Evaluating the amino acid CF3-bicyclopentylglycine as a new label for solid-state 19 F-NMR structure analysis of membrane-bound peptides.

The conformation, alignment and dynamic behavior of membrane-bound peptides is readily accessible by solid-state (19)F-NMR spectroscopy, but it has been difficult to incorporate suitable (19)F-labelled amino acids into synthetic peptides. To avoid the drawbacks of previously used labels, we have rationally designed and synthesized a novel amino acid that suits all theoretical and practical requirements for peptide synthesis and subsequent (19)F-NMR structure analysis [Mikhailiuk et. al, Angew. Chem. 2006, 118, 5787-5789]. The enantiomerically pure L-form of 3-(trifluoromethyl)bicyclopent-[1.1.1]-1-ylglycine (CF(3)-Bpg) carries a CF(3) group that is rigidly attached to the peptide backbone and does not racemize during peptide synthesis. It could be demonstrated for several different peptides that their biological activity is usually not affected by a single label, nor the conformation, as monitored by circular dichroism. Here, we carry out a more detailed structure analysis to evaluate the potential and reliability of CF(3)-Bpg for solid-state NMR, using the well-known alpha-helical antimicrobial peptide PGLa as a test case. We have collected several orientational constraints from the anisotropic (19)F--(19)F dipolar couplings of CF(3)-Bpg in various positions of PGLa embedded in lipid bilayers. These resulting structural parameters are then compared with those previously determined from 4-CF(3)-phenylglycine and 3,3,3-d(3)-alanine labels on the same peptide. The analysis confirms that CF(3)-Bpg does not perturb the alpha-helical conformation of PGLa. Likewise, the helix alignment is shown to follow the established concentration-dependent pattern in realigning from a surface-bound S-state to an obliquely tilted T-state. Hence, the advantages of CF(3)-Bpg over all previously used (19)F-labeled side chains are evident, as they combine ease of chemical incorporation and peptide purification with high NMR sensitivity and absent background signals, allowing a straightforward analysis of the dipolar splittings with no need for chemical shift referencing without any ambiguity in the sign of the couplings.     

Probing aromatic, hydrophobic, and steric effects on the self-assembly of an amyloid-β fragment peptide

Aromatic amino acids have been shown to promote self-assembly of amyloid peptides, although the basis for this amyloid-inducing behavior is not understood. We adopted the amyloid-β 16-22 peptide (Aβ(16-22), Ac-KLVFFAE-NH(2)) as a model to study the role of aromatic amino acids in peptide self-assembly. Aβ(16-22) contains two consecutive Phe residues (19 and 20) in which Phe 19 side chains form interstrand contacts in fibrils while Phe 20 side chains interact with the side chain of Va l18. The kinetic and thermodynamic effect of varying the hydrophobicity and aromaticity at positions 19 and 20 by mutation with Ala, Tyr, cyclohexylalanine (Cha), and pentafluorophenylalanine (F(5)-Phe) (order of hydrophobicity is Ala < Tyr < Phe < F(5)-Phe < Cha) was characterized. Ala and Tyr position 19 variants failed to undergo fibril formation at the peptide concentrations studied, but Cha and F(5)-Phe variants self-assembled at dramatically enhanced rates relative to wild-type. Cha mutation was thermodynamically stabilizing at position 20 (ΔΔG = -0.2 kcal mol(-1) relative to wild-type) and destabilizing at position 19 (ΔΔG = +0.2 kcal mol(-1)). Conversely, F(5)-Phe mutations were strongly stabilizing at both positions (ΔΔG = -1.3 kcal mol(-1) at 19, ΔΔG = -0.9 kcal mol(-1) at 20). The double Cha and F(5)-Phe mutants showed that the thermodynamic effects were additive (ΔΔG = 0 kcal mol(-1) for Cha 19,20 and -2.1 kcal mol(-1) for F(5)-Phe 19,20). These results indicate that sequence hydrophobicity alone does not dictate amyloid potential, but that aromatic, hydrophobic, and steric considerations collectively influence fibril formation.     

Solution Synthesis,Conformational Analysis,and Antimicrobial Activity of Three Alamethicin F50/5 Analogs Bearing a Trifluoroacetyl Label

We prepared, by solution‐phase methods, and fully characterized three analogs of the membrane‐active peptaibiotic alamethicin F50/5, bearing a single trifluoroacetyl (Tfa) label at the N‐terminus, at position 9 (central region) or at position 19 (C‐terminus), and with the three Gln at positions 7, 18, and 19 replaced by Glu(OMe) residues. To add the Tfa label at position 9 or 19, a γ‐trifluoroacetylated α,γ‐diaminobutyric acid (Dab) residue was incorporated as a replacement for the original Val⁹ or Glu(OMe)¹⁹ amino acid. We performed a detailed conformational analysis of the three analogs (using FT‐IR absorption, CD, 2D‐NMR, and X‐ray diffraction), which clearly showed that Tfa labeling does not introduce any dramatic backbone modification in the predominantly α‐helical structure of the parent peptaibiotic. The results of an initial solid‐state ¹⁹F‐NMR study on one of the analogs favor the conclusion that the Tfa group is a very promising reporter for the analysis of peptaibiotic? membrane interactions. Finally, we found that the antimicrobial activities of the three newly synthesized analogs depend on the position of the Tfa label in the peptide sequence.     

Sunflower Seed Aminopeptidase-Catalyzed Hydrolysis of Phe-Xaa Dipeptides — Exploring the S1′ Specificity of the Enzyme

The S₁′ substrate specificity of the sunflower seed major aminopeptidase was studied with a series of dipeptide substrates with phenylalanine at P₁ and a hydrophobic amino acid at P₁′ position. The kinetic parameters of hydrolysis are significantly affected by the structure, side chain hydrophobicity and configuration of the P₁′ moiety. Its binding during enzyme-substrate complex formation takes place at a hydrophobic site of limited size following an extraction mechanism as seen from the applied structure-activity correlation. Attempts to establish such dependencies for the catalytic step of the reaction reveal the presence of additional S₁′-P₁′ enzyme-substrate interactions of greater complexity.     

Determination of the absolute configuration of the glucosinolate methyl sulfoxide group reveals a stereospecific biosynthesis of the side chain

Glucosinolates are plant metabolites containing an anionic nitrogeneous thioglucosidic core structure and a structurally diverse amino acid-derived side chain, which after hydrolysis by thioglucohydrolases (myrosinases) afford biological active degradation products such as nitriles and isothiocyanates. Structural diversity in glucosinolates is partially due to enzymatic modifications occurring on the preformed core structure, like the recently described oxidation of sulfides to sulfoxides catalyzed by a flavin monooxygenase identified in Arabidopsis thaliana. The enzyme product, 4-methylsulfinylbutylglucosinolate, bears a chiral sulfoxide group in its side chain. We have analyzed the epimeric purity of 4-methylsulfinylbutylglucosinolate by NMR methods using a chiral lanthanide shift reagent. The absolute configuration of the sulfoxide group has been established by comparing the ¹H NMR spectra of the two sulfoximine diastereomers of natural 4-methylsulfinylbutylglucosinolate. According to our data, 4-methylsulfinylbutylglucosinolate isolated from broccoli and A. thaliana is a pure epimer and its sulfoxide group has the R_S configuration. The product of the A. thaliana flavin monooxygenase has these same properties demonstrating that the enzyme is stereospecific and supporting its involvement in glucosinolate side chain formation.     

Carbon-13 NMR chemical shifts of the common amino acid residues measured in aqueous solutions of the linear tetrapeptides H-Gly-Gly- X-L- Ala-OH

The ¹³C nmr chemical shifts of the common amino acid residues were measured in D₂O solutions of the linear tetrapeptides H-Gly-Gly-X-L -Ala-OH. For Asp, Glu, Lys, Tyr and His, the titration shifts arising from the ionization of te amino acid side chains were also obtained. These data are compared with the corresponding ¹³C chemical shifts in the protected tetrapeptides CF₃CO-Gly-Gly-X-L -Ala-OCH₃, the linear pentapeptides H-Gly-Gly-X-Gly-Gly-OH, and the free amino acids. On this basism the selection of suitable “random coil” ¹³C chemical shifts for conformational studies of polypeptides chain is discussed.     

A new amphipathy scale: I. Determination of the scale from molecular dynamics data

Two new amphipathy scales elaborated from molecular dynamics data are presented. Their applications contribute for the identification of the hydrophobic or hydrophilic regions in proteins solely from the primary structure. The new amphipathy coefficients (AC) reflect the side chain/solvent molecules configurational energies. A polar (water) and an apolar solvent, CCl₄, were used resulting in the two AC_water and AC_CCl4 scales. These solvents were chosen to simulate the aqueous phases and the transmembrane ambients of cellular membranes where the membrane proteins act. The new amphipathy scales were compared with some previous scales determined by different methods, which were also compared between them, indicating more than 90% of the correlation coefficients are less than 0.9: the scales are strictly dependent on the methodologies used in their determination. The AC_CCl4 scale is related with the size of side chain amino acids while AC_water is related with the hydrophobicity of side chain amino acids. The quality of the scales was confirmed by an example of application where AC_water was able to identify correctly the transmembrane, hydrophobic regions of a membrane protein. These results also indicate that water is an important factor responsible for the tertiary structure of membrane proteins.     

Labeling of subunit b of the ATP synthase from Escherichia coli with a photoreactive phospholipid analogue

Purified ATP synthase (F1F0) from Escherichia coli K12 was labeled with the hydrophobic photoreactive label 1-palmitoyl 2-(2-azido-4-nitro)benzoyl sn-glycero-3-[3H]phosphocholine in reconstituted proteoliposomes. The F0-subunit b was predominantly labeled. A very low amount of label was detected on the other F0-subunits a and c. The label in subunit b could be traced back by proteolytic digestion to the NH2-terminal fragment 1 to 53 which contains the stretch of hydrophobic amino acid residues 1 to 32. By sequencing the intact protein, the distribution of label among the amino acids in this segment was determined. Cysteine 21 was predominantly labeled. Other labeled amino acids occurred at the NH2-terminal (Asn-2) and at position 26 (tryptophan). Due to the restricted mobility of the label in the lipid bilayer, these residues are suggested to be located in or close to the polar head of the lipid bilayer. These results will be compared with predictions for the arrangement of the polypeptide b derived from the hydrophobicity profile.     

Development of short and highly potent self‐assembling elastin‐derived pentapeptide repeats containing aromatic amino acid residues

Tropoelastin is the primary component of elastin, which forms the elastic fibers that make up connective tissues. The hydrophobic domains of tropoelastin are thought to mediate the self‐assembly of elastin into fibers, and the temperature‐mediated self‐assembly (coacervation) of one such repetitive peptide sequence (VPGVG) has been utilized in various bio‐applications. To elucidate a mechanism for coacervation activity enhancement and to develop more potent coacervatable elastin‐derived peptides, we synthesized two series of peptide analogs containing an aromatic amino acid, Trp or Tyr, in addition to Phe‐containing analogs and tested their functional characteristics. Thus, position 1 of the hydrophobic pentapeptide repeat of elastin (X¹P²G³V⁴G⁵) was substituted by Trp or Tyr. Eventually, we acquired a novel, short Trp‐containing elastin‐derived peptide analog (WPGVG)₃ with potent coacervation ability. From the results obtained during this process, we determined the importance of aromaticity and hydrophobicity for the coacervation potency of elastin‐derived peptide analogs. Generally, however, the production of long‐chain synthetic polypeptides in quantities sufficient for commercial use remain cost‐prohibitive. Therefore, the identification of (WPGVG)₃, which is a 15‐mer short peptide consisting simply of five natural amino acids and shows temperature‐dependent self‐assembly activity, might serve as a foundation for the development of various kinds of biomaterials. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

The effect of the cosolvent trifluoroethanol on a tryptophan side chain orientation in the hydrophobic core of troponin C

The unique biophysical properties of tryptophan residues have been exploited for decades to monitor protein structure and dynamics using a variety of spectroscopic techniques, such as fluorescence and nuclear magnetic resonance (NMR). We recently designed a tryptophan mutant in the regulatory N‐domain of cardiac troponin C (F77W‐cNTnC) to study the domain orientation of troponin C in muscle fibers using solid‐state NMR. In our previous study, we determined the NMR structure of calcium‐saturated mutant F77W‐V82A‐cNTnC in the presence of 19% 2,2,2‐trifluoroethanol (TFE). TFE is a widely used cosolvent in the biophysical characterization of the solution structures of peptides and proteins. It is generally assumed that the structures are unchanged in the presence of cosolvents at relatively low concentrations, and this has been verified for TFE at the level of the overall secondary and tertiary structure for several calcium regulatory proteins. Here, we present the NMR solution structure of the calcium saturated F77W‐cNTnC in presence of its biological binding partner troponin I peptide (cTnI_144–163) and in the absence of TFE. We have also characterized a panel of six F77W‐cNTnC structures in the presence and absence TFE, cTnI_144–163, and the extra mutation V82A, and used ¹⁹F NMR to characterize the effect of TFE on the F77(5fW) analog. Our results show that although TFE did not perturb the overall protein structure, TFE did induce a change in the orientation of the indole ring of the buried tryptophan side chain from the anticipated position based upon homology with other proteins, highlighting the potential dangers of the use of cosolvents.     

Sulfate metabolism of rat P0 glycoprotein: some observations

It has been known for some time that P₀, the major intrinsic protein in PNS myelin, contains sulfate. The position of sulfate has been described for beef PNS myelin, but rat PNS myelin differs somewhat from that of the beef, therefore an investigation of the location of sulfate in rat P₀ was undertaken. Weanling rat nerves were incubated with [³H] amino acid mixture and [³⁵S]O₄, and purified myelin was prepared, and the proteins separated on polyacrylamide gels. The bulk of the [³⁵S]O₄ was incorporated into P₀, but smaller peaks of sulfate label were found in the higher molecular weight proteins. With tunicamycin in the incubation mixture, sulfate incorporation was inhibited. Incubation of the labeled myelin mixture with endo F or glycanase resulted in total loss of sulfate label on P₀, therefore all of the [³⁵S]O₄ was incorporated into the oligosaccharide chain, with none on the polypeptide. Castanospermine and deoxymannojirimycin inhibited [³⁵S]O₄ incorporation into P₀, but no inhibition was exerted by swainsonine. These results indicate that sulfate resides in the core of the oligosaccharide chain, with none in the terminal region. Such a structure would correlate with the lack of an HNK-1 epitope, absent in the rat, but found in P₀ of many species.Abbreviations Used Endo H endoglycosidase H - Endo F endoglycosidase F - GalNAc N-acetyl galactosamine - GlcNAc N-acetyl glucosamine - MAG myelin-associated glycoprotein - Man mannose Special issue dedicated to Dr. Marjorie B. Lees.     

Probing the interactions of an acyl carrier protein domain from the 6-deoxyerythronolide B synthase

The assembly‐line architecture of polyketide synthases (PKSs) provides an opportunity to rationally reprogram polyketide biosynthetic pathways to produce novel antibiotics. A fundamental challenge toward this goal is to identify the factors that control the unidirectional channeling of reactive biosynthetic intermediates through these enzymatic assembly lines. Within the catalytic cycle of every PKS module, the acyl carrier protein (ACP) first collaborates with the ketosynthase (KS) domain of the paired subunit in its own homodimeric module so as to elongate the growing polyketide chain and then with the KS domain of the next module to translocate the newly elongated polyketide chain. Using NMR spectroscopy, we investigated the features of a structurally characterized ACP domain of the 6‐deoxyerythronolide B synthase that contribute to its association with its KS translocation partner. Not only were we able to visualize selective protein–protein interactions between the two partners, but also we detected a significant influence of the acyl chain substrate on this interaction. A novel reagent, CF₃‐S‐ACP, was developed as a ¹⁹F NMR spectroscopic probe of protein–protein interactions. The implications of our findings for understanding intermodular chain translocation are discussed.     

Simultaneous NMR assignment of backbone and side chain amides in large proteins with IS-TROSY

A new strategy for the simultaneous NMR assignment of both backbone and side chain amides in large proteins with isotopomer-selective transverse-relaxation-optimized spectroscopy (IS-TROSY) is reported. The method considers aspects of both the NMR sample preparation and the experimental design. First, the protein is dissolved in a buffer with 50%H₂O/50%D₂O in order to promote the population of semideuterated NHD isotopomers in side chain amides of Asn/Gln residues. Second, a ¹³C′-coupled 2D ¹⁵N–¹H IS-TROSY spectrum provides a stereospecific distinction between the geminal protons in the E and Z configurations of the carboxyamide group. Third, a suite of IS-TROSY-based triple-resonance NMR experiments, e.g. 3D IS-TROSY-HNCA and 3D IS-TROSY-HNCACB, are designed to correlate aliphatic carbon atoms with backbone amides and, for Asn/Gln residues, at the same time with side chain amides. The NMR assignment procedure is similar to that for small proteins using conventional 3D HNCA/3D HNCACB spectra, in which, however, signals from NH₂ groups are often very weak or even missing due to the use of broad-band proton decoupling schemes and NOE data have to be used as a remedy. For large proteins, the use of conventional TROSY experiments makes resonances of side chain amides not observable at all. The application of IS-TROSY experiments to the 35-kDa yeast cytosine deaminase has established a complete resonance assignment for the backbone and stereospecific assignment for side chain amides, which otherwise could not be achieved with existing NMR experiments. Thus, the development of IS-TROSY-based method provides new opportunities for the NMR study of important structural and biological roles of carboxyamides and side chain moieties of arginine and lysine residues in large proteins as well as amino moieties in nucleic acids.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Synthesis and application of N‐[1‐(4‐(4‐fluorophenyl)‐2,6‐dioxocyclohexylidene)ethyl] (Fde)‐protected amino acids for optimization of solid‐phase peptide synthesis using gel‐phase 19F NMR spectroscopy

N‐[1‐(4‐(4‐fluorophenyl)‐2,6‐dioxocyclohexylidene)ethyl] (Fde) protected amino acids have been prepared and applied in solid‐phase peptide synthesis monitored by gel‐phase ¹⁹F NMR spectroscopy. The Fde protective group could be cleaved with 2% hydrazine or 5% hydroxylamine solution in DMF as determined with gel‐phase ¹⁹F NMR spectroscopy. The dipeptide Ac‐L ‐Val‐L ‐Val‐NH₂ 12 was constructed using Fde‐L ‐Val‐OH and no noticeable racemization took place during the amino acid coupling with N,N′‐diisopropylcarbodiimide and 1‐hydroxy‐7‐azabenzotriazole or Fde deblocking. To extend the scope of Fde protection, the hydrophobic nonapeptide LLLLTVLTV from the signal sequence of mucin MUC1 was successfully prepared using Fde‐L ‐Leu‐OH at diagnostic positions. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Site-specific ¹⁹F NMR chemical shift and side chain relaxation analysis of a membrane protein labeled with an unnatural amino acid

Site‐specific ¹⁹F chemical shift and side chain relaxation analysis can be applied on large size proteins. Here, one‐dimensional ¹⁹F spectra and T₁, T₂ relaxation data were acquired on a SH3 domain in aqueous buffer containing 60% glycerol, and a nine‐transmembrane helices membrane protein diacyl‐glycerol kinase (DAGK) in dodecyl phosphochoine (DPC) micelles. The high quality of the data indicates that this method can be applied to site‐specifically analyze side chain internal mobility of membrane proteins or large size proteins.     

N‐terminal β‐strand swapping in a consensus‐derived alternative scaffold driven by stabilizing hydrophobic interactions

The crystal structure of an N‐terminal β‐strand‐swapped consensus‐derived tenascin FN3 alternative scaffold has been determined. A comparison with the unswapped structure reveals that the side chain of residue F88 orients differently and packs more tightly with the hydrophobic core of the domain. Dimer formation also results in the burial of a hydrophobic patch on the surface of the domain. Thus, it appears that tighter packing of F88 in the hydrophobic core and burial of surface hydrophobicity provide the driving forces for the N‐terminal β‐strand swapping, leading to the formation of a stable compact dimer. Proteins 2014; 82:1527–1533. © 2014 Wiley Periodicals, Inc.     

Molecular Analysis of the Putative Inactivation Particle in the Inactivation Gate of Brain Type IIA Na+ Channels

Fast Na⁺ channel inactivation is thought to involve binding of phenylalanine 1489 in the hydrophobic cluster IFM in L_III-IV of the rat brain type IIA Na⁺ channel. We have analyzed macroscopic and single channel currents from Na⁺ channels with mutations within and adjacent to hydrophobic clusters in L_III-IV. Substitution of F1489 by a series of amino acids disrupted inactivation to different extents. The degree of disruption was closely correlated with the hydrophilicity of the amino acid at position 1489. These mutations dramatically destabilized the inactivated state and also significantly slowed the entry into the inactivated state, consistent with the idea that F1489 forms a hydrophobic interaction with a putative receptor during the fast inactivation process. Substitution of a phe residue at position 1488 or 1490 in mutants lacking F1489 did not restore normal inactivation, indicating that precise location of F1489 is critical for its function. Mutations of T1491 disrupted inactivation substantially, with large effects on the stability of the inactivated state and smaller effects on the rate of entry into the inactivated state. Mutations of several other hydrophobic residues did not destabilize the inactivated state at depolarized potentials, indicating that the effects of mutations at F1489 and T1491 are specific. The double mutant YY1497/8QQ slowed macroscopic inactivation at all potentials and accelerated recovery from inactivation at negative membrane potentials. Some of these mutations in L_III-IV also affected the latency to first opening, indicating coupling between L_III-IV and channel activation. Our results show that the amino acid residues of the IFM hydrophobic cluster and the adjacent T1491 are unique in contributing to the stability of the inactivated state, consistent with the designation of these residues as components of the inactivation particle responsible for fast inactivation of Na⁺ channels.     

Novel interleukin-5 inhibitors based on hydroxyethylaminomethyl-4H-chromen-4-one scaffold

Hydroxyethylaminomethyl-4H-chromenones were previously discovered as fairly strong IL-5 inhibitor. For determination of detail structure activity relationship, N-substituted hydroxyethylaminomethylchromenones 4a–n were prepared and evaluated for their IL-5 inhibitory activity. Shifting the hydrophobic group to nitrogen from 1-position of hydroxyethylamino moiety of hydroxyethylaminomethyl-4H-chromenones enhances the activity. The increment in bulkiness or hydrophobicity of alkyl side chain at amino group increases the activity. The same level of activity of 5-(cyclohexylmethoxy)-3-(N-benzyl-2-hydroxyethylaminomethyl)-4H-chromenone analogs regardless of hydrophobic or hydrophilic substituents at 4th position of phenyl ring might infer the existence of tunnel structure in the putative receptor for accepting these side chains.     

A Residue-specific Shift in Stability and Amyloidogenicity of Antibody Variable Domains

Variable (V) domains of antibodies are essential for antigen recognition by our adaptive immune system. However, some variants of the light chain V domains (V_L) form pathogenic amyloid fibrils in patients. It is so far unclear which residues play a key role in governing these processes. Here, we show that the conserved residue 2 of V_L domains is crucial for controlling its thermodynamic stability and fibril formation. Hydrophobic side chains at position 2 stabilize the domain, whereas charged residues destabilize and lead to amyloid fibril formation. NMR experiments identified several segments within the core of the V_L domain to be affected by changes in residue 2. Furthermore, molecular dynamic simulations showed that hydrophobic side chains at position 2 remain buried in a hydrophobic pocket, and charged side chains show a high flexibility. This results in a predicted difference in the dissociation free energy of ∼10 kJ mol⁻¹, which is in excellent agreement with our experimental values. Interestingly, this switch point is found only in V_L domains of the κ family and not in V_Lλ or in V_H domains, despite a highly similar domain architecture. Our results reveal novel insight into the architecture of variable domains and the prerequisites for formation of amyloid fibrils. This might also contribute to the rational design of stable variable antibody domains.     

Callyaerins A–F and H,new cytotoxic cyclic peptides from the Indonesian marine sponge Callyspongia aerizusa

Bioassay guided fractionation of the EtOAc fraction of the sponge Callyspongia aerizusa yielded seven new cytotoxic cyclic peptides callyaerins A–F (1–6) and H (8). Their structures were determined using extensive 1D (¹H, ¹³C and DEPT) and 2D (COSY, HMQC, HMBC, TOCSY, and ROESY) NMR and mass spectral (ESI and HRESI-TOF) data. All compounds were cyclic peptides containing ring systems of 5–9 amino acids and side chains of 2–5 amino acids in length. An unusual (Z)-2,3-diaminoacrylic acid unit provided the template for ring closure and afforded the linkage to the peptidic side chain which was always initiated with a proline moiety. All peptides contained three or more proline residues and the remaining residues were predominantly hydrophobic residues with all amino acids present in the l form. Callyaerins A–F (1–6) and H (8) showed biological activity in antibacterial assays and in various cytotoxicity assays employing different tumour cell-lines (L5178Y, HeLa, and PC12). Callyaerins E (5) and H (8) exhibited strong activity against the L5178Y cell line with ED₅₀ values of 0.39 and 0.48 μM, respectively. On the other hand, callyaerin A (1) showed strong inhibitory properties towards C. albicans.