Antibacterial and membrane‐damaging activities of β‐bungarotoxin B chain

This study investigates whether the B chain of β‐bungarotoxin exerted antibacterial activity against Escherichia coli (Gram‐negative bacteria) and Staphylococcus aureus (Gram‐positive bacteria) via its membrane‐damaging activity. The B chain exhibited a growth inhibition effect on E. coli but did not show a bactericidal effect on S. aureus. The B‐chain bactericidal action on E. coli positively correlated with an increase in membrane permeability in the bacterial cells. Lipopolysaccharide (LPS) layer destabilization and lipoteichoic acid (LTA) biosynthesis inhibition in the cell wall increased the B‐chain bactericidal effect on E. coli and S. aureus. The B chain induced leakage and fusion in E. coli and S. aureus membrane‐mimicking liposomes. Compared with LPS, LTA notably suppressed the membrane‐damaging activity and fusogenicity of the B chain. The B chain showed similar binding affinity with LPS and LTA, whereas LPS and LTA binding differently induced B‐chain conformational change as evidenced by the circular dichroism spectra. Taken together, our data indicate that the antibacterial action of the B chain is related to its ability to induce membrane permeability and suggest that the LPS‐induced and LTA‐induced B‐chain conformational change differently affects the bactericidal action of the B chain. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

Temperature‐dependent hemolytic activity of membrane pore‐forming peptide toxin,tolaasin

Tolaasin, a pore‐forming peptide toxin produced by Pseudomonas tolaasii, causes brown blotch disease on cultivated mushrooms. Hemolysis using red blood cells was measured to evaluate the cytotoxicity of tolaasin. To investigate the mechanism of tolaasin‐induced cell disruption, we studied the effect of temperature on the hemolytic process. At 4 °C, poor binding of the tolaasin molecules to the erythrocyte membrane was observed and most of the tolaasin molecules stayed in the solution. However, once tolaasin bound to erythrocytes at 37 °C and the temperature was decreased, complete hemolysis was observed even at 4 °C. These results indicate that tolaasin binding to cell membrane is temperature‐sensitive while tolaasin‐induced membrane disruption is less sensitive to temperature change. The effect of erythrocyte concentration was measured to understand the membrane binding and pore‐forming properties of tolaasin. The percentage of hemolysis measured by both hemoglobin release and cell lysis decreased as erythrocyte concentration increased in the presence of a fixed amount of tolaasin. The result shows that hemolysis is dependent on the amount of tolaasin and multiple binding of tolaasin is required for the hemolysis of a single cell. In analysis of dose‐dependence, the hemolysis was proportional to the tenth power of the amount of tolaasin, implying that tolaasin‐induced hemolysis can be explained by a multi‐hit model. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Solution NMR resonance assignment strategies for β‐barrel membrane proteins

Membrane proteins in detergent micelles are large and dynamic complexes that present challenges for solution NMR investigations such as spectral overlap and line broadening. In this study, multiple methods are introduced to facilitate resonance assignment of β‐barrel membrane proteins using Opa₆₀ from Neisseria gonorrhoeae as a model system. Opa₆₀ is an eight‐stranded β‐barrel with long extracellular loops (～63% of the protein) that engage host receptors and induce engulfment of the bacterium. The NMR spectra of Opa₆₀ in detergent micelles exhibits significant spectral overlap and resonances corresponding to the loop regions had variable line widths, which interfered with a complete assignment of the protein. To assign the β‐barrel residues, trypsin cleavage was used to remove much of the extracellular loops while preserving the detergent solubilized β‐barrel. The removal of the loop resonances significantly improved the assignment of the Opa₆₀ β‐barrel region (97% of the resonances corresponding to the β‐barrel and periplasmic turns were assigned). For the loop resonance assignments, two strategies were implemented; modulating temperature and synthetic peptides. Lowering the temperature broadened many peaks beyond detection and simplified the spectra to only the most dynamic regions of the loops facilitating 27 loop resonances to be assigned. To further assign functionally important and unstructured regions of the extracellular loops, a synthetic 20 amino acid peptide was synthesized and had nearly complete spectral overlap with the full‐length protein allowing 17 loop resonances to be assigned. Collectively, these strategies are effective tools that may accelerate solution NMR structure determination of β‐barrel membrane proteins.     

Identification of the membrane penetrating domain of Vibrio cholerae cytolysin as a beta-barrel structure

Vibrio cholerae cytolysin (VCC) is an oligomerizing pore-forming toxin that is related to cytolysins of many other Gram-negative organisms. VCC contains six cysteine residues, of which two were found to be present in free sulphydryl form. The positions of two intramolecular disulphide bonds were mapped, and one was shown to be essential for correct folding of protoxin. Mutations were created in which the two free cysteines were deleted, so that single cysteine substitution mutants could be generated for site-specific labelling. Employment of polarity-sensitive fluorophores identified amino acid side-chains that formed part of the pore-forming domain of VCC. The sequence commenced at residue 311, and was deduced to form a beta-barrel in the assembled oligomer with the subsequent odd-numbered residues facing the lipid bilayer and even-numbered residues facing the lumen. Pro328/Lys329 were tentatively identified as the position at which the sequence turns back into the membrane and where the antiparallel beta-strand commences. This was deduced from fluorimetric analyses combined with experiments in which the pore was reversibly occluded by derivatization of sulphydryl groups with a bulky moiety. Our data support computer-based predictions that the membrane-permeabilizing amino acid sequence of VCC is homologous to the beta-barrel-forming sequence of staphylococcal cytolysins and identify the beta-barrel as a membrane-perforating structure that is highly conserved in evolution.     

Unfolding distinguishes the Vibrio cholerae cytolysin precursor from the mature form of the toxin

Vibrio cholerae cytolysin (VCC) is a potent cytolytic toxin that induces colloid osmotic lysis of its target eukaryotic cells by forming transmembrane oligomeric β-barrel channels. VCC is secreted by the bacteria as an inactive precursor (Pro-VCC) and is subsequently activated by proteolytic removal of an N-terminal "Pro-domain", thus generating the active form of the toxin (Mature-VCC). Earlier studies have indicated an intramolecular chaperone-like role of the Pro-domain favoring efficient secretion of the toxin from the periplasm into the extracellular space. However, the exact role of the Pro-domain in the VCC structure--function mechanism remains unclear. Here, we have compared the Pro-VCC and Mature-VCC molecules in terms of their structural and conformational properties. We have studied unfolding of the two variants of the VCC molecule in response to an array of denaturing conditions, including low-pH, chemical denaturant and heat-induced unfolding. Pro-VCC shows a more profound tendency to unfold in response to such denaturing conditions compared to Mature-VCC. Biophysical characterization of the isolated Pro-domain further suggests that the increased unfolding propensity of Pro-VCC does not arise because of an increased level of unfolding of the Pro-region itself. Altogether, our results imply that a natively folded architecture of the Pro-VCC molecule with sufficient structural and conformational plasticity presumably allows it to adopt a suitable configuration that is possibly required for its efficient secretion and subsequent proteolytic maturation under physiological conditions.     

Cholesterol‐β1AR interaction versus cholesterol‐β2AR interaction

Two 8‐µs all‐atom molecular dynamics simulations have been performed on the two highly homologous G protein‐coupled receptor (GPCR) subtypes, β₁‐ and β₂‐adrenergic receptors, which were embedded in a lipid bilayer with randomly dispersed cholesterol molecules. During the simulations, cholesterol molecules accumulate to different surface regions of the two receptors, suggesting the subtype specificity of cholesterol–β‐adrenergic receptor interaction and providing some clues to the physiological difference of the two subtypes. Meanwhile, comparison between the two receptors in interacting with cholesterols shed some new light on general determinants of cholesterol binding to GPCRs. Our results indicate that although the concave surface, charged residues and aromatic residues are important, neither of these stabilizing factors is indispensable for a cholesterol interaction site. Different combinations of these factors lead to the diversified binding modes of cholesterol binding to the receptors. Our long‐time simulations, for the first time, revealed the pathway of a cholesterol molecule entering the consensus cholesterol motif (CCM) site, and the binding process of cholesterol to CCM is accompanied by a side chain flipping of the conserved Trp4.50. Moreover, the simulation results suggest that the I‐/V‐/L‐rich region on the extracellular parts of helix 6 might be an alternatively conserved cholesterol‐binding site for the class‐A GPCRs. Proteins 2014; 82:760–770. © 2013 Wiley Periodicals, Inc.     

The autophagic pathway: a cell survival strategy against the bacterial pore-forming toxin Vibrio cholerae cytolysin

Vibrio cholerae is the causative agent of cholera in humans. In addition to the criticalvirulence factors cholera toxin and toxin coregulated pilus, V. cholerae secretes V.cholerae cytolysin (VCC), a pore-forming exotoxin able to induce cell lysis and extensivevacuolation. We have shown that this vacuolation is related to the activation of autophagyin response to VCC action. Furthermore, we found that the autophagic pathway wasrequired to protect cells upon VCC intoxication. Based on additional data presented here,we propose a model aimed to explain the mechanism of cell protection. We postulatethat VCC-induced autophagic vacuoles, which display features of multivesicular bodies and enclose the toxin, are implicated in cell defense through VCC degradation involvingfusion with lysosomes.     

A pore‐forming toxin enables Serratia a nonlytic egress from host cells

Several pathogens co‐opt host intracellular compartments to survive and replicate, and they thereafter disperse progeny to prosper in a new niche. Little is known about strategies displayed by Serratia marcescens to defeat immune responses and disseminate afterwards. Upon invasion of nonphagocytic cells, Serratia multiplies within autophagosome‐like vacuoles. These Serratia‐containing vacuoles (SeCV) circumvent progression into acidic/degradative compartments, avoiding elimination. In this work, we show that ShlA pore‐forming toxin (PFT) commands Serratia escape from invaded cells. While ShlA‐dependent, Ca²⁺ local increase was shown in SeCVs tight proximity, intracellular Ca²⁺ sequestration prevented Serratia exit. Accordingly, a Ca²⁺ surge rescued a ShlA‐deficient strain exit capacity, demonstrating that Ca²⁺ mobilization is essential for egress. As opposed to wild‐type‐SeCV, the mutant strain‐vacuole was wrapped by actin filaments, showing that ShlA expression rearranges host actin. Moreover, alteration of actin polymerization hindered wild‐type Serratia escape, while increased intracellular Ca²⁺ reorganized the mutant strain‐SeCV actin distribution, restoring wild‐type‐SeCV phenotype. Our results demonstrate that, by ShlA expression, Serratia triggers a Ca²⁺ signal that reshapes cytoskeleton dynamics and ends up pushing the SeCV load out of the cell, in an exocytic‐like process. These results disclose that PFTs can be engaged in allowing bacteria to exit without compromising host cell integrity.     

Residues involved in the pore‐forming activity of the Clostridium perfringens iota toxin

Clostridium perfringens iota toxin is a binary toxin that is organized into enzyme (Ia) and binding (Ib) components. Ib forms channels in lipid bilayers and mediates the transport of Ia into the target cells. Here we show that Ib residues 334–359 contain a conserved pattern of alternating hydrophobic and hydrophilic residues forming two amphipathic β‐strands involved in membrane insertion and channel formation. This stretch of amino acids shows remarkable structural and functional analogies with the β‐pore‐forming domain of C. perfringens epsilon toxin. Several mutations within the two amphipathic β‐strands affected pore formation, single‐channel conductance and ion selectivity (S339E‐S341E, Q345H N346E) confirming their involvement in channel formation. F454 of Ib corresponds to the Φ‐clamp F427 of anthrax protective antigen and F428 of C2II binary toxins. The mutation F454A resulted in a loss of cytotoxicity and strong increase in single‐channel conductance (500 pS as compared with 85 pS in 1 M KCl) with a slight decrease in cation selectivity, indicating that the Φ‐clamp is highly conserved and crucial for binary toxin activity. In contrast, the mutants Q367D, N430D, L443E had no or only minor effects on Ib properties, while T360I, T360A and T360W caused a dramatic effect on ion selectivity and single‐channel conductance, indicating gross disturbance of the oligomer structure. This suggests that, at least in the iota toxin family, T360 has a structural role in the pore organization. Moreover, introduction of charged residues within the channel (S339E‐S341E) or in the vestibule (Q367D, N430D and L443E) had virtually no effect on chloroquine or Ia binding, whereas F454A, T360I, T360A and T360W strongly decreased the chloroquine and Ia affinity to Ib. These results support that distinct residues within the vestibule interact with chloroquine and Ia or are responsible for channel structure, while the channel lining amino acids play a less important role.     

Endosomal pH favors shedding of membrane‐inserted amyloid‐β peptide

Amyloid‐β peptides (Aβs) are generated in a membrane‐embedded state by sequential processing of amyloid precursor protein (APP). Although shedding of membrane‐embedded Aβ is essential for its secretion and neurotoxicity, the mechanism behind shedding regulation is not fully elucidated. Thus, we devised a Langmuir film balance‐based assay to uncover this mechanism. We found that Aβ shedding was enhanced under acidic pH conditions and in lipid compositions resembling raft microdomains, which are directly related to the microenvironment of Aβ generation. Furthermore, Aβ shedding efficiency was determined by the length of the C‐terminal membrane‐spanning region, whereas pH responsiveness appears to depend on the N‐terminal ectodomain. These findings indicate that Aβ shedding may be directly coupled to its generation and represents an unrecognized control mechanism regulating the fate of membrane‐embedded products of APP processing.     

Involvement and necessity of the Cpx regulon in the event of aberrant β‐barrel outer membrane protein assembly

The Cpx and σ^E regulons help maintain outer membrane integrity; the Cpx pathway monitors the biogenesis of cell surface structures, such as pili, while the σ^E pathway monitors the biogenesis of β‐barrel outer membrane proteins (OMPs). In this study we revealed the importance of the Cpx regulon in the event of β‐barrel OMP mis‐assembly, by utilizing mutants expressing either a defective β‐barrel OMP assembly machinery (Bam) or assembly defective β‐barrel OMPs. Analysis of specific mRNAs showed that ΔcpxR bam double mutants failed to induce degP expression beyond the wild type level, despite activation of the σ^E pathway. The synthetic conditional lethal phenotype of ΔcpxR in mutant Bam or β‐barrel OMP backgrounds was reversed by wild type DegP expressed from a heterologous plasmid promoter. Consistent with the involvement of the Cpx regulon in the event of aberrant β‐barrel OMP assembly, the expression of cpxP, the archetypal member of the cpx regulon, was upregulated in defective Bam backgrounds or in cells expressing a single assembly‐defective β‐barrel OMP species. Together, these results showed that both the Cpx and σ^E regulons are required to reduce envelope stress caused by aberrant β‐barrel OMP assembly, with the Cpx regulon principally contributing by controlling degP expression.     

Pore formation by Vibrio cholerae cytolysin requires cholesterol in both monolayers of the target membrane

Vibrio cholerae cytolysin (VCC) forms oligomeric transmembrane pores in cholesterol-rich membranes. To better understand this process, we used planar bilayer membranes. In symmetric membranes, the rate of the channel formation by VCC has a superlinear dependency on the cholesterol membrane fraction. Thus, more than one cholesterol molecule can facilitate VCC-pore formation. In asymmetric membranes, the rate of pore formation is limited by the leaflet with the lower cholesterol content. Methyl-beta-cyclodextrin, which removes cholesterol from membranes, rapidly inhibits VCC pore formation, even when it is added to the side opposite that of VCC addition. The results suggest that cholesterol in both membrane leaflets aid VCC-pore formation and that either leaflet can function as a kinetic bottleneck with respect to the rate of pore-formation.     

Osmoprotective polymer additives attenuate the membrane pore‐forming activity of antimicrobial peptoids

Antimicrobial peptides (AMPs) are critical components of the innate immune system and exhibit bactericidal activity against a broad spectrum of bacteria. We investigated the use of N‐substituted glycine peptoid oligomers as AMP mimics with potent antimicrobial activity. The antimicrobial mechanism of action varies among different AMPs, but many of these peptides can penetrate bacterial cell membranes, causing cell lysis. We previously hypothesized that amphiphilic cyclic peptoids may act through a similar pore formation mechanism against methicillin‐resistant Staphylococcus aureus (MRSA). Peptoid‐induced membrane disruption is observed by scanning electron microscopy and results in a loss of membrane integrity. We demonstrate that the antimicrobial activity of the peptoids is attenuated with the addition of polyethylene glycol osmoprotectants, signifying protection from a loss of osmotic balance. This decrease in antimicrobial activity is more significant with larger osmoprotectants, indicating that peptoids form pores with initial diameters of ～2.0–3.8 nm. The initial membrane pores formed by cyclic peptoid hexamers are comparable in diameter to those formed by larger and structurally distinct AMPs. After 24 h, the membrane pores expand to >200 nm in diameter. Together, these results indicate that cyclic peptoids exhibit a mechanism of action that includes effects manifested at the cell membrane of MRSA. © 2014 Wiley Periodicals, Inc. Biopolymers 103: 227–236, 2015.     

Inflammasome activation by Pseudomonas aeruginosa's ExlA pore‐forming toxin is detrimental for the host

During acute Pseudomonas aeruginosa infection, the inflammatory response is essential for bacterial clearance. Neutrophil recruitment can be initiated following the assembly of an inflammasome within sentinel macrophages, leading to activation of caspase‐1, which in turn triggers macrophage pyroptosis and IL‐1β/IL‐18 maturation. Inflammasome formation can be induced by a number of bacterial determinants, including Type III secretion systems (T3SSs) or pore‐forming toxins, or, alternatively, by lipopolysaccharide (LPS) via caspase‐11 activation. Surprisingly, previous studies indicated that a T3SS‐induced inflammasome increased pathogenicity in mouse models of P. aeruginosa infection. Here, we investigated the immune reaction of mice infected with a T3SS‐negative P. aeruginosa strain (IHMA879472). Virulence of this strain relies on ExlA, a secreted pore‐forming toxin. IHMA879472 promoted massive neutrophil infiltration in infected lungs, owing to efficient priming of toll‐like receptors, and thus enhanced the expression of inflammatory proteins including pro‐IL‐1β and TNF‐α. However, mature‐IL‐1β and IL‐18 were undetectable in wild‐type mice, suggesting that ExlA failed to effectively activate caspase‐1. Nevertheless, caspase‐1/11 deficiency improved survival following infection with IHMA879472, as previously described for T3SS+ bacteria. We conclude that the detrimental effect associated with the ExlA‐induced inflammasome is probably not due to hyperinflammation, rather it stems from another inflammasome‐dependent process.     

Lithium induces follicular atresia in rat ovary through a GSK‐3β/β‐catenin dependent mechanism

Lithium chloride (LiCl) is a drug used to treat bipolar disorder, but has side effects in the female reproductive system. Although lithium is known to decrease folliculogenesis and induce follicular atresia in rodent ovaries, its cellular and molecular effects in the ovary have not yet been addressed. To investigate these effects, 23‐day‐old immature female rats were injected with 10 IU pregnant mare serum gonadotropin (PMSG), followed by injections of 250 mg/kg LiCl every 12 hr for four doses. Ovaries were removed 40 and 48 hr after PMSG administration and prepared for histology, immunohistochemistry, Western blotting, and DNA laddering analysis. Our results showed that in the ovaries of LiCl‐treated rats, few antral but more atretic follicles were present compared to those of the control rats. The induction of atresia by LiCl was further confirmed by the presence of DNA fragmentation, accompanied by a reduced level of 17β‐estradiol in the serum. At the cellular level, lithium significantly decreased the number of proliferating cell nuclear antigen (PCNA)‐positive cells and conversely increased the number of TUNEL‐positive cells in the granulosa layer of the antral follicles. At the molecular level, lithium increased the level of phosphorylated glycogen synthase kinase‐3β, and unexpectedly decreased the expression of active (stabilized) β‐catenin. Altogether, our results indicate that lithium disrupts the balance between proliferation and apoptosis in granulosa cells, leading to follicular atresia possibly through the reduction in both the stabilized β‐catenin and 17β‐estradiol synthesis. Mol. Reprod. Dev. 80: 286–296, 2013. © 2013 Wiley Periodicals, Inc.     

Dissection of β‐barrel outer membrane protein assembly pathways through characterizing BamA POTRA 1 mutants of Escherichia coli

BamA of Escherichia coli is an essential component of the hetero‐oligomeric machinery that mediates β‐barrel outer membrane protein (OMP) assembly. The C‐ and N‐termini of BamA fold into trans‐membrane β‐barrel and five soluble POTRA domains respectively. Detailed characterization of BamA POTRA 1 missense and deletion mutants revealed two competing OMP assembly pathways, one of which is followed by the archetypal trimeric β‐barrel OMPs, OmpF and LamB, and is dependent on POTRA 1. Interestingly, our data suggest that BamA also requires its POTRA 1 domain for proper assembly. The second pathway is independent of POTRA 1 and is exemplified by TolC. Site‐specific cross‐linking analysis revealed that the POTRA 1 domain of BamA interacts with SurA, a periplasmic chaperone required for the assembly of OmpF and LamB, but not that of TolC and BamA. The data suggest that SurA and BamA POTRA 1 domain function in concert to assist folding and assembly of most β‐barrel OMPs except for TolC, which folds into a unique soluble α‐helical barrel and an OM‐anchored β‐barrel. The two assembly pathways finally merge at some step beyond POTRA 1 but presumably before membrane insertion, which is thought to be catalysed by the trans‐membrane β‐barrel domain of BamA.     

The TPR domain of BepA is required for productive interaction with substrate proteins and the β‐barrel assembly machinery complex

BepA (formerly YfgC) is an Escherichia coli periplasmic protein consisting of an N‐terminal protease domain and a C‐terminal tetratricopeptide repeat (TPR) domain. We have previously shown that BepA is a dual functional protein with chaperone‐like and proteolytic activities involved in membrane assembly and proteolytic quality control of LptD, a major component of the outer membrane lipopolysaccharide translocon. Intriguingly, BepA can associate with the BAM complex: the β‐barrel assembly machinery (BAM) driving integration of β‐barrel proteins into the outer membrane. However, the molecular mechanism of BepA function and its association with the BAM complex remains unclear. Here, we determined the crystal structure of the BepA TPR domain, which revealed the presence of two subdomains formed by four TPR motifs. Systematic site‐directed in vivo photo‐cross‐linking was used to map the protein–protein interactions mediated by the BepA TPR domain, showing that this domain interacts both with a substrate and with the BAM complex. Mutational analysis indicated that these interactions are important for the BepA functions. These results suggest that the TPR domain plays critical roles in BepA functions through interactions both with substrates and with the BAM complex. Our findings provide insights into the mechanism of biogenesis and quality control of the outer membrane.     

The modular nature of the β‐barrel assembly machinery,illustrated in Borrelia burgdorferi

Diderm bacteria have an outer membrane that provides defense against environmental factors including antibiotics. Understanding the process of outer membrane biogenesis is, therefore, of critical importance in order to envisage new treatments of these bacterial pathogens. Borrelia burgdorferi is the pathogen responsible for Lyme disease. Its outer membrane contains integral, β‐barrel proteins as well as swathes of externally exposed lipoproteins. Previous work has demonstrated that the β‐barrel assembly machine (BAM complex) in B. burgdorferi and other Spirochetes shares several similarities with the BAM complex in other bacterial lineages, such as the Proteobacteria that includes Escherichia coli. However, Iqbal et al. ( 2016 ) have identified the inner membrane protein TamB as a subunit of the BAM complex in Spirochetes. This latest study highlights the modular nature of the BAM complex, and suggests that in some bacterial lineages the BAM complex and translocation and assembly module (the TAM) function as a single unit.     

Syntheses and anti‐tubercular activity of β‐substituted and α,β‐disubstituted peptidyl β‐aminoboronates and boronic acids

Tuberculosis is still affecting millions of people worldwide, and new resistant strains of Mycobacterium tuberculosis are being found. It is therefore necessary to find new compounds for treatment. In this paper, we report the synthesis and in vitro testing of peptidyl β‐aminoboronic acids and β‐aminoboronates with anti‐tubercular activity. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

The effect of β‐methylation on the conformation of α, β‐dehydrophenylalanine: a DFT study

Dehydroamino acids are non‐coded amino acids that offer unique conformational properties. Dehydrophenylalanine (ΔPhe) is most commonly used to modify bioactive peptides to constrain the topography of the phenyl ring in the side chain, which commonly serves as a pharmacophore. The Ramachandran maps (in the gas phase and in CHCl₃ mimicking environments) of ΔPhe analogues with methyl groups at the β position of the side chain as well as at the C‐terminal amide were calculated using the B3LYP/6‐31 + G** method. Unexpectedly, β‐methylation alone results in an increase of conformational freedom of the affected ΔPhe residue. However, further modification by introducing an additional methyl group at C‐terminal methyl amide results in a steric crowding that fixes the torsion angle ψ of all conformers to the value 123°, regardless of the Z or E position of the phenyl ring. The number of conformers is reduced and the accessible conformational space of the residues is very limited. In particular, (Z)‐Δ(βMe)Phe with the tertiary C‐terminal amide can be classified as the amino acid derivative that has a single conformational state as it seems to adopt only the β conformation. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.