Occurrence Of Amino Acids Research Articles

Hassallidin A, a Glycosylated Lipopeptide with Antifungal Activity from the Cyanobacterium Hassallia sp.

Hassallidin A (1), a new antifungal glycosylated lipopeptide, was isolated from an epilithic cyanobacterium collected in Bellano, Italy, identified as Tolypothrix (basionym Hassallia) species. Chemical, mass spectrometric, and spectroscopic analyses, including one- and two-dimensional NMR, were performed to determine an esterified eight-residue cyclic peptide linked with a carbohydrate and a fatty acid residue. Chiral GC-MS analysis revealed the occurrence of the nonproteinogenic amino acids D-allo-Thr, D-Thr, D-Tyr, D-Gln, and dehydroaminobutyric acid (Dhb) within the peptide moiety. The additional components of hassallidin A could be identified as alpha,beta-dihydroxytetradecanoic acid (Dht) and mannose. This is the first report on a cyclic peptide of cyanobacterial origin that contains both a fatty acid and a carbohydrate moiety. Compound 1 exhibits antifungal activity against Aspergillus fumigatus and Candida albicans with MIC values of 4.8 microg/mL for both test organisms.

Amphipathic α‐helices in proteins: Results from analysis of protein structures

Amphipathic alpha-helices play a crucial role in mediating the interaction of peptides and proteins with membranes. We have analyzed protein structures for the occurrence of 18-residue amphipathic helices. We find several of these alpha-helices having average hydrophobic moments and average hydrophobicities that would favor their interaction with membranes. We have analyzed the distribution of net charge, helix length, normalized frequency of occurrence, and propensities of the 20 amino acids in the delineated 18-residue helices. We have observed distinct differences in the frequencies of occurrence of polar and hydrophobic amino acids at positions 1-18 in amphipathic and nonamphipathic helices. There are also differences in propensities of the 20 amino acids to occur at positions 1-18 of amphipathic and nonamphipathic helices. Synthetic peptides corresponding to some of these surface-seeking helices do possess antibacterial and/or hemolytic activities. Knowledge of the distribution of charges in 18-residue surface-seeking amphipathic alpha-helices, as well as propensity of occurrence of amino acids at various positions, would be useful inputs in the de novo design of amphipathic peptides.

Measuring d-amino acid-containing neuropeptides with capillary electrophoresis

Neuropeptides are heavily posttranslationally modified (PTM) gene products that are often characterized by a variety of mass spectrometric approaches. Recently, the occurrence of amino acids in the D-form has been documented in several neuropeptides. As this modification has no associated mass shift, this particular PTM is difficult to evaluate using mass spectrometry (MS) alone. Here we demonstrate several approaches using capillary electrophoresis (CE) with absorbance and laser-induced fluorescence (LIF) for the separation of native and derivatized molluscan peptides containing D-amino acids. The combination of peptide derivatization followed by CE/LIF is well suited for single cell measurements because of its ability to characterize the peptides in such small samples. In order to verify this approach, the D-Trp-containing peptide NdWFa (NH2-Asn-D-Trp-Phe-CONH2), present in individual neurons from the marine mollusk Aplysia californica, has been characterized. The mass spectra show that NdWFa and/or NWFa are present in specific neurons; CE/LIF analysis of these cells demonstrates that NdWFa is the dominant form of the peptide.

Glycine amide hydrolysis with water and OH radical: a comparative DFT study

Abstract The only indisputable evidence on the occurrence of amino acids outside our planet is their detection in carbonaceous chondrites. However only a small fraction was found in chemically free state, whereas hydrolyzable derivatives of unknown composition (possibly amides) is a major source of the detectable amino acids. The difference between carbonaceous chondrites and the icy bodies (interstellar ices and comets) is the basic component, which is water in the latter case. Thus, it is reasonable to expect for a higher content of hydrolyzed derivatives (free amino acids as final products) in the icy bodies, as compared to the one in carbonaceous chondrites. As a preliminary test for energetic feasibility of this type of reactions, we studied theoretically the reaction of glycine amide hydrolysis, at the B3LYP/6-31++(d,p) level. We found that a very high activation barrier makes this reaction prohibited. Besides molecular water, a similar hydrolytic effect might be produced by hydroxyl radicals (OH) generated under the ionizing action of cosmic rays. Therefore we tested the reaction of glycine amide with OH radical as well. Nevertheless, although the barrier height decreased significantly, by ca. 30 kcal mol −1 , it remained positive.

Qualitative differences between day- and night-time rhizodeposition in maize ( Zea mays L.) as investigated by pyrolysis-field ionization mass spectrometry

Rhizodeposition is an important pathway of atmospheric C-input to soil, however, quantity and quality of plant rhizodeposition are insufficiently known. Therefore, the composition and diurnal dynamics of water-soluble root-derived substances and products of their interaction with sandy soil were investigated in maize plants ( Zea mays L.) by pyrolysis-field ionization mass spectrometry (Py-FIMS). In both night- and day-rhizodeposits the C, N and S concentrations were larger by factors ranging from 3.0 to 9.7 than the samples from non-cropped soil. The rhizodeposition was larger during the day than during the night-time and the composition of these deposits was different. The largest differences in the Py-FI mass spectra resulted from signals assigned to amino acids (aspartic acid, asparagine, glutamic acid, leucine, isoleucine, hydroxyproline and phenylalanine) and carbohydrates, in particular pentoses, which were exuded in the photosynthetic period. Marker signals in the Py-FI mass spectra and the curves of their thermal volatilization provided unequivocal evidence for the occurrence of free amino acids in the day-rhizodeposits. Other compounds detected in the Py-FI mass spectra were interpreted as constituents of rhizodeposits (lipids, suberin, fatty acids) or products of the interaction of rhizodeposits and microbial metabolites with stable soil organic matter (lignin dimers and alkylaromatics). It was concluded that the diurnal dynamics in the molecular-chemical composition between day- and night-rhizodeposits resulted from the exudation carbohydrates and amino acids during the photosynthetic period, the deposition of other root-derived compounds such as lipids, suberin and fatty acids, and the microbial metabolism of all available organic compounds in the rhizosphere. Furthermore, applications of the presented approach in C-turnover and phytoremediation research, and for risk assessment of genetically modified crops are proposed.

The contributions of specific amino acid side chains to signal intensities of peptides in matrix-assisted laser desorption/ionization mass spectrometry.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) analysis of complex peptide mixtures is often hampered by signal suppression effects as well as certain intrinsic properties of specific peptides that influence the desorption/ionization behavior. The present systematic study reports on the relationship between the occurrence of certain amino acids in peptides and the intensities of the related ions which appear during MALDI-MS analysis for both tryptic digests of proteins and synthetic peptide mixtures. The analysis of the tryptic digests revealed that the peptide sequences of the most intense peaks detected by MALDI-MS contained significantly higher proportions of arginine, phenylalanine, proline, and leucine than the average values for the measured proteins. The relationship between the relative signal intensities and amino acid compositions of peptides was studied in more detail by the partial least squares (PLS) method using equimolar mixtures of 144 well-characterized synthetic peptides. The regression coefficients clearly indicated that the presence of arginine, phenylalanine, leucine and proline tend to enhance the desorption/ionization process which results in higher MALDI-MS peak intensities. Furthermore, it was shown that the impact of arginine depends strongly on the identity of adjacent amino acids.

Hydrophobicity Analysis of Protein Primary Structures to Identify Helical Regions

A wavelet based approach for the hydrophobicity analysis of protein primary structures is proposed to predict the presence of alpha helices in the secondary structure. The information about hydropathy profile periodicity content together with a score of probability of occurrence of a single amino acid allows the localization of alpha helices. The accuracy is comparable to other consolidated predictors based on different techniques (i.e.: neural networks, hidden markov models). This method is particularly suitable to capture the amphiphilic character of the helical structures.

Representation of mutation pressure and selection pressure by PAM matrices

This paper analyses the relationship between the mutation data matrix 1PAM/PET91, representing the effect of both mutation and selection pressures exerted on 16130 homologous proteins of different organisms, and a mutation probability matrix (1PAM/MPM) representing the effect of pure mutation pressure on protein coding of the Borrelia burgdorferi genome. The 1PAM/PMP matrix was derived with the help of computer simulations, which used empirical nucleotide substitution rates found for the B. burgdorferi genome. Here, it is shown that the frequency of amino acid occurrence is strongly related to their effective survival time. We found that the shorter the turnover time of an amino acid under pure mutation pressure, the lower its fraction in the proteins coded by the genome and the more protected by selection pressure is its position in proteins. Results of analyses suggest that during evolution the mutational pressure has been optimised to some extent to the selection requirements.

Use of selective Trp side chain labeling to characterize protein-protein and protein-ligand interactions by NMR spectroscopy.

Recent studies on amino acid occurrence in protein binding sites suggest that only a reduced number of residues are responsible for most interaction energy in protein-protein and protein-ligand interactions. Above all, tryptophan (Trp) seems to be the most frequent residue in protein's hot spots. Here we report a novel, efficient, and cost-effective method to selectively incorporate specific isotope labels into the side chains of Trp residues in recombinant proteins. We show that the method proposed allows selective NMR observation of Trp side chains that enables studies of ligand binding, protein-protein interactions, hydrogen binding, protein folding, and side chain dynamics. Examples with the protein BIR3 will be given.

Exceptional pairs of amino acid neighbors in α-helices

Amino acids seem to have specific preferences for various locations in α-helices. These specific preferences, called singlet local propensity (SLP), have been determined by calculating the preference of occurrence of each amino acid in different positions of the α-helix. We have studied the occurrence of amino acids, single or pairs, in different positions, singlet or doublet, of α-helices in a database of 343 non-homologous proteins representing a unique superfamily from the SCOP database with a resolution better than 2.5 Å from the Protein Data Bank. The preference of single amino acids for various locations of the helix was shown by the relative entropy of each amino acid with respect to the background. Based on the total relative entropy of all amino acids occurring in a single position, the Ncap position was found to be the most selective position in the α-helix. A rigorous statistical analysis of amino acid pair occurrences showed that there are exceptional pairs for which, the observed frequency of occurrence in various doublet positions of the α-helix is significantly different from the expected frequency of occurrence in that position. The doublet local propensity (DLP) was defined as the preference of occurrences of amino acid pairs in different doublet positions of the α-helix. For most amino acid pairs, the observed DLP (DLPO) was nearly equal to the expected DLP (DLPE), which is the product of the related SLPs. However, for exceptional pairs of amino acids identified above, the DLPO and DLPE values were significantly different. Based on the relative values of DLPO and DLPE, exceptional amino acid pairs were divided into two categories. Those, for which the DLPO values are higher than DLPE, should have a strong tendency to pair together in the specified position. For those pairs which the DLPO values are less than DLPE, there exists a hindrance in neighboring of the two amino acids in that specific position of the α-helix. These cases have been identified and listed in various tables in this paper. The amount of mutual information carried by the exceptional pairs of amino acids was significantly higher than the average mutual information carried by other amino acid pairs. The average mutual information conveyed by amino acid pairs in each doublet position was found to be very small but non-zero.

Random insertion and deletion mutagenesis.

AbstractRandom mutagenesis combined with high-throughput screening is a versatile strategy for improving protein functions or creating artificial enzymes (1,2). Several methods for introducing random mutations in vitro have been reported (3). Among these, error-prone PCR mutagenesis, based on inaccurate copying by DNA polymerase, is the most commonly used technique to introduce random point mutations (4). However, the error-prone PCR method has an inherent drawback of biased occurrence of amino acids as the result of single base replacements in the triplet codons. For example, mutation from AUG (Met) to UGG (Trp) is unlikely to take place. To achieve a non-biased random replacement on the amino acid level, oligonucleotide-directed mutagenesis (5) and cassette mutagenesis (6,7) have been carried out. These methods are limited to a defined region of the gene and can not introduce mutations at random positions. To introduce codon-based mutations at various positions, the split-and-mix method (8) or other synthetic methods of constructing DNAs using dinucleotide or trinucleotide units have been attempted (9–11). These synthetic methods may be applicable only to introduce mutations within a narrow range of the target gene.KeywordsKlenow FragmentCeric Ammonium NitrateMagnesium AcetateLigation BufferRandom Point MutationThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Environment-dependent and position-specific frequencies of amino acid occurrences in .ALPHA.-helices

By using the method to define a local structural motif of proteins by the Delaunay tessellation proposed by Wako and Yamato (Protein Eng. 11, 981-990 (1998)), we analyzed environment-dependent and position-specific frequencies of amino-acid occurrences in α-helices. In that method the three-dimensional structure of a protein molecule is uniquely divided into non-overlapping Delaunay tetrahedrons, each vertex of which is occupied by one of the comprising residues. A code is then assigned to each tetrahedron so as to characterize the local structure containing it. The tetrahedrons located in the interior of the α-helices are assigned 36 kinds of codes. The differences in the codes reflect the existence and absence of four residues surrounding the relevant region of the α-helix. In other words, the environment of the α-helix can be differentiated by these codes. Accordingly, we analyzed the frequencies of amino acid occurrences on each vertex of the tetrahedrons for each of these codes. Such data provide information about possible amino acid substitutions specific to a vertex position (i.e., a position in the α-helix) for a given code (i.e., environment around the α-helix). Furthermore, the principal component analysis was carried out to reveal general features of the amino acid occurrences in the α-helices. In relation to these results, such frequencies at the N- and C-terminals of the α-helix are also discussed.

Information-Theoretic Analysis of Protein Sequences Shows that Amino Acids Self-cluster

We analyse for each of 20 amino acids X the statistics of spacings between consecutive occurrences of X within the well-characterized Saccharomyces cerevisiae genome. The occurrences of amino acids may exhibit near random, clustered or smoothed out behaviour, like one-dimensional stochastic processes along the protein chain. If amino acids are distributed randomly within a sequence, then they follow a Poisson process, and a histogram of the number of observations of each gap size would asymptotically follow a negative exponential distribution. The novelty of the present approach lies in the use of differential geometric methods to quantify information on sequencing of amino acids and groups of amino acids, via the sequences of intervals between their occurrences. The differential geometry arises from an information-theoretic distance function on the two-dimensional space of stochastic processes subordinate to gamma distributions-which latter include the random process as a special case. We find that maximum-likelihood estimates of parametric statistics show that all 20 amino acids tend to cluster, some substantially. In other words, the frequencies of short gap lengths tend to be higher and the variance of the gap lengths is greater than expected by chance. This may be because localizing amino acids with the same properties may favour secondary structure formation or transmembrane domains. Gap sizes of 1 or 2 are generally disfavoured, 1 strongly so. The only exceptions to this are Gln and Ser, as a result of poly(Gln) or poly(Ser) sequences. There are preferences for gaps of 4 and 7 that can be attributed to alpha -helices. In particular, a favoured gap of 7 for Leu is found in coiled coils. Our method contributes to the characterization of whole sequences by extracting and quantifying stable stochastic features.

Information-Theoretic Analysis of Protein Sequences Shows that Amino Acids Self-cluster

We analyse for each of 20 amino acids X the statistics of spacings between consecutive occurrences of X within the well-characterized Saccharomyces cerevisiae genome. The occurrences of amino acids may exhibit near random, clustered or smoothed out behaviour, like one-dimensional stochastic processes along the protein chain. If amino acids are distributed randomly within a sequence, then they follow a Poisson process, and a histogram of the number of observations of each gap size would asymptotically follow a negative exponential distribution. The novelty of the present approach lies in the use of differential geometric methods to quantify information on sequencing of amino acids and groups of amino acids, via the sequences of intervals between their occurrences. The differential geometry arises from an information-theoretic distance function on the two-dimensional space of stochastic processes subordinate to gamma distributions—which latter include the random process as a special case. We find that maximum-likelihood estimates of parametric statistics show that all 20 amino acids tend to cluster, some substantially. In other words, the frequencies of short gap lengths tend to be higher and the variance of the gap lengths is greater than expected by chance. This may be because localizing amino acids with the same properties may favour secondary structure formation or transmembrane domains. Gap sizes of 1 or 2 are generally disfavoured, 1 strongly so. The only exceptions to this are Gln and Ser, as a result of poly(Gln) or poly(Ser) sequences. There are preferences for gaps of 4 and 7 that can be attributed to α -helices. In particular, a favoured gap of 7 for Leu is found in coiled coils. Our method contributes to the characterization of whole sequences by extracting and quantifying stable stochastic features.

Decreasing the ring size of a cyclic nonribosomal peptide antibiotic by in-frame module deletion in the biosynthetic genes.

Many natural products of therapeutical and biotechnological importance are nonribosomally synthesized peptides. Structural hallmarks of this class of compounds are the occurrence of unusual amino acids, mostly cyclic peptide backbones, and numerous further modifications such as acylation, heterocyclic ring formation, and glycosylation. Because of their structural complexity, chemical synthesis is usually an unattractive route to these molecules. In contrast, genetic engineering of the biosynthesis genes emerges as a potentially powerful approach to the combinatorial biosynthesis of useful analogues of the lead compounds. Nonribosomal peptide synthetases (NRPSs) carry out a sequential multistep assembly and modification of the peptides in a thiotemplate process described by the multiple carrier model. The modular architecture of NRPSs suggests straightforward methods for the reprogramming of these enzymes by exchange of catalytic subunits. However, many of the reported engineering attempts faced low product yields or even inactive hybrid enzymes. Using a new approach to obtain hybrid NRPSs, we show here that the deletion of an entire module in an NRPS assembly line caused the secretion of the predicted peptide antibiotic variant with a decreased ring size. Furthermore, a module exchange resulted in a significantly higher product yield than that observed in previous studies.

Role of paired basic residues of protein C-termini in phospholipid binding.

It is a well known phenomenon that the occurrence of several distinct amino acids at the C-terminus of proteins is non-random. We have analysed all Saccharomyces cerevisiae proteins predicted by computer databases and found lysine to be the most frequent residue both at the last (-1) and at the penultimate amino acid (-2) positions. To test the hypothesis that C-terminal basic residues efficiently bind to phospholipids we randomly expressed GST-fusion proteins from a yeast genomic library. Fifty-four different peptide fragments were found to bind phospholipids and 40% of them contained lysine/arginine residues at the (-1) or (-2) positions. One peptide showed high sequence similarity with the yeast protein Sip18p. Mutational analysis revealed that both C-terminal lysine residues of Sip18p are essential for phospholipid-binding in vitro. We assume that basic amino acid residues at the (-1) and (-2) positions in C-termini are suitable to attach the C-terminus of a given protein to membrane components such as phospholipids, thereby stabilizing the spatial structure of the protein or contributing to its subcellular localization. This mechanism could be an additional explanation for the C-terminal amino acid bias observed in proteins of several species.

Diversity of microcystin genes within a population of the toxic cyanobacterium Microcystis spp. in Lake Wannsee (Berlin, Germany).

In order to find out how many genotypes determine microcystin production of Microcystis spp. in field populations, single colonies (clones) were sampled from Lake Wannsee (Berlin, Germany), characterized morphologically, and subsequently analyzed by PCR for a region within the mcyB gene encoding the activation of one amino acid during microcystin biosynthesis. The different morphospecies varied considerably in the proportion of microcystin-producing genotypes. Most colonies (73%) of M. aeruginosa contained this gene whereas only 16% of the colonies assigned to M. ichthyoblabe and no colonies of M. wesenbergii gave a PCR product of the mcyB gene. Restriction fragment length polymorphism revealed seven restriction profiles showing low variability in nucleotide sequence within each restriction type (0.4-4%) and a low to high variability (1.6-38%) between restriction types. In addition, the sequences of amino acids within the mcyB gene were analyzed to compare the specificity of the amino acid activation during microcystin biosynthesis between restriction types and with the occurrence of amino acids in microcystin variants as detected by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Most of the microcystin-producing colonies showed high similarity in the sequence of amino acids and contained microcystin-LR (LR refers to leucine and arginine in the variable positions of the heptapeptide), microcystin-RR, and microcystin-YR, as well as other variants in minor concentrations. It is concluded that the gene product found for most of the microcystin-producing colonies in the lake is rather unspecific and the diversity of microcystin variants in the lake results from activation of various amino acids during microcystin biosynthesis in the same genotypes.

Catabolism of amino acids by lactic acid bacteria isolated from Cheddar cheese

Abstract Fifty five percent of 152 lactic acid bacteria (LAB) isolated from 9 mature Cheddar cheeses were able to catabolise protein hydrolysates and individual amino acids in the Biolog MT1 TM microplate assay. A more detailed study of 31 isolates confirmed a requirement for α -ketoglutaric acid and inter- and intra-species differences in the range and number of amino acids utilised. GCMS analysis of cell-free supernatants of 29 LAB isolates grown on a low-carbohydrate medium containing lactalbumin hydrolysate identified more than 90 compounds. Putative metabolites of valine, leucine, isoleucine, phenylalanine and sulphur-containing amino acids were detected although the metabolite profiles were isolate-specific. The occurrence and activity of amino acid converting enzymes was also monitored in the LAB isolates. Methionine, branched-chain and aromatic amino acid aminotransferase activities were detected with α -ketoglutarate as acceptor. There was no conclusive evidence for a widespread occurrence of deaminase, decarboxylase and demethiolase activities. The majority of isolates examined formed thiols from methionine via an aminotransferase-mediated reaction.

SPINE: an integrated tracking database and data mining approach for identifying feasible targets in high-throughput structural proteomics.

High-throughput structural proteomics is expected to generate considerable amounts of data on the progress of structure determination for many proteins. For each protein this includes information about cloning, expression, purification, biophysical characterization and structure determination via NMR spectroscopy or X-ray crystallography. It will be essential to develop specifications and ontologies for standardizing this information to make it amenable to retrospective analysis. To this end we created the SPINE database and analysis system for the Northeast Structural Genomics Consortium. SPINE, which is available at bioinfo.mbb.yale.edu/nesg or nesg.org, is specifically designed to enable distributed scientific collaboration via the Internet. It was designed not just as an information repository but as an active vehicle to standardize proteomics data in a form that would enable systematic data mining. The system features an intuitive user interface for interactive retrieval and modification of expression construct data, query forms designed to track global project progress and external links to many other resources. Currently the database contains experimental data on 985 constructs, of which 740 are drawn from Methanobacterium thermoautotrophicum, 123 from Saccharomyces cerevisiae, 93 from Caenorhabditis elegans and the remainder from other organisms. We developed a comprehensive set of data mining features for each protein, including several related to experimental progress (e.g. expression level, solubility and crystallization) and 42 based on the underlying protein sequence (e.g. amino acid composition, secondary structure and occurrence of low complexity regions). We demonstrate in detail the application of a particular machine learning approach, decision trees, to the tasks of predicting a protein's solubility and propensity to crystallize based on sequence features. We are able to extract a number of key rules from our trees, in particular that soluble proteins tend to have significantly more acidic residues and fewer hydrophobic stretches than insoluble ones. One of the characteristics of proteomics data sets, currently and in the foreseeable future, is their intermediate size ( approximately 500-5000 data points). This creates a number of issues in relation to error estimation. Initially we estimate the overall error in our trees based on standard cross-validation. However, this leaves out a significant fraction of the data in model construction and does not give error estimates on individual rules. Therefore, we present alternative methods to estimate the error in particular rules.

Cover Picture

The cover picture shows the reaction between zinc oxide (upper right) and the α-amino acid isovaline, whose formula is depicted in front of a fragment of the Murchison meteorite (upper left). One of the products of this reaction is an unprecedented, cyclic zinc isovalinato hexamer. The occurrence of amino acids, for example isovaline, in certain primitive meteorites gives rise to the assumption that metal amino acidates may already have formed on early Earth some 4×109 years ago. At higher temperatures, solid metal amino acidates decompose to yield large numbers of organic compounds. In prebiotic times, the necessary thermal energy could have been provided by volcanic activities and meteorite impacts. These points are discussed in more detail by Strasdeit et al. on p. 1133 ff. (We thank J. Hurley for the permission to use the picture of the Murchison fragment.)