4‐Cyano‐α‐methyl‐l‐phenylalanine as a Spectroscopic Marker for the Investigation of PeptaibioticMembrane Interactions

Two analogs of the ten‐amino acid residue, membrane‐active lipopeptaibiotic trichogin GA IV, mono‐labeled with 4‐cyano‐α‐methyl‐L ‐phenylalanine, a potentially useful fluorescence and IR absorption probe of the local microenvironment, were synthesized by the solid‐phase methodology and conformationally characterized. The single modification was incorporated either at the N‐terminus (position 1) or near the C‐terminus (position 8) of the peptide main chain. In both cases, the replaced amino acid was the equally helicogenic α‐aminoisobutyric acid (Aib) residue. We performed a solution conformational analysis by use of FT‐IR absorption, CD, and 2D‐NMR spectroscopies. The results indicate that both labeled analogs essentially maintain the overall helical propensity of the naturally occurring lipopeptaibiotic. Peptide? membrane interactions were assessed by fluorescence and ATR‐IR absorption techniques. Analogies and differences between the two peptides were highlighted. Taken together, our data confirm literature results that some of the spectroscopic parameters of the 4‐cyanobenzyl chromophore are sensitive markers of the local microenvironment.     

An HPLC‐ESMS study on the solid‐phase assembly of C‐terminal proline peptides

DKP formation is a serious side reaction during the solid‐phase synthesis of peptide acids containing either Pro or Gly at the C‐terminus. This side reaction not only leads to a lower overall yield, but also to the presence in the reaction crude of several deletion peptides lacking the first amino acids. For the preparation of protected peptides using the Fmoc/tBu strategy, the use of a ClTrt‐Cl‐resin with a limited incorporation of the C‐terminal amino acid is the method of choice. The use of resins with higher loading levels leads to more impure peptide crudes. The use of HPLC‐ESMS is a useful method for analysing complex samples, such as those formed when C‐terminal Pro peptides are prepared by non‐optimized solid‐phase strategies. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Site‐specific labeling of synthetic peptide using the chemoselective reaction between N‐methoxyamino acid and isothiocyanate

Site‐specific labeling of synthetic peptides carrying N‐methoxyglycine (MeOGly) by isothiocyanate is demonstrated. A nonapeptide having MeOGly at its N‐terminus was synthesized by the solid‐phase method and reacted with phenylisothiocyanate under various conditions. In acidic solution, the reaction specifically gave a peptide having phenylthiourea structure at its N‐terminus, leaving side chain amino group intact. The synthetic human β‐defensin‐2 carrying MeOGly at its N‐terminus or the side chain amino group of Lys¹⁰ reacted with phenylisothiocyanate or fluorescein isothiocyanate also at the N‐methoxyamino group under the same conditions, demonstrating that this method is generally useful for the site‐specific labeling of linear synthetic peptides as well as disulfide‐containing peptides. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Micelle bound structure and DNA interaction of brevinin‐2‐related peptide,an antimicrobial peptide derived from frog skin

Brevinin‐2‐related peptide (BR‐II), a novel antimicrobial peptide isolated from the skin of frog, Rana septentrionalis, shows a broad spectrum of antimicrobial activity with low haemolytic activity. It has also been shown to have antiviral activity, specifically to protect cells from infection by HIV‐1. To understand the active conformation of the BR‐II peptide in membranes, we have investigated the interaction of BR‐II with the prokaryotic and eukaryotic membrane‐mimetic micelles such as sodium dodecylsulfate (SDS) and dodecylphosphocholine (DPC), respectively. The interactions were studied using fluorescence and circular dichroism (CD) spectroscopy. Fluorescence experiments revealed that the N‐terminus tryptophan residue of BR‐II interacts with the hydrophobic core of the membrane mimicking micelles. The CD results suggest that interactions with membrane‐mimetic micelles induce an α‐helix conformation in BR‐II. We have also determined the solution structures of BR‐II in DPC and SDS micelles using NMR spectroscopy. The structural comparison of BR‐II in the presence of SDS and DPC micelles showed significant conformational changes in the residues connecting the N‐terminus and C‐terminus helices. The ability of BR‐II to bind DNA was elucidated by agarose gel retardation and fluorescence experiments. The structural differences of BR‐II in zwitterionic versus anionic membrane mimics and the DNA binding ability of BR‐II collectively contribute to the general understanding of the pharmacological specificity of this peptide towards prokaryotic and eukaryotic membranes and provide insights into its overall antimicrobial mechanism. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

An improved synthesis of (2S, 4S)‐ and (2S, 4R)‐2‐amino‐4‐methyldecanoic acids: assignment of the stereochemistry of culicinins

An improved synthesis of (2S, 4S)‐ and (2S, 4R)‐2‐amino‐4‐methyldecanoic acids was accomplished using a glutamate derivative as starting material and Evans' asymmetric alkylation as the decisive step. The NMR data of the two diastereomers were measured and compared with those of the natural product. As a result, the stereochemistry of this novel amino acid unit in culicinins was assigned as (2S, 4R). Copyright © 2011 European Peptide Society and John Wiley & Sons, Ltd.     

Kinetic improvement of an algal diacylglycerol acyltransferase 1 via fusion with an acyl‐CoA binding protein

Microalgal oils in the form of triacylglycerols (TAGs) are broadly used as nutritional supplements and biofuels. Diacylglycerol acyltransferase (DGAT) catalyzes the final step of acyl‐CoA‐dependent biosynthesis of TAG, and is considered a key target for manipulating oil production. Although a growing number of DGAT1s have been identified and over‐expressed in some algal species, the detailed structure?function relationship, as well as the improvement of DGAT1 performance via protein engineering, remain largely untapped. Here, we explored the structure?function features of the hydrophilic N‐terminal domain of DGAT1 from the green microalga Chromochloris zofingiensis (CzDGAT1). The results indicated that the N‐terminal domain of CzDGAT1 was less disordered than those of the higher eukaryotic enzymes and its partial truncation or complete removal could substantially decrease enzyme activity, suggesting its possible role in maintaining enzyme performance. Although the N‐terminal domains of animal and plant DGAT1s were previously found to bind acyl‐CoAs, replacement of CzDGAT1 N‐terminus by an acyl‐CoA binding protein (ACBP) could not restore enzyme activity. Interestingly, the fusion of ACBP to the N‐terminus of the full‐length CzDGAT1 could enhance the enzyme affinity for acyl‐CoAs and augment protein accumulation levels, which ultimately drove oil accumulation in yeast cells and tobacco leaves to higher levels than the full‐length CzDGAT1. Overall, our findings unravel the distinct features of the N‐terminus of algal DGAT1 and provide a strategy to engineer enhanced performance in DGAT1 via protein fusion, which may open a vista in generating improved membrane‐bound acyl‐CoA‐dependent enzymes and boosting oil biosynthesis in plants and oleaginous microorganisms.     

Isolation and partial characterization of a bacteriocin produced by Lactobacillus plantarum BM‐1 isolated from a traditionally fermented Chinese meat product

Lactobacillus plantarum BM‐1 isolated from a traditionally fermented Chinese meat product was found to produce a novel bacteriocin that is active against a wide range of gram‐positive and gram‐negative bacteria. Production of the bacteriocin BM‐1 started early in the exponential phase and its maximum activity (5120 AU/mL) was recorded early during the stationary phase (16 hr). Bacteriocin BM‐1 is sensitive to proteolytic enzymes but stable in the pH range of 2.0–10.0 and heat‐resistant (15 min at 121°C). This bacteriocin was purified through pH‐mediated cell adsorption–desorption and cation‐exchange chromatography on an SP Sepharose Fast Flow column. The molecular weight of the purified bacteriocin BM‐1 was determined to be 4638.142 Da by electrospray ionization Fourier transform mass spectrometry. Furthermore, the N‐terminal amino acid sequence was obtained through automated Edman degradation and found to comprise the following 15 amino acid residues: H₂N‐Lys‐Tyr‐Tyr‐Gly‐Asn‐Gly‐Val‐Tyr‐Val‐Gly‐Lys‐His‐Ser‐Cys‐Ser. Comparison of this sequence with that of other bacteriocins revealed that bacteriocin BM‐1 contains the consensus YGNGV amino acid motif near the N‐terminus. Based on its physicochemical characteristics, molecular weight, and N‐terminal amino acid sequence, plantaricin BM‐1 is a novel class IIa bacteriocin.     

Studies on the Selective Trifluoroacetolytic Scission of Native Peptaibols and Model Peptides Using HPLC and ESI‐CID‐MS

Representative members of a group of linear, N‐acylated polypeptide antibiotics (peptaibols) containing α‐aminoisobutyric acid (Aib) and, in part, isovaline (Iva), as well as proteinogenic amino acids and a C‐terminal‐bonded 2‐amino alcohol, were treated with anhydrous trifluoroacetic acid (TFA) at 37° for 0.5–26 h. The resulting fragments were separated by HPLC and characterized by electrospray ionization collision‐induced dissociation mass spectrometry (ESI‐CID‐MS). The following 16–20‐residue peptaibols were investigated: natural, microheterogeneous mixtures of antiamoebins and alamethicin F50, uniform paracelsin A, and synthetic trichotoxin A50/E. In the natural peptides, bonds formed between Aib (Iva) and Pro (Hyp) were rapidly and selectively cleaved within 0.5 h. Furthermore, TFA esters of the C‐terminal amino alcohols were formed. Depending on time, release of C‐terminal tri‐ and tetrapeptides as well as amino acids from the major fragments was observed. Synthetic homooligopeptides, namely Z‐ and Ac‐(Aib)₁₀‐O^tBu and Z‐(Aib)₇‐O^tBu, were analyzed for comparison. On treatment with TFA, a regular series of Z‐(Aib)_10–5‐OH from Z‐(Aib)₁₀‐O^tBu were detected within 0.5 h, and, after 3 h, release of a regular series of Z‐(Aib)_7–3‐OH from Z‐(Aib)₇‐O^tBu were observed. Moreover, concomitant release of the series of H‐(Aib)_10–3‐OH from the decapeptide occurred. From these data, a repetitive cleavage mechanism via intermediate formation of C‐terminal oxazolones on trifluoroacetolysis is proposed. Furthermore, their formation and stability in native peptaibols are correlated with subtle structural differences in protein amino acids linked to Aib. From the conspicuous concordance of the formation and abundance of regular series of trifluoroacetolytic fragments and of positive ions of the b‐series in CID‐MS, the generation of intermediate oxazolonium ions in both gas and liquid phase is concluded.     

Structural analysis of the human cannabinoid receptor one carboxyl‐terminus identifies two amphipathic helices

Recent research has implicated the C‐terminus of G‐protein coupled receptors in key events such as receptor activation and subsequent intracellular sorting, yet obtaining structural information of the entire C‐tail has proven a formidable task. Here, a peptide corresponding to the full‐length C‐tail of the human CB1 receptor (residues 400–472) was expressed in E.coli and purified in a soluble form. Circular dichroism (CD) spectroscopy revealed that the peptide adopts an α‐helical conformation in negatively charged and zwitterionic detergents (48–51% and 36–38%, respectively), whereas it exhibited the CD signature of unordered structure at low concentration in aqueous solution. Interestingly, 27% helicity was displayed at high peptide concentration suggesting that self‐association induces helix formation in the absence of a membrane mimetic. NMR spectroscopy of the doubly labeled (¹⁵N‐ and ¹³C‐) C‐terminus in dodecylphosphocholine (DPC) identified two amphipathic α‐helical domains. The first domain, S401‐F412, corresponds to the helix 8 common to G protein‐coupled receptors while the second domain, A440‐M461, is a newly identified structural motif in the distal region of the carboxyl‐terminus of the receptor. Molecular modeling of the C‐tail in DPC indicates that both helices lie parallel to the plane of the membrane with their hydrophobic and hydrophilic faces poised for critical interactions. © 2009 Wiley Periodicals, Inc. Biopolymers 91: 565–573, 2009. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Efficient one‐pot synthesis of N‐ethyl amino acids

Mono‐N‐ethylated α‐amino acid esters are obtained in high yields using reductive amination procedures. Formation of imine is achieved by excess of acetaldehyde, followed by removal of acetaldehyde and reduction by NaBH(OAc)₃. The elaborated one‐pot synthesis allows for the efficient synthesis of side‐chain protected amino acid derivatives. Copyright © 1999 European Peptide Society and John Wiley & Sons, Ltd.     

Structure of the Escherichia coli ArnA N‐formyltransferase domain in complex with N5‐formyltetrahydrofolate and UDP‐Ara4N

ArnA from Escherichia coli is a key enzyme involved in the formation of 4‐amino‐4‐deoxy‐l ‐arabinose. The addition of this sugar to the lipid A moiety of the lipopolysaccharide of pathogenic Gram‐negative bacteria allows these organisms to evade the cationic antimicrobial peptides of the host immune system. Indeed, it is thought that such modifications may be responsible for the repeated infections of cystic fibrosis patients with Pseudomonas aeruginosa. ArnA is a bifunctional enzyme with the N‐ and C‐terminal domains catalyzing formylation and oxidative decarboxylation reactions, respectively. The catalytically competent cofactor for the formylation reaction is N¹⁰‐formyltetrahydrofolate. Here we describe the structure of the isolated N‐terminal domain of ArnA in complex with its UDP‐sugar substrate and N⁵‐formyltetrahydrofolate. The model presented herein may prove valuable in the development of new antimicrobial therapeutics.     

β‐amino acid substitution to investigate the recognition of angiotensin II (AngII) by angiotensin converting enzyme 2 (ACE2)

In spite of the important role of angiotensin converting enzyme 2 (ACE2) in the cardiovascular system, little is known about the substrate structural requirements of the AngII–ACE2 interaction. Here we investigate how changes in angiotensin II (AngII) structure affect binding and cleavage by ACE2. A series of C3 β‐amino acid AngII analogs were generated and their secondary structure, ACE2 inhibition, and proteolytic stability assessed by circular dichroism (CD), quenched fluorescence substrate (QFS) assay, and LC‐MS analysis, respectively. The β‐amino acid‐substituted AngII analogs showed differences in secondary structure, ACE2 binding and proteolytic stability. In particular, three different subsets of structure‐activity profiles were observed corresponding to substitutions in the N‐terminus, the central region and the C‐terminal region of AngII. The results show that β‐substitution can dramatically alter the structure of AngII and changes in structure correlated with ACE2 inhibition and/or substrate cleavage. β‐amino acid substitution in the N‐terminal region of AngII caused little change in structure or substrate cleavage, while substitution in the central region of AngII lead to increased β‐turn structure and enhanced substrate cleavage. β‐amino acid substitution in the C‐terminal region significantly diminished both secondary structure and proteolytic processing by ACE2. The β‐AngII analogs with enhanced or decreased proteolytic stability have potential application for therapeutic intervention in cardiovascular disease. Copyright © 2010 John Wiley & Sons, Ltd.     

The structure of S. lividans acetoacetyl‐CoA synthetase shows a novel interaction between the C‐terminal extension and the N‐terminal domain

The adenosine monoposphate‐forming acyl‐CoA synthetase enzymes catalyze a two‐step reaction that involves the initial formation of an acyl adenylate that reacts in a second partial reaction to form a thioester between the acyl substrate and CoA. These enzymes utilize a Domain Alternation catalytic mechanism, whereby a ～110 residue C‐terminal domain rotates by 140° to form distinct catalytic conformations for the two partial reactions. The structure of an acetoacetyl‐CoA synthetase (AacS) is presented that illustrates a novel aspect of this C‐terminal domain. Specifically, several acetyl‐ and acetoacetyl‐CoA synthetases contain a 30‐residue extension on the C‐terminus compared to other members of this family. Whereas residues from this extension are disordered in prior structures, the AacS structure shows that residues from this extension may interact with key catalytic residues from the N‐terminal domain. Proteins 2015; 83:575–581. © 2014 Wiley Periodicals, Inc.     

Alteration of fatty acid chain length of Chlamydomonas reinhardtii by simultaneous expression of medium‐chain‐specific thioesterase and acyl carrier protein

Biofuel from fatty acids with chain lengths of 8–15 (C8–C15) have properties similar to those of conventional diesel and jet fuels, thus, can save time and reduce costs for the refurbishment of engines and maintenance of oiling facilities. Most oil‐producing algae yield C16–C18 fatty acids; however, the manipulation of algae using genetic engineering is a promising approach to obtain C8–C15 fatty acids. The introduction of a medium‐chain‐specific thioesterase (TE) is expected to effectively alter algae to produce medium‐chain fatty acids (MCFAs). TE is the main determinant of fatty acid chain length as it releases fatty acids from the acyl carrier protein (ACP) in the fatty acid elongation cycle. In a previous study, the introduction of heterologous C8–C12‐specific TEs into Chlamydomonas reinhardtii did not increase the yield of MCFAs. This effect was attributed to a low affinity of the heterologous TEs to C. reinhardtii ACP. Therefore, we introduced both the C10–C14‐specific TE gene and the ACP gene from the land plant Cuphea lanceolata into C. reinhardtii. We measured free fatty acids (FFAs) and triacylglycerols (TAGs) in the transformants using liquid chromatography–mass spectrometry. The production of C12:0 and C14:0, chain length 12 and 14 without unsaturation, FFAs was not significantly increased in any of the tested strains. However, we found a slight but significant increase in TAG‐containing MCFAs in both TE only and TE–ACP transformants. The increased production rate of C14:0‐containing TAGs ranged from 1.25‐ to 1.58‐fold, indicating the ability of medium‐chain‐specific TE to increase MCFAs. These results suggest that the selection of specific TEs is important when modifying eukaryotic algae to produce MCFAs.     

Synthesis of stable C‐linked ferrocenyl amino acids and their use in solution‐phase peptide synthesis

Incorporation of ferrocenyl group to peptides is an efficient method to alter their hydrophobicity. Ferrocenyl group can also act as an electrochemical probe when incorporated onto functional peptides. Most often, ferrocene is incorporated onto peptides post‐synthesis via amide, ester or triazole linkages. Stable amino acids containing ferrocene as a C‐linked side chain are potentially useful building units for the synthesis of ferrocene‐containing peptides. We report here an efficient route to synthesize ferrocene‐containing amino acids that are stable and can be used in peptide synthesis. Coupling of 2‐ferrocenyl‐1,3‐dithiane and iodides derived from aspartic acid or glutamic acid using n‐butyllithium leads to the incorporation of a ferrocenyl unit to the δ‐position or ε‐position of an α‐amino acid. The reduction or hydrolysis of the dithiane group yields an alkyl or an oxo derivative. The usability of the synthesized amino acids is demonstrated by incorporating one of the amino acids in both C‐terminus and N‐terminus of tripeptides in solution phase. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

Pyrazole amino acids: hydrogen bonding directed conformations of 3‐amino‐1H‐pyrazole‐5‐carboxylic acid residue

A series of model compounds containing 3‐amino‐1H‐pyrazole‐5‐carboxylic acid residue with N‐terminal amide/urethane and C‐terminal amide/hydrazide/ester groups were investigated by using NMR, Fourier transform infrared, and single‐crystal X‐ray diffraction methods, additionally supported by theoretical calculations. The studies demonstrate that the most preferred is the extended conformation with torsion angles ? and ψ close to ±180°. The studied 1H‐pyrazole with N‐terminal amide/urethane and C‐terminal amide/hydrazide groups solely adopts this energetically favored conformation confirming rigidity of that structural motif. However, when the C‐terminal ester group is present, the second conformation with torsion angles ? and ψ close to ±180° and 0°, respectively, is accessible. The conformational equilibrium is observed in NMR and Fourier transform infrared studies in solution in polar environment as well as in the crystal structures of other related compounds. The observed conformational preferences are clearly related to the presence of intramolecular interactions formed within the studied residue. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.     

A cleavable self‐assembling tag strategy for preparing proteins and peptides with an authentic N‐terminus

Recombinant protein expression and purification remains a central need for biotechnology. Herein, the authors report a streamlined protein and peptide purification strategy using short self‐assembling peptides and a C‐terminal cleavage intein. In this strategy, the fusion protein is first expressed as an aggregate induced by the self‐assembling peptide. Upon simple separation, the target protein or peptide with an authentic N‐terminus is then released in the solution by intein‐mediated cleavage. Different combinations of four self‐assembling peptides (ELK16, L₆KD, FK and FR) with three inteins (Sce VMA, Mtu ΔI‐CM and Ssp DnaB) were explored. One protein and two peptides were used as model polypeptides to test the strategy. The intein Mtu ΔI‐CM, which has pH‐shift inducible cleavage, was found to work well with three self‐assembling peptides (L₆KD, FR, FK). Using this intein gave a yield of protein or peptide comparable with that from other more established strategies, such as the Trx‐strategy, but in a simpler and more economical way. This strategy provides a simple and efficient method by which to prepare proteins and peptides with an authentic N‐terminus, which is especially effective for peptides of 30‐100 amino acids in length that are typically unstable and susceptible to degradation in Escherichia coli.     

Temperature‐induced changes in lipid biomarkers and mycosporine‐like amino acids in the psychrophilic dinoflagellate Peridinium aciculiferum

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C5 conserved region of hydrophilic C‐terminal part of Saccharomyces cerevisiae Nha1 antiporter determines its requirement of Erv14 COPII cargo receptor for plasma‐membrane targeting

Erv14, a conserved cargo receptor of COPII vesicles, helps the proper trafficking of many but not all transporters to the yeast plasma membrane, for example, three out of five alkali‐metal‐cation transporters in Saccharomyces cerevisiae. Among them, the Nha1 cation/proton antiporter, which participates in cell cation and pH homeostasis, is a large membrane protein (985 aa) possessing a long hydrophilic C‐terminus (552 aa) containing six conserved regions (C1–C6) with unknown function. A short Nha1 version, lacking almost the entire C‐terminus, still binds to Erv14 but does not need it to be targeted to the plasma membrane. Comparing the localization and function of ScNha1 variants shortened at its C‐terminus in cells with or without Erv14 reveals that only ScNha1 versions possessing the complete C5 region are dependent on Erv14. In addition, our broad evolutionary conservation analysis of fungal Na⁺/H⁺ antiporters identified new conserved regions in their C‐termini, and our experiments newly show C5 and other, so far unknown, regions of the C‐terminus, to be involved in the functionality and substrate specificity of ScNha1. Taken together, our results reveal that also relatively small hydrophilic parts of some yeast membrane proteins underlie their need to interact with the Erv14 cargo receptor.     

Site‐specific conjugation of the quencher on peptide's N‐terminal for the synthesis of a targeted non‐spreading activatable optical probe

Optical imaging offers high sensitivity and portability at low cost. The design of ‘smart’ or ‘activatable’ probes can decrease the background noise and increase the specificity of the signal. By conjugating a fluorescent dye and a compatible quencher on each side of an enzyme's substrate, the signal remains in its ‘off ’ state until it reaches the area where a specific enzyme is expressed. However, the signal can leak from that area unless the dye is attached to a molecule able to bind to a specific target also presented in that area. The aim of this study was to (i) specifically conjugate the quencher on the α‐amino group of the peptide's N‐terminus, (ii) conjugate the dye on the ε‐amino group of a lysine in C‐terminus, and (iii) conjugate the carboxyl group of the peptide's C‐terminus to an amino group present on an antibody, using carbodiimide chemistry. The use of protecting groups, such as Boc or Fmoc, to allow site‐specific conjugation, presents several drawbacks including ‘on beads labeling’, additional steps required for deprotection and removal from the resin, decreased yield, and dye degradation. A method of preferential labeling of α‐amino N‐terminal group in slightly acidic solution, proposed by Selo et al. (1996) has partially solved the problem. The present study reports improvements of the method allowing to (i) avoid the homo‐bilabeling, (ii) increase the yield of the N‐terminal labeling by two folds, and (iii) decrease the cost by 44‐fold. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.