Integrating the intrinsic conformational preferences of noncoded α-amino acids modified at the peptide bond into the noncoded amino acids database

Recently, we reported a database (Noncoded Amino acids Database; http://recerca.upc.edu/imem/index.htm) that was built to compile information about the intrinsic conformational preferences of nonproteinogenic residues determined by quantum mechanical calculations, as well as bibliographic information about their synthesis, physical and spectroscopic characterization, the experimentally established conformational propensities, and applications (Revilla-López et al., J Phys Chem B 2010;114:7413-7422). The database initially contained the information available for α-tetrasubstituted α-amino acids. In this work, we extend NCAD to three families of compounds, which can be used to engineer peptides and proteins incorporating modifications at the--NHCO--peptide bond. Such families are: N-substituted α-amino acids, thio-α-amino acids, and diamines and diacids used to build retropeptides. The conformational preferences of these compounds have been analyzed and described based on the information captured in the database. In addition, we provide an example of the utility of the database and of the compounds it compiles in protein and peptide engineering. Specifically, the symmetry of a sequence engineered to stabilize the 3(10)-helix with respect to the α-helix has been broken without perturbing significantly the secondary structure through targeted replacements using the information contained in the database.     

The structure of β-FeOOH·Cln (akaganeite) and its uptake of amino acids

Yellowish brown precipitates in samples of Red Sea hot brine have recently been identified as -FeOOH·Cl_n or akaganéite. Synthetic -FeOOH·Cl_n has been prepared by hydrolysis of FeCl₃-solution for transmission electron microscope studies of the structure of this iron (III) oxide hydroxide. Extreme care has been taken to avoid destabilization of the crystalline compound during preparation of ultrathin sections for the uptake of micrographs. The results indicate that previously reported and debated structures in the size range of 40–60 Å of the somatoidic akaganéite crystals are no attifacts but instead a superimposed structure that is sensitive to washing and drying procedures. This superimposed structure might be important for the uptake of amino acids by -FeOOH·Cl_n and may perhaps also be suitable for catalytic effects such as prebiotic synthesis of polypeptides.     

Synthesis and antimicrobial activity against Pseudomonas aeruginosa of macrocyclic β-hairpin peptidomimetic antibiotics containing N-methylated amino acids

Antimicrobial resistance among Gram-negative bacteria is a growing problem, fueled by the paucity of new antibiotics that target these microorganisms. One novel family of macrocyclic β-hairpin-shaped peptidomimetics was recently shown to act specifically against Pseudomonas spp. by a novel mechanism of action, targeting the outer membrane protein LptD, which mediates lipopolysaccharide transport to the cell surface during outer membrane biogenesis. Here we explore the mode of binding of one of these β-hairpin peptidomimetics to LptD in Pseudomonas aeruginosa, by examining the effects on antimicrobial activity following N-methylation of individual peptide bonds. An N-methyl scan of the cyclic peptide revealed that residues on both sides of the β-hairpin structure at a non-hydrogen bonding position likely mediate hydrogen-bonding interactions with the target LptD. Structural analyses by NMR spectroscopy further reinforce the conclusion that the folded β-hairpin structure of the peptidomimetic is critical for binding to the target LptD. Finally, new NMe analogues with potent activity have been identified, which opens new avenues for optimization in this family of antimicrobial peptides.     

Increase in the production of β-carotene in recombinant Escherichia coli cultured in a chemically defined medium supplemented with amino acids

Escherichia coli DH5α strain was selected as the recombinant host, and a chemically defined medium supplemented with amino acids was used instead of a complex medium for the efficient production of β-carotene. In a fed-batch culture using glycerol with a chemically defined medium supplemented with amino acids, the concentration, specific content, and productivity of β-carotene were 2,470 mg/l, 72 mg/g cells, and 77 mg/l h after 32 h, respectively. These values were, respectively, 43, 33, and 26 % higher than those obtained using the complex medium. This is the highest β-carotene production that has been reported among the recombinant cells to date.     

Synthesis and binding properties of endomorphin-2analogs containing α-hydroxymethyl amino acids

Novel endomorphin-2 analogs containing the unusual amphiphilic amino acid (R)- and (S)--hydroxymethyltyrosine in position 1 and (R)- and (S)--hydroxymethylphenylalanine in the positions 3 and 4 were synthesized via the solid-phase method. The binding characteristics of the synthetic analogs may suggest that -hydroxymethyl substitution of aminoacid residues influences the conformation of a peptide much more than simply increasing the local amphiphilic character of the peptide.     

Synthesis of a burkholdine analogue containing β-hydroxytyrosine

Synthesis of a β-OHTyr-containing Bk analogue, a cyclic octalipopeptide with antifungal activities, is described. Since β-OHTyr-containing peptides generally are unstable in strong acidic conditions, synthesis of β-HOTyr-containing peptides by SPPS have rarely been reported. To overcome this problem, we found that using distilled TFA removed the protecting groups of side chains of β-OHTyr-containing Bk analogue, which was prepared by Fmoc-SPPS.

Abbreviations: β-OHTyr: β-hydroxytyrosine; β-OHAsn: β-hydroxyasparagine; Bk: burkholdine; FAA: fatty acyl amino acid; β-MeOTyr: β-methoxytyrosine; SPPS: solid phase peptide synthesis; MIC: minimun inhibitory concentration; DMF: dimethyl formamide; DIPEA: diisopropylethylamine; DIPC: diisopropylcarbodiimide; HOBt: 1-hydroxybenzotriazole; Fmoc: 9-fluorenylmethyloxycarbonyl; HFIP: 1,1,1,3,3,3-hexafluoropropan-2-ol; TFA: trifluoroacetic acid; LAP: N-lauryl ?3-amino-4-carbamolypropanoic acid; HPLC: high performance liquid chromatography; ESI-TOFMS: electrospray ionization-time of flight mass spectrometry; Bn: benzyl; Boc: t-butyloxycatbonyl; 2-CTC: 2-chlorotritylchloride.     

Two γ-Glutamylpeptides of acetylenic amino acids in Tricholomopsis rutilans

Two γ-glutamylpeptides, γ-L-glutamyl-L-2-aminohex-4-ynoic acid and γ-L-glutamyl-L-erythro-2-amino-3-hydroxyhex-4-ynoic acid,     

Solution structure of β2-microglobulin and insights into fibrillogenesis

The solution structure of human β₂-microglobulin (β₂-m) was determined by ¹H NMR spectroscopy and restrained modeling calculations. Compared to the crystal structure of type I major histocompatibility complex (MHC-I), where the protein is associated to the heavy-chain component, several differences are observed, i.e., increased separation between strands A and B, displacements of strand C′ and loop DE, shortening of strands D and E. These modifications can be considered as the prodromes of the amyloid transition. Even minor charge changes in response to pH, as is the case with H31 imidazole protonation, trigger the transition that starts with unpairing of strand A. The same mechanism accounts for the partial unfolding and fiber formation subsequent to Cu²⁺ binding which is shown to occur primarily at H31. Solvation of the protected regions in MHC-I decreases the tertiary packing by breaking the contiguity of the surface hydrophobic patches via surface charge cluster. Mutants or truncated forms of β₂-m can be designed to remove the instability from H31 titration or to enhance the instability through surface charge suppression. By monitoring the conformational evolution of wild-type protein and variants thereof, either in response or absence of external perturbation, valuable insights into intermediate structure and fibrillogenesis mechanisms are gained.     

Studies on the rules of β-strand alignment in a protein β-sheet structure

To further disclose the underlying mechanisms of protein β-sheet formation, studies were made on the rules of β-strands alignment forming β-sheet structure using statistical and machine learning approaches. Firstly, statistical analysis was performed on the sum of β-strands between each β-strand pairs in protein sequences. The results showed a propensity of near-neighbor pairing (or called “first come first pair”) in the β-strand pairs. Secondly, based on the same dataset, the pairwise cross-combinations of real β-strand pairs and four pseudo-β-strand contained pairs were classified by support vector machine (SVM). A novel feature extracting approach was designed for classification using the average amino acid pairing encoding matrix (APEM). Analytical results of the classification indicated that a segment of β-strand had the ability to distinguish β-strands from segments of α-helix and coil. However, the result also showed that a β-strand was not strongly conserved to choose its real partner from all the alternative β-strand partners, which was corresponding with the ordination results of the statistical analysis each other. Thus, the rules of “first come first pair” propensity and the non-conservative ability to choose real partner, were possible important factors affecting the β-strands alignment forming β-sheet structures.     

CuI-promoted one-pot synthesis of N-Boc protected β-ketotriazole amino acids: application in the synthesis of new class of dipeptidomimetics

One-pot click chemistry of N(α)-Boc-bromomethylketones, NaN3 and propiolic acid affords N-Boc protected 1,4-disubstituted 1,2,3-β-ketotriazole acids in good to excellent yield. The use of CuI as catalyst and DMSO as solvent leads the click reaction to efficient, practical and column-free preparation of the title compounds. The utility of the resulting unnatural amino acids as building blocks to prepare triazole possessing peptidomimetics is also delineated.     

Structure and evolution of the stigmapophysis—A unique repose wing-coupling structure in Psocodea

The gain of foldable wings is regarded as one of the key innovations enabling the present-day diversity of neopteran insects. Wing folding allows compact housing of the wings and shields the insect body from damage. Wing-fixing systems have evolved in some insects, probably to increase the durability of the shielding function by the wings. Bark lice (Psocodea) are known to possess a unique wing-to-wing repose coupling system, but a detailed morphological and evolutionary study of this system is lacking. In this study, we examined this repose coupling structure by SEM in 32 species including representatives of all three suborders of bark lice (Trogiomorpha, Troctomorpha and Psocomorpha). We concluded that the repose wing-coupling apparatus independently evolved twice within Psocodea. In Trogiomorpha, the apparatus is located on the subcostal vein of the forewing and is composed of elongated rib-like structures. In Troctomorpha and Psocomorpha, in contrast, the repose coupling structure is located on the radius vein of the forewing and is formed by a swollen vein. These morphological and developmental differences in the repose coupling structures also provide phylogenetic information at different systematic levels.     

Functional analysis of conserved aromatic amino acids in the discoidin domain of Paenibacillus β-1,3-glucanase

The 190-kDa Paenibacillus β-1,3-glucanase (LamA) contains a catalytic module of the glycoside hydrolase family 16 (GH16) and several auxiliary domains. Of these, a discoidin domain (DS domain), present in both eukaryotic and prokaryotic proteins with a wide variety of functions, exists at the carboxyl-terminus. To better understand the bacterial DS domain in terms of its structure and function, this domain alone was expressed in Escherichia coli and characterized. The results indicate that the DS domain binds various polysaccharides and enhances the biological activity of the GH16 module on composite substrates. We also investigated the importance of several conserved aromatic residues in the domain's stability and substrate-binding affinity. Both were affected by mutations of these residues; however, the effect on protein stability was more notable. In particular, the forces contributed by a sandwiched triad (W1688, R1756, and W1729) were critical for the presumable β-sandwich fold.     

Incorporation of β-amino acids into dihydrofolate reductase by ribosomes having modifications in the peptidyltransferase center

Ribosomes containing modifications in three regions of 23S rRNA, all of which are in proximity to the ribosomal peptidyltransferase center (PTC), were utilized previously as a source of S-30 preparations for in vitro protein biosynthesis experiments. When utilized in the presence of mRNAs containing UAG codons at predetermined positions + β-alanyl–tRNA_CUA, the modified ribosomes produced enhanced levels of full length proteins via UAG codon suppression. In the present study, these earlier results have been extended by the use of substituted β-amino acids, and direct evidence for β-amino acid incorporation is provided. Presently, five of the clones having modified ribosomes are used in experiments employing four substituted β-amino acids, including α-methyl-β-alanine, β,β-dimethyl-β-alanine, β-phenylalanine, and β-(p-bromophenyl)alanine. The β-amino acids were incorporated into three different positions (10, 18 and 49) of Escherichia coli dihydrofolate reductase (DHFR) and their efficiencies of suppression of the UAG codons were compared with those of β-alanine and representative α-l-amino acids. The isolated proteins containing the modified β-amino acids were subjected to proteolytic digestion, and the derived fragments were characterized by mass spectrometry, establishing that the β-amino acids had been incorporated into DHFR, and that they were present exclusively in the anticipated peptide fragments. DHFR contains glutamic acid in position 17, and it has been shown previously that Glu-C endoproteinase can hydrolyze DHFR between amino acids residues 17 and 18. The incorporation of β,β-dimethyl-β-alanine into position 18 of DHFR prevented this cleavage, providing further evidence for the position of incorporation of the β-amino acid.     

Specific amino acids responsible for the cold adaptedness of Micrococcus antarcticus β-glucosidase BglU

Psychrophilic enzymes display efficient activity at moderate or low temperatures (4–25 °C) and are therefore of great interest in biotechnological industries. We previously examined the crystal structure of BglU, a psychrophilic β-glucosidase from the bacterium Micrococcus antarcticus, at 2.2 Å resolution. In structural comparison and sequence alignment with mesophilic (BglB) and thermophilic (GlyTn) counterpart enzymes, BglU showed much lower contents of Pro residue and of charged amino acids (particularly positively charged) on the accessible surface area. In the present study, we investigated the roles of specific amino acid residues in the cold adaptedness of BglU. Mutagenesis assays showed that the mutations G261R and Q448P increased optimal temperature (from 25 to 40–45 °C) at the expense of low-temperature activity, but had no notable effects on maximal activity or heat lability. Mutations A368P, T383P, and A389E significantly increased optimal temperature (from 25 to 35–40 °C) and maximal activity (~1.5-fold relative to BglU). Thermostability of A368P and A389E increased slightly at 30 °C. Mutations K163P, N228P, and H301A greatly reduced enzymatic activity—almost completely in the case of H301A. Low contents of Pro, Arg, and Glu are important factors contributing to BglU’s psychrophilic properties. Our findings will be useful in structure-based engineering of psychrophilic enzymes and in production of mutants suitable for a variety of industrial processes (e.g., food production, sewage treatment) at cold or moderate temperatures.

     

Synthesis and structural studies of new analogues of PTH(1–11) containing Cα-tetra-substituted amino acids in position 8

The N-terminal 1–34 fragment of parathyroid hormone (PTH) is fully active in vitro and in vivo and it can reproduce all biological responses characteristic of the native intact PTH. Recently, analogues of PTH(1–11) fragments with helicity-enhancing substitutions have been demonstrated to yield potent analogues of PTH(1–34). The work describes the synthesis, biological activity and structure of analogues of the best modified PTH sequence H-Aib-Val-Aib-Glu-Ile-Gln-Leu-Nle-His-Gln-Har-NH₂ (I). In particular, the effect of the Ala/Aib substitution at positions 1 and 3 as well as of the replacement of Nle in position 8 with d-Nle, l-(αMe)-Nle and d-(αMe)-Nle was studied. The resulting peptides were characterized structurally by CD spectroscopy, solution NMR and MD, and in vitro for activity with respect to the cognate receptor, parathyroid hormone receptor.     

Role of negatively charged amino acids in β4 F-loop in activation and desensitization of α3β4 rat neuronal nicotinic receptors

The role of negatively charged amino acids in the F-loop of the β4 subunit in channel activation and desensitization was studied using the patch-clamp technique. The selected amino acids were changed to their neutral analogs via point mutations. Whole-cell currents were recorded in COS cells transiently transfected with the α3β4 nicotinic acetylcholine receptor. The application of acetylcholine (ACh), nicotine (Nic), cytisine (Cyt), carbamylcholine (CCh) and epibatidine (Epi) to cells clamped at − 40 mV produced inward currents which displayed biphasic desensitization. The EC₅₀ of Epi and Nic were increased by a factor of 3-6 due to mutations D191N or D192N. Only Epi remained an agonist in the double-mutated receptors with EC₅₀ increased 17-fold. The interaction of the receptors with the competitive antagonist (+)tubocurarine (TC) was weakened almost 3-fold in the double-mutated receptors. The mutations increased the proportion of the slower desensitization component and increased the response plateau, resulting in decreased receptor desensitization. The double mutation substantially accelerated the return from long-term desensitization induced by Epi.     

Abnormal subcellular distribution of β-glucuronidase in mice with a genetic alteration in enzyme structure

Liver -glucuronidase is structurally altered in inbred strain PAC so that a peptide subunit with a more basic isoelectric point, GUS-SN, is produced. This allele of -glucuronidase was transferred to strain C57BL/6J by 12 backcross matings to form the congenic line B6 · PAC-Gus ⁿ. Liver -glucuronidase activity was halved in males of the congenic strain compared to normal males. The lowered activity was specifically accounted for by a decrease in the lysosomal component. There was no alteration in the concentration of microsomal activity. This alteration in the subcellular distribution of -glucuronidase in Gus ⁿ/Gus ⁿ mice was confirmed by two independent gel electrophoretic systems which separate microsomal and lysosomal components. -Glucuronidase activity was likewise approximately halved in mutant spleen, lung, and brain, organs which contain exclusively or predominantly lysosomal -glucuronidase. The loss of liver lysosomal -glucuronidase activity was shown by immunotitration to be due to a decrease in the number of -glucuronidase molecules in lysosomes of the congenic strain. The Gus ⁿ structural alteration likely causes the lowered lysosomal -glucuronidase activity since the two traits remain in congenic animals. Heterozygous Gus ⁿ/Gus ^b animals had intermediate levels of liver -glucuronidase. Also, the effect was specific, in that three other lysosomal enzymes were not reproducibly lower in Gus ⁿ/Gus ⁿ mice. Gus ⁿ is, therefore, an unusual example of a mutation which causes a change in the subcellular distribution of a two-site enzyme.This work was supported by National Institutes of Health Grants GM-33559 and GM-33160 and National Science Foundation Grant PCM-8215808.     

Enzymatic transformation of desacetyl-lanatoside A into digitoxin by β-glucosidase action in cyclodextrin solutions

Summary The enzymatic transformation of desacetyl-lanatoside A (DLA) to its secondary glycoside, digitoxin, in solutions of -and -cyclodextrins is effected using of -glucosidase from barley. Due to the interaction of cyclodextrins (CyDs) with desacetyl-lanatoside A, an increase in solubility of the latter of 24.5 and 230 times was observed for -cyclodextrin and -cyclodextrin, respectively. Kinetic studies of the enzymatic transformation gave for -glucosidase the values K_M=3.3×10^–4 mol. dm^–3 and V_max=0.557 mol mg^–1 min^–1 when the substrate was the deacetyl-lanatoside A complex with -cyclodextrin, while in the case of the complex with -cyclodextrin these values were K_M=5.45×10^–4 mol dm^–3 and V_max=0.896 mol mg^–1 min^–1.     

Binding of β4γ5 by adenosine A1 and A2A receptors determined by stable isotope labeling with amino acids in cell culture and mass spectrometry

Characterization of G protein βγ dimer isoform expression in different cellular contexts has been impeded by low levels of protein expression, broad isoform heterogeneity, and antibodies of limited specificity, sensitivity, or availability. As a new approach, we used quantitative mass spectrometry to characterize native βγ dimers associated with adenosine A(1):α(i1) and adenosine A(2A):α(S) receptor fusion proteins expressed in HEK-293 cells. Cells expressing A(1):α(i1) were cultured in media containing [(13)C(6)]Arg and [(13)C(6)]Lys and βγ labeled with heavy isotopes purified. Heavy βγ was combined with either recombinant βγ purified from Sf9 cells, βγ purified from the A(2A):α(S) expressed in HEK-293 cells cultured in standard media, or an enriched βγ fraction from HEK-293 cells. Samples were separated by SDS-PAGE, protein bands containing β and γ were excised, digested with trypsin, and separated by HPLC, and isotope ratios were analyzed by mass spectrometry. Three β isoforms, β(1), β(2), and β(4), and seven γ isoforms, γ(2), γ(4), γ(5), γ(7), γ(10), γ(11), and γ(12), were identified in the analysis. β(1) and γ(5) were most abundant in the enriched βγ fraction, and this βγ profile was generally mirrored in the fusion proteins. However, both A(2A):α(S) and A(1):α(i1) bound more β(4) and γ(5) compared to the enriched βγ fraction; also, more β(4) was associated with A(2A):α(S) than A(1):α(i1). Both fusion proteins also contained less γ(2), γ(10), and γ(12) than the enriched βγ fraction. These results suggest that preferences for particular βγ isoforms may be driven in part by structural motifs common to adenosine receptor family members.     

Ileal digestibility of nutrients and amino acids in low quality soybean meal sources treated with β-mannanase for growing pigs

Apparent ileal digestibility (AID) of energy, dry matter (DM), nitrogen (N) and amino acids and standardized ileal digestibility (SID) of amino acids in low quality soybean meals with different CP concentration (SBM 44% CP and SBM 48% CP) with or without 400 U β-mannanase/kg supplementation were evaluated in 20 cannulated barrows ((Landrace×Yorkshire)×Duroc) with an average BW of 25.08±3.42 kg. A N-free diet was used to determine basal endogenous losses of amino acids. The supplementation of β-mannanase improved (P<0.05) AID of DM compared with non-supplemented diet. However, enzyme supplementation did not improve (P>0.05) AID of N and energy. The type of SBM (SBM 44% CP v. SBM 48% CP) had no effect on AID of DM, N and energy. β-mannanase improved (P<0.05) AID of sum of essential amino acids, arginine, histidine, lysine, valine and glycine. The SID of lysine was higher (P<0.05) in enzyme supplemented than in non-supplemented diets. Larger AID and SID of threonine and proline (P<0.05) were observed in SBM 48% CP than in SBM 44% CP. In conclusion, the supplementation of enzyme improved AID of arginine, histidine, lysine, valine and glycine, but it did not cause marked difference in SID of these amino acids except for lysine. The low nutrient digestibility of the SBM sources used in the present experiment might have favoured the positive effect of β-mannanase supplementation.