A common theme in the amino acid sequences of actin and many actin-binding proteins?

The amino acid sequences of several actin regulatory proteins have recently been determined. Do these proteins function by mimicking actin-actin interaction sites?     

Stability and amino acid preferences of type VIII reverse turn: the most common turn in peptides?

Free energies of the alpha(r)beta and betabeta conformations of 14 tetrapeptides, based on the sequence SALN and protein X-ray structures, were calculated using molecular dynamics simulations and MM-PBSA calculations. The alphaalpha conformations of five of the tetrapeptides were also studied. SALN has been earlier shown by molecular dynamics simulations and NMR spectroscopy to have a tendency to form an alpha(r)beta turn. The gas-phase energy of the molecular mechanical force field (CHARMM), the electrostatic and non-polar solvation free energies and solute entropies were used to explain the free energy differences of the alphaalpha, betabeta and alpha(r)beta conformations of the peptides. The alpha(r)beta conformation of SALN and SATN was predicted to be slightly more stable than the extended conformation (betabeta), in agreement with experimental results. The SALN mutants SAIN, SAVN, SATN, SSIN and MSHV, were also predicted to be potential alpha(r)beta turn-forming peptides. We report also revised positional potentials for the type VIII turn, based on a non-homologous set of protein structures. This protein databank analysis confirms the main results of the earlier analyses and reveals several new amino acid residues with a significant positional preference. The results of this work led us to suggest that the alpha(r)beta turn may be the most common turn type in peptides. Such turns may be readily formed in aqueous solution and thereby play important roles in the protein folding process by serving as an initiation point for structure formation.     

γ-Aminobutyric acid and related amino acids in plant immune responses: Emerging mechanisms of action

The entanglement between primary metabolism regulation and stress responses is a puzzling and fascinating theme in plant sciences. Among the major metabolites found in plants, γ-aminobutyric acid (GABA) fulfils important roles in connecting C and N metabolic fluxes through the GABA shunt. Activation of GABA metabolism is known since long to occur in plant tissues following biotic stresses, where GABA appears to have substantially different modes of action towards different categories of pathogens and pests. While it can harm insects thanks to its inhibitory effect on the neuronal transmission, its capacity to modulate the hypersensitive response in attacked host cells was proven to be crucial for host defences in several pathosystems. In this review, we discuss how plants can employ GABA's versatility to effectively deal with all the major biotic stressors, and how GABA can shape plant immune responses against pathogens by modulating reactive oxygen species balance in invaded plant tissues. Finally, we discuss the connections between GABA and other stress-related amino acids such as BABA (β-aminobutyric acid), glutamate and proline.     

Relationship between structure and amino acid sequence of strongly twisted and coiled β-hairpins in globular proteins

β-Hairpins are widespread in proteins, and it is possible to find them both within β-sheets and separately. In this work, a comparative analysis of amino acid sequences of β-strands within strongly twisted β-hairpins from different structural protein subclasses has been conducted. Strongly twisted and coiled β-hairpin generates in the space a right double helix out of β-strands that are connected by a loop region (connections). The frequencies of amino acid residues on the internal (concave) and external (convex) surfaces of strongly twisted β-hairpins have been determined (220 β-hairpins from nonhomologous proteins were studied). The concave surface of these β-hairpins is mainly generated by hydrophobic residues, while the convex surface by hydrophilic residues; accordingly, the alternation of hydrophobic internal and hydrophilic external residues is observed in their amino acid sequences. Amino acid residues of glycine and alanine (especially in places of the largest twisting of the strands) were anomalously frequently found in internal positions of strongly twisted and coiled β-hairpins. It was established that internal positions never contain the proline residues, while external positions in the twisting region contain them in a relatively large amount. It was demonstrated that at least one amino acid residue in α_L- or ε-conformation is required for generation of relatively short (up to 7 amino acid residues) connection. As a rule, these positions are occupied by glycines. Thus, not only the alternation of hydrophobic and hydrophilic amino acid residues, but also the presence of one or two glycine residues in the connection region and the excess of glycines and alanines in the places of the largest strand twisting on the concave surface, as well as the presence of prolines on the convex surface, are required to generate a strongly twisted and coiled β-hairpin.     

Synthesis and in vitro stability of amino acid prodrugs of 6-β-naltrexol for microneedle-enhanced transdermal delivery

A small library of amino acid ester prodrugs of 6-β-naltrexol (NTXOL, 1) was prepared in order to investigate the candidacy of these prodrugs for microneedle-enhanced transdermal delivery. Six amino acid ester prodrugs were synthesized (6a–f). 6b, 6d, and 6e were stable enough at skin pH (pH 5.0) to move forward to studies in 50% human plasma. The lead compound (6e) exhibited the most rapid bioconversion to NTXOL in human plasma (t_1/2 = 2.2 ± 0.1 h).     

Pattern of amino acid substitutions in transmembrane domains of β-barrel membrane proteins for detecting remote homologs in bacteria and mitochondria

β-barrel membrane proteins play an important role in controlling the exchange and transport of ions and organic molecules across bacterial and mitochondrial outer membranes. They are also major regulators of apoptosis and are important determinants of bacterial virulence. In contrast to β-helical membrane proteins, their evolutionary pattern of residue substitutions has not been quantified, and there are no scoring matrices appropriate for their detection through sequence alignment. Using a Bayesian Monte Carlo estimator, we have calculated the instantaneous substitution rates of transmembrane domains of bacterial β-barrel membrane proteins. The scoring matrices constructed from the estimated rates, called bbTM for β-barrel Transmembrane Matrices, improve significantly the sensitivity in detecting homologs of β-barrel membrane proteins, while avoiding erroneous selection of both soluble proteins and other membrane proteins of similar composition. The estimated evolutionary patterns are general and can detect β-barrel membrane proteins very remote from those used for substitution rate estimation. Furthermore, despite the separation of 2-3 billion years since the proto-mitochondrion entered the proto-eukaryotic cell, mitochondria outer membrane proteins in eukaryotes can also be detected accurately using these scoring matrices derived from bacteria. This is consistent with the suggestion that there is no eukaryote-specific signals for translocation. With these matrices, remote homologs of β-barrel membrane proteins with known structures can be reliably detected at genome scale, allowing construction of high quality structural models of their transmembrane domains, at the rate of 131 structures per template protein. The scoring matrices will be useful for identification, classification, and functional inference of membrane proteins from genome and metagenome sequencing projects. The estimated substitution pattern will also help to identify key elements important for the structural and functional integrity of β-barrel membrane proteins, and will aid in the design of mutagenesis studies.     

A new model of weak acid permeation through membranes revisited: does Overton still rule?

According to a recent publication by Thomae, A. V., H. Wunderli-Allenspach, and S. D. Kr?mer (2005. Biophys. J. 89:1802-1811), membrane bilayers are well-permeable to the charged species of aromatic carboxylic acids. At physiological pH, the anions were claimed to be the major diffusing species. In contrast, calculation of the Born energy barrier predicts a 10(5)-fold higher permeability for the uncharged (protonated) form. To test the new model, we now have measured both the current carried by the salicylate anion through solvent-free planar membranes and the amount of protons transported by the neutral species. The corresponding membrane permeabilities of the charged and protonated forms were 4 x 10(-7) cm/s and 1.2 cm/s. These data are in perfect agreement with literature data gathered in the last three decades (compare, e.g., Gutknecht, J., and D. C. Tosteson. 1973. Science. 182:1258-1261). They indicate that the report by Thomae at al. represents an experimental artifact. The well-documented role of neutral species in the permeation process of weak acids and bases across artificial and natural membranes is not in question. Overton still rules.     

A new enzymatic reaction for producing new-type amino acids by Escherichia coli: Production of α-amino-β-(1-indoline) propionic acid from indoline and l-Serine

We found that some reaction products were produced from indole-mimic compounds, such as indoline (2,3-dihydroindole), indazole, 7-azaindole and 3-indazolinone, with l-serine by the catalytic action of the lyophilized cells of Escherichia coli T4-3 (a mutant defective in indole-3-glycerolphosphate synthase [EC 4.1.1.48]) cultured in a tryptophan-limited medium.A main product from indoline and l-serine was isolated and identified as a-amino-β-(1-indoline) propionic acid (AIP) from data obtained by paperchromatography, elemental analysis, UV, IR, ¹H-NMR and mass spectrometry.The reaction conditions and the requirements for the reaction were also studied.AIP was produced only in the case of using l-serine, l-serine methylester and l-serine ethylester as the amino acid source.On the enzyme concerned AIP production, studies were carried out by using the mutant strains of E. coli defective in the enzyme(s) of tryptophan operon. Tryptophan synthase [EC 4.2.1.20], particularly its B protein, was presumed to be a possible candidate.     

β-Neo-endorphin,a new hypothalamic “big” Leu-enkephalin of porcine origin: Its purification and the complete amino acid sequence

A new hypothalamic “big” Leu-enkephalin of porcine origin, designated as β-neo-endorphin, has been isolated from a side fraction, obtained in our previous isolation of α-neo-endorphin and PH-8P (dynorphin[1–8]). The complete amino acid sequence has been elucidated to be : Tyr-Gly-Gly-Phe-Leu-Arg-Lys-Tyr-Pro. The sequence has been confirmed by the comparison of natural β-neo-endorphin with a synthetic peptide. In addition, β-neo-endorphin exhibits potent opioid activity in guinea-pig ileum assay.     

Urinary amino acid analysis: A comparison of iTRAQ®–LC–MS/MS,GC–MS,and amino acid analyzer

Urinary amino acid analysis is typically done by cation-exchange chromatography followed by post-column derivatization with ninhydrin and UV detection. This method lacks throughput and specificity. Two recently introduced stable isotope ratio mass spectrometric methods promise to overcome those shortcomings. Using two blinded sets of urine replicates and a certified amino acid standard, we compared the precision and accuracy of gas chromatography/mass spectrometry (GC–MS) and liquid chromatography–tandem mass spectrometry (LC–MS/MS) of propyl chloroformate and iTRAQ^® derivatized amino acids, respectively, to conventional amino acid analysis. The GC–MS method builds on the direct derivatization of amino acids in diluted urine with propyl chloroformate, GC separation and mass spectrometric quantification of derivatives using stable isotope labeled standards. The LC–MS/MS method requires prior urinary protein precipitation followed by labeling of urinary and standard amino acids with iTRAQ^® tags containing different cleavable reporter ions distinguishable by MS/MS fragmentation. Means and standard deviations of percent technical error (%TE) computed for 20 amino acids determined by amino acid analyzer, GC–MS, and iTRAQ^®–LC–MS/MS analyses of 33 duplicate and triplicate urine specimens were 7.27 ± 5.22, 21.18 ± 10.94, and 18.34 ± 14.67, respectively. Corresponding values for 13 amino acids determined in a second batch of 144 urine specimens measured in duplicate or triplicate were 8.39 ± 5.35, 6.23 ± 3.84, and 35.37 ± 29.42. Both GC–MS and iTRAQ^®–LC–MS/MS are suited for high-throughput amino acid analysis, with the former offering at present higher reproducibility and completely automated sample pretreatment, while the latter covers more amino acids and related amines.     

Single amino acid substitutions producing instability of globular proteins. Calculation of their frequencies in the entire mutational spectra of the α- and β-subunits of human hemoglobin

Summary The frequencies of substitutions resulting in protein instability were calculated by a method estimating changes in stability produced by amino acid substitutions. The method takes into account the accessibility of an amino acid position to a solvent and changes in the specificity of amino acid interactions. When tested on human mutant hemoglobins, the method yielded predictions with a preciseness of 80%. The consideration of the evolutionary homologous proteins in the analysis allowed us to estimate the evolutionary constraints imposed on stability of their spatial structure. With these limitations, approximately 50% of amino acid substitutions in the entire mutational spectra of the - and -subunits of human hemoglobin were found to damage the spatial structure of the globular proteins.     

Heteronuclear relayed E.COSY revisited: Determination of 3J(Hα,Cγ) couplings in Asx and aromatic residues in proteins

Constant-time 3D heteronuclear relayed E.COSY [Schmidt et al. (1996) J. Biomol. NMR, 7, 142–152], as based on generic 2D small-flip-angle HMQC-COSY [Schmidt et al. (1995) J. Biomol. NMR, 6, 95–105], has been modified to allow for quantitative determination of heteronuclear three-bond ³ J(H,C) couplings. The method is applicable to amino acid spin topologies with carbons in the position which lack attached protons, i.e. to asparagine, aspartate, and aromatic residues in uniformly ¹³C-enriched proteins. The pulse sequence critically exploits heteronuclear triple-quantum coherence (HTQC) of CH₂ moieties involving geminal H proton pairs, taking advantage of improved multiple-quantum relaxation properties, at the same time avoiding scalar couplings between those spins involved in multiple-quantum coherence, thus yielding E.COSY-type multiplets with a splitting structure that is simpler than with the original scheme. Numerical least-squares 2D line-shape simulation is used to extract ³ J(H,C) coupling constants which are of relevance to side-chain ₁ dihedral-angle conformations in polypeptides. Methods are demonstrated with recombinant ¹⁵N,¹³C-enriched ribonuclease T1 and Desulfovibrio vulgaris flavodoxin with bound oxidized FMN.     

Critical amino acids responsible for converting specificities of proteins and for enhancing enzyme evolution are located around β-turn potentials: Data-based prediction

Various reports have described that amino acid substitutions can alter substrate, positional, inhibitory, and target gene specificities of proteins. By using the method of Chou and Fasman, the present work predicts that critical amino acids for converting these specificities are located around β-turns. Residues responsible for the alterations of substrate specificities of trypsin,l-lactate dehydrogenase, aspartate aminotransferase, β-lactamase, and cytochrome P-450 are found to exist within regions predicted as β-turns. The ratios of hydroxylation and oxygenation positions of substrates by cytochrome P-450 and lipoxygenase, respectively, are varied by changes of the protein structures, probably around turn conformations. Inhibitory specificities of bovine pancreatic trypsin inhibitor and α₁-antitrypsin and target gene specificity of glucocorticoid receptor are converted by changing turn structures. Occurrence of β-turn probabilities can be predicted around the amino acid alteration positions of an evolutionally antecedent protein of a nylon degradation enzyme. These findings will have relevance to work on protein engineering and enzyme evolution.     

The nucleotide and deduced amino acid sequences of a cDNA encoding a new species of Pregnancy-Specific β1-Glycoprotein (PSβG)

We screened a cDNA library of a human placenta with cDNA for nonspecific cross-reacting antigen, a member of the carcinoembryonic antigen gene family. One of the positive clones, PS34, was found to encode a 426 amino acid protein belonging to pregnancy-specific β₁-glycoprotein (PSβG). The mature PS34 protein consisted of domains, N, A1, A2, B2 and C. The domain-N of PS34 showed sequence similarities of 79.8–83.5% to those of the PSβG members so far reported, indicating PS34 is a new member of PSβG and also of the carcinoembryonic antigen gene family.     

Production of rosmarinic acid and a new rosmarinic acid 3′-O-β-D-glucoside in suspension cultures of the hornwort Anthoceros agrestis Paton

Cell suspension cultures of the hornwort Anthoceros agrestis Paton (Anthocerotaceae) were cultivated and characterized in CB-media containing 2 and 4% sucrose. The suspension cells accumulated rosmarinic acid up to 5.1% of the cell dry weight as well as caffeoyl-4'-hydroxyphenyllactate. Moreover, a more hydrophilic compound was detected which was isolated and identified as rosmarinic acid 3'-O-beta-D-glucoside, a new rosmarinic acid derivative. This new rosmarinic acid derivative was found up to 1.0% of the cell dry weight in suspension cells of A. agrestis.