that the English language highly likely follows Zipf''s law, a special case of power law. We found that the rank-frequency plot of SCSs in proteins exhibits a similar distribution when low-rank tails are excluded. In comparison with natural English and “compressed” English without spaces between words, amino acid sequences of proteins show larger linear ranges and smaller exponents with heavier low-rank tails, demonstrating that the SCS distribution in proteins is largely scale-free. A distribution pattern of SCSs in proteins is similar among species, but species-specific features are also present. Based on the availability scores of SCSs, we found that sequence motifs are enriched in high-availability sites (i.e., “key words”) and vice versa. In fact, the highest availability peak within a given protein sequence often directly corresponds to a sequence motif. The amino acid composition of high-availability sites within motifs is different from that of entire motifs and all protein sequences, suggesting the possible functional importance of specific SCSs and their compositional amino acids within motifs. We anticipate that our availability-based word decoding approach is complementary to sequence alignment approaches in predicting functionally important sites of unknown proteins from their amino acid sequences.     

Developmental regulation of amino acid transport in Neurospora crassa

Conidia of Neurospora crassa exhibit an ability to transport various amino acids against a concentration gradient. The conidial transport system has previously been characterized in terms of kinetics, competitions, and genetic control. This study describes the development of a new and highly active transport capability which is elaborated during the early stages of development but prior to evident germination. It has been named “postconidial” transport activity and represents as much as 20-fold greater initial rates as compared to those observed with conidia. Development of the postconidial transport activity requires protein synthesis and can be partially repressed when the substrate amino acid is present during the developmental preincubation period. A mutant has been utilized which exhibits normal conidial but fails to develop normal postconidial transport activity for any amino acid examined. Although temperature optimum and pH dependence are similar in conidial and postconidial systems, there is evidence that the new activity is not a simple amplification of an existing capability. This is reflected as a change in competition patterns between particular amino acids as development proceeds.     

YjeH Is a Novel Exporter of l-Methionine and Branched-Chain Amino Acids in Escherichia coli

Amino acid efflux transport systems have important physiological functions and play vital roles in the fermentative production of amino acids. However, no methionine exporter has yet been identified in Escherichia coli. In this study, we identified a novel amino acid exporter, YjeH, in E. coli. The yjeH overexpression strain exhibited high tolerance to the structural analogues of l-methionine and branched-chain amino acids, decreased intracellular amino acid levels, and enhanced export rates in the presence of a Met-Met, Leu-Leu, Ile-Ile, or Val-Val dipeptide, suggesting that YjeH functions as an exporter of l-methionine and the three branched-chain amino acids. The export of the four amino acids in the yjeH overexpression strain was competitively inhibited in relation to each other. The expression of yjeH was strongly induced by increasing cytoplasmic concentrations of substrate amino acids. Green fluorescent protein (GFP)-tagged YjeH was visualized by total internal reflection fluorescence microscopy to confirm the plasma membrane localization of YjeH. Phylogenetic analysis of transporters indicated that YjeH belongs to the amino acid efflux family of the amino acid/polyamine/organocation (APC) superfamily. Structural modeling revealed that YjeH has the typical “5 + 5” transmembrane α-helical segment (TMS) inverted-repeat fold of APC superfamily transporters, and its binding sites are strictly conserved. The enhanced capacity of l-methionine export by the overexpression of yjeH in an l-methionine-producing strain resulted in a 70% improvement in titer. This study supplements the transporter classification and provides a substantial basis for the application of the methionine exporter in metabolic engineering.     

Variations in the Electrostatic Landscape of Class II Human Leukocyte Antigen Molecule Induced by Modifications in the Myelin Basic Protein Peptide: A Theoretical Approach

The receptor-ligand interactions involved in the formation of the complex between Class II Major Histocompatibility Complex molecules and antigenic peptides, which are essential for establishing an adaptive immunological response, were analyzed in the Class II Human Leukocyte Antigen (HLA) - Myelin Basic Protein (MBP) peptide complex (HLA-DRβ1*1501-MBP) using a multipolar molecular electrostatic potential approach. The Human Leukocyte Antigen - peptide complex system was divided into four pockets together with their respective peptide fragment and the corresponding occupying amino acid was replaced by each of the remaining 19 amino acids. Partial atomic charges were calculated by a quantum chemistry approach at the Hatree Fock/3-21*G level, to study the behavior of monopole, dipole and quadrupole electrostatic multipolar moments. Two types of electrostatic behavior were distinguished in the pockets'' amino acids: “anchoring” located in Pocket 1 and 4, and “recognition” located in Pocket 4 and 7. According to variations in the electrostatic landscape, pockets were ordered as: Pocket 1>Pocket 9≫Pocket 4≈Pocket 7 which is in agreement with the binding ability reported for Class II Major Histocompatibility Complex pockets. In the same way, amino acids occupying the polymorphic positions β13R, β26F, β28D, β9W, β74A, β47F and β57D were shown to be key for this Receptor-Ligand interaction. The results show that the multipolar molecular electrostatic potential approach is appropriate for characterizing receptor-ligand interactions in the MHC–antigenic peptide complex, which could have potential implications for synthetic vaccine design.     

Meta-carboxy-substituted aromatic amino acids and γ-glutamyl peptides: Chemical characters for classification in the Iridaceae

Free amino acids and γ-glutamyl peptides have been examined in 116 species of Iridaceae. 3-(3-Carboxyphenyl)-alanine, 3′-carboxyphenylglycine and γ-glutamyl peptides occur widely in the subfamily Iridoideae but have not been found in the two other subfamilies, Ixioideae and Sisyrinchoideae. The two aromatic amino acids occur in varying concentrations in species within the Irideae and Tigrideae. The γ-glutamyl peptides are distributed in a distinct pattern within the Irideae. The results are discussed in relation with botanical classification of the subfamily Irioideae and with existing knowledge on the occurrence, biosynthesis and degradation of the aromatic amino acids and the γ-glutamyl peptides.     

Pre-proparathyroid hormone: analysis of radioactive tryptic peptides and amino acid sequence.

The amino acid sequence of bovine pre-proparathyroid hormone has been partially determined by analysis of the polypeptide labeled selectively with radioactive amino acids. Analysis of tryptic peptides containing methionine or lysine indicated that parathyroid hormone, proparathyroid hormone, and pre-proparathyroid hormone had several common peptides. Two lysine-containing peptides present in proparathyroid hormone but not in parathyroid hormone were also present in pre-proparathyroid hormone. In addition, pre-proparathyroid hormone contained several additional lysine- and methionine-containing peptides not present in parathyroid hormone or proparathyroid hormone. Analysis by repetitive Edman degradation of the polypeptide labeled with lysine, methionine, and other amino acids indicated that pre-proparathyroid hormone contained 25 additional amino acids at the amino terminus of proparathyroid hormone; the identities of 17 of the 25 amino acids have been established. An unusual feature found was the presence of methionyl-methionyl at the amino terminus and the presence of 5 methionines within the first 14 amino acids.     

Identifying the ligated amino acid of archaeal tRNAs based on positions outside the anticodon

Proper recognition of tRNAs by their aminoacyl-tRNA synthetase is essential for translation accuracy. Following evidence that the enzymes can recognize the correct tRNA even when anticodon information is masked, we search for additional nucleotide positions within the tRNA molecule that potentially contain information for amino acid identification. Analyzing 3936 sequences of tRNA genes from 86 archaeal species, we show that the tRNAs’ cognate amino acids can be identified by the information embedded in the tRNAs’ nucleotide positions without relying on the anticodon information. We present a small set of six to 10 informative positions along the tRNA, which allow for amino acid identification accuracy of 90.6% to 97.4%, respectively. We inspected tRNAs for each of the 20 amino acid types for such informative positions and found that tRNA genes for some amino acids are distinguishable from others by as few as one or two positions. The informative nucleotide positions are in agreement with nucleotide positions that were experimentally shown to affect the loaded amino acid identity. Interestingly, the knowledge gained from the tRNA genes of one archaeal phylum does not extrapolate well to another phylum. Furthermore, each species has a unique ensemble of nucleotides in the informative tRNA positions, and the similarity between the sets of positions of two distinct species reflects their evolutionary distance. Hence, we term this set of informative positions a “tRNA cipher.” It is tempting to suggest that the diverging code identified here might also serve the aminoacyl tRNA synthetase in the task of tRNA recognition.     

De Novo Synthesis of Amino Acids by the Ruminal Bacteria Prevotella bryantii B14, Selenomonas ruminantium HD4, and Streptococcus bovis ES1

The influence of peptides and amino acids on ammonia assimilation and de novo synthesis of amino acids by three predominant noncellulolytic species of ruminal bacteria, Prevotella bryantii B₁4, Selenomonas ruminantium HD4, and Streptococcus bovis ES1, was determined by growing these bacteria in media containing ¹⁵NH₄Cl and various additions of pancreatic hydrolysates of casein (peptides) or amino acids. The proportion of cell N and amino acids formed de novo decreased as the concentration of peptides increased. At high concentrations of peptides (10 and 30 g/liter), the incorporation of ammonia accounted for less than 0.16 of bacterial amino acid N and less than 0.30 of total N. At 1 g/liter, which is more similar to peptide concentrations found in the rumen, 0.68, 0.87, and 0.46 of bacterial amino acid N and 0.83, 0.89, and 0.64 of total N were derived from ammonia by P. bryantii, S. ruminantium, and S. bovis, respectively. Concentration-dependent responses were also obtained with amino acids. No individual amino acid was exhausted in any incubation medium. For cultures of P. bryantii, peptides were incorporated and stimulated growth more effectively than amino acids, while cultures of the other species showed no preference for peptides or amino acids. Apparent growth yields increased by between 8 and 57%, depending on the species, when 1 g of peptides or amino acids per liter was added to the medium. Proline synthesis was greatly decreased when peptides or amino acids were added to the medium, while glutamate and aspartate were enriched to a greater extent than other amino acids under all conditions. Thus, the proportion of bacterial protein formed de novo in noncellulolytic ruminal bacteria varies according to species and the form and identity of the amino acid and in a concentration-dependent manner.     

Carbon Dioxide Fixation by Lupin Root Nodules: II. Studies with C-labeled Glucose, the Pathway of Glucose Catabolism, and the Effects of Some Treatments That Inhibit Nitrogen Fixation

Labeling studies using detached lupin (Lupinus angustifolius) nodules showed that over times of less than 3 minutes, label from [3,4-¹⁴C]glucose was incorporated into amino acids, predominantly aspartic acid, to a much greater extent than into organic acids. Only a slight preferential incorporation was observed with [1-¹⁴C]- and [6-¹⁴C]glucose, while with [U-¹⁴C]-glucose more label was incorporated into organic acids than into amino acids at all labeling times. These results are consistent with a scheme whereby the “carbon skeletons” for amino acid synthesis are provided by the phosphoenolpyruvate carboxylase reaction.     

Eurocin,a New Fungal Defensin: STRUCTURE,LIPID BINDING,AND ITS MODE OF ACTION*

Antimicrobial peptides are a new class of antibiotics that are promising for pharmaceutical applications because they have retained efficacy throughout evolution. One class of antimicrobial peptides are the defensins, which have been found in different species. Here we describe a new fungal defensin, eurocin. Eurocin acts against a range of Gram-positive human pathogens but not against Gram-negative bacteria. Eurocin consists of 42 amino acids, forming a cysteine-stabilized α/β-fold. The thermal denaturation data point shows the disulfide bridges being responsible for the stability of the fold. Eurocin does not form pores in cell membranes at physiologically relevant concentrations; it does, however, lead to limited leakage of a fluorophore from small unilamellar vesicles. Eurocin interacts with detergent micelles, and it inhibits the synthesis of cell walls by binding equimolarly to the cell wall precursor lipid II.     

Structure-Activity Study of the Lantibiotic Mutacin II from Streptococcus mutans T8 by a Gene Replacement Strategy

Mutacin II, elaborated by group II Streptococcus mutans, is a ribosomally synthesized and posttranslationally modified polypeptide antibiotic containing unusual thioether and didehydro amino acids. To ascertain the role of specific amino acid residues in mutacin II antimicrobial activity, we developed a streptococcal expression system that facilitates the replacement of the mutA gene with a single copy of a mutated variant gene. As a result, variants of mutacin II can be designed and expressed. The system was tested by constructing the following mutant peptides: ΔN1, V7A, P9A, T10A, T10S, C15A, C26A, and C27A. All of these mutacin II variants except ΔN1 and T10A, which were not secreted, were isolated, and their identities were verified by mass spectrometry. Variants P9A, C15A, C26A, and C27A failed to exert antimicrobial activity. Because the P9A and T10A variants comprise the “hinge” region of mutacin II, these observations suggest that in addition to the thioether and didehydro amino acids, the hinge region is essential for biological activity and biosynthesis or export of the peptide. Tandem mass spectrometry of the N-terminal part of the wild-type molecule and its C15A variant confirmed that the threonine at position 10 is dehydrated and present as a didehydrobutyrine residue. This analysis of the active T10S variant further suggested that a didehydro amino acid at this position is specific for antimicrobial activity and that the biosynthetic machinery does not discriminate between threonine and serine. In contrast, the lack of production of mutacin variants with alanine substituted for threonine at position 10, as well as the deletion of asparagine at the N terminus (ΔN1), indicates that specific residues in the propeptide may be crucial for certain steps in the biosynthetic pathway of this lantibiotic.     

Amino Acid Synthesis in Photosynthesizing Spinach Cells : EFFECTS OF AMMONIA ON POOL SIZES AND RATES OF LABELING FROM CO(2)

Isolated cells from leaves of Spinacia oleracea have been maintained in a state capable of high rates of photosynthetic CO₂ fixation for more than 60 hours. The incorporation of ¹⁴CO₂ under saturating CO₂ conditions into carbohydrates, carboxylic acids, and amino acids, and the effect of ammonia on this incorporation have been studied. Total incorporation, specific radioactivity, and pool size have been determined as a function of time for most of the protein amino acids and for γ-aminobutyric acid. The measurements of specific radio-activities and of the approaches to ¹⁴C “saturation” of some amino acids indicate the presence and relative sizes of metabolically active and passive pools of these amino acids.     

The SAF-box domain of chromatin protein DEK

DEK is an abundant chromatin protein in metazoans reaching copy numbers of several millions/nucleus. Previous work has shown that human DEK, a protein of 375 amino acids, has two functional DNA-binding domains, of which one resides in a central part of the molecule and contains sequences corresponding to the scaffold attachment factor-box (SAF-box) domain as found in a growing number of nuclear proteins. Isolated SAF-box peptides (amino acids 137–187) bind weakly to DNA in solution, but when many SAF-box peptides are brought into close proximity on the surface of Sephadex beads, cooperative effects lead to a high affinity to DNA. Furthermore, a peptide (amino acids 87–187) that includes a sequence on the N-terminal side of the SAF-box binds efficiently to DNA. This peptide prefers four-way junction DNA over straight DNA and induces supercoils in relaxed circular DNA just like the full-length DEK. Interestingly, however, the 87–187 amino acid peptide introduces negative supercoils in contrast to the full-length DEK, which is known to introduce positive supercoils. We found that two adjacent regions (amino acids 68–87 and 187–250) are necessary for the formation of positive supercoils. Our data contribute to the ongoing characterization of the abundant and ubiquitous DEK chromatin protein.     

Development of purification process for dual‐function recombinant human heavy‐chain ferritin by the investigation of genetic modification impact on conformation

Ferritin is a promising drug delivery platform and has been functionalized through genetic modifications. This work has designed and expressed a dual‐functional engineered human heavy‐chain ferritin (HFn) with the inserted functional peptide PAS and RGDK to extend half‐life and improve tumor targeted drug delivery. A facile and cost‐effective two‐step purification pathway for recombinant HFn was developed. The genetic modification was found to affect HFn conformation, and therefore varied the purification performance. Heat‐acid precipitation followed by butyl fast flow hydrophobic interaction chromatography (HIC) has been developed to purify HFn and modified HFns. Nucleic acid removal reached above 99.8% for HFn and modified HFns. However, HFn purity reached above 95% and recovery yield (overall) above 90%, compared with modified HFns purity above 82% and recovery yield (overall) above 58%. It is interesting to find that the inserted functional peptides significantly changed the molecule conformation, where a putative turnover of the E‐helix with the inserted functional peptides formed a “flop” conformation, in contrast with the “flip” conformation of HFn. It could be the cause of fragile stability of modified HFns, and therefore less tolerant to heat and acid condition, observed by the lower recovery yield in heat‐acid precipitation.     

Studies of the proteins, peptides and free amino acids of mature bovine enamel

1. The organic matrix of enamel from erupted bovine teeth has been found to be composed mostly of small peptides containing principally aspartic acid, glycine, glutamic acid and serine. 2. A small amount of higher-molecular-weight components has been isolated by various procedures. One non-diffusible fraction was found to be heterogeneous in the ultracentrifuge, and composed principally of material that by gel filtration indicated a molecular weight greater than 30000. These components were largely carbohydrate in nature (glycoproteins and glycopeptides), containing only small amounts of amino acids.     

Equilibrium of the Cis-Trans Isomerisation of the Peptide Bond with N-alkyl Amino Acids Measured by 2D NMR

The conformational cis-trans equilibrium around the peptide bond in model tripeptides has been determined by 2D NMR methods (HOHAHA, ROESY). The study was limited to three different N-substituted amino acids in position 2, namely Pro (proline), Tic (1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid), and N-MePhe (N-methylphenylalanine). In all cases the amino acid in position 1 was tyrosine and in position 3, phenylalanine. The results of our studies show that the cis-trans ratio depends mostly on the configuration of the amino acids forming the peptide bond undergoing the cis-trans isomerisation. The amino acid following the sequence (in position 3) does not have much influence on the cis-trans isomerisation, indicating that there is no interaction of the side chains between these amino acids. The model peptides with the L-Tyr-L-AA-(L- or D-)Phe (where AA is N-substituted amino acid) chiralities give 80–100% more of the cis form in comparison to the corresponding peptides with the D-Tyr-L-AA-(L-or D-)Phe chiralities. These results indicate that the incorporation of N-substituted amino acids in small peptides with the same chirality as the precedent amino acid involved in the peptide bond undergoing the cis/trans isomerisation moves the equilibrium to a significant amount of the cis form.     

The decisive step in betaxanthin biosynthesis is a spontaneous reaction1

Experiments were performed to confirm that the aldimine bond formation is a spontaneous reaction, because attempts to find an enzyme catalyzing the last decisive step in betaxanthin biosynthesis, the aldimine formation, failed. Feeding different amino acids to betalain-forming hairy root cultures of yellow beet (Beta vulgaris L. subsp. vulgaris “Golden Beet”) showed that all amino acids (S- and R-forms) led to the corresponding betaxanthins. We observed neither an amino acid specificity nor a stereoselectivity in this process. In addition, increasing the endogenous phenylalanine (Phe) level by feeding the Phe ammonia-lyase inhibitor 2-aminoindan 2-phosphonic acid yielded the Phe-derived betaxanthin. Feeding amino acids or 2-aminoindan 2-phosphonic acid to hypocotyls of fodder beet (B. vulgaris L. subsp. vulgaris “Altamo”) plants led to the same results. Furthermore, feeding cyclo-3-(3,4-dihydroxyphenyl)-alanine (cyclo-Dopa) to these hypocotyls resulted in betanidin formation, indicating that the decisive step in betacyanin formation proceeds spontaneously. Finally, feeding betalamic acid to broad bean (Vicia faba L.) seedlings, which are known to accumulate high levels of Dopa but do not synthesize betaxanthins, resulted in the formation of dopaxanthin. These results indicate that the condensation of betalamic acid with amino acids (possibly including cyclo-Dopa or amines) in planta is a spontaneous, not an enzyme-catalyzed reaction.     

Effect of site-specific amino acid D-isomerization on β-sheet transition and fibril formation profiles of Tau microtubule-binding repeat peptides

d-amino acid-containing proteins have been found in several human tissues, and the spontaneous accumulation of d-amino acids in proteins is thought to be involved in age-dependent diseases including dementia. Tau, a microtubule-associated protein, is a major component of neurofibrillary tangles in Alzheimer's disease. Site-specific amino acid D-isomerization in Tau has been observed in the brains of patients with Alzheimer's disease. Here, we conducted amino acid D-isomerization at specific sites in microtubule-binding repeat peptides of Tau (Tau R2 and R3) and examined the effects on Tau structure and fibril formation. Our results demonstrate that amino acid D-isomerization in Tau R2 peptides decreased the rates of β-sheet transition and fibril formation compared with those of the wild-type peptide composed of all l-amino acids. In contrast, Tau R3 peptides that had undergone amino acid D-isomerization at either Asp314, Ser316, or Ser324 showed increased rates of β-sheet transition and fibril formation compared with those of the wild-type Tau R3 peptide.     

A comparison of the ability of normal liver,a premalignant liver,a solid hepatoma and the zajdela ascitic hepatoma,to take up amino acidsin vitro

Summary The net total uptake of several amino acids at low (0.8–3.1 moles/liter) as well as high (800–1200 moles/liter) extracellular concentrations, by normal rat liver, a premalignant liver, a solid hepatoma, and the Zajdela ascitic hepatoma cells, has been compared under conditions in which protein synthesis continues. At low amino acid concentrations, the initial (3 min) total uptake of the various amino acids in the Zajdela cells, was 3–10 (average 7) times more, and the intracellular concentration of the labeled amino acids taken up 14–45 (average 31) times more, than in normal liver. At the high amino acid concentrations, the total uptake in the Zajdela cells, at 60–120 min was 2–5 (average 3.5) times more, and the intracellular concentration of the amino acids taken up 8–19 (average 13) times more, than in normal liver; the corresponding values for the premalignant liver and the solid hepatoma were in between those for normal liver and the Zajdela cells. Further, the rate of the total uptake of amino acids, their intracellular concentration, the proportion of the amino acid taken up utilized for protein synthesis, the rate of incorporation of the amino acid taken up into protein, and the cellular growth rate, seemed to be correlated in the four cell/tissue preparations studied. In most cases, the rate of the net uptake fell drastically with time, the uptake virtually stopping after 90–180 min, probably due to lack of serum in the incubation medium.