Semisynthesis of carboxy-terminal fragments of thermolysin

Enzyme-catalyzed synthesis of two polypeptide fragments, one of which is obtained by chemical synthesis, in the presence of proteolytic enzymes and in aqueous organic solvents constitutes a convenient procedure for the synthesis of proteins and their analogs. This novel semisynthetic procedure was investigated for preparing COOH-terminal fragments of the metallo-protease thermolysin. Fragment 205–316, obtained by autolysis of the protein in the presence of EDTA, was first cleaved selectively with Staphylococcus aureus V8 protease at the level of the single Glu³⁰² residue into fragments 205–302 and 303–316. Upon incubation for 2–5 days of fragment 205–302 with a 5–fold excess of peptide 303–316, prepared by solid phase synthesis, with V8-protease in 0.1M ammonium acetate, pH6.0, containing 50% glycerol as organic cosolvent, enzyme-catalyzed reformation of the peptide bond was achieved in yields up to ñ90% (based on fragment 205–302). The same procedure was used to prepare also the thermolysin fragments 205–315 and 205–311 by enzymatic coupling of fragment 205–302 to peptide 303–315 or 303–311, these last prepared by proteolytic digestion of the synthetic peptide 303–316. This procedure of semisynthesis opens up an approach for the site-directed modification of the tetrahelical COOH-terminal fragment 205–316 of thermolysin at the level of its helical segment encompassing residues 301–312 in the native, intact protein. Such analogs will be useful for examining structure-folding-stability relationships in this folded fragment possessing domain-like characteristics.     

Domain characteristics of the cyanogen bromide fragment 121–316 of thermolysin

The molecule of thermolysin was shown by X-ray crystallography to be composed of two structural domains of equal size comprising residues 1–157 and 158–316. In order to explore the possibility that these domains correspond to globular fragments able to refold autonomously, we have investigated the conformational and stability properties of fragment 121–316, which was obtained by limited chemical cleavage of thermolysin with cyanogen bromide. As judged by far-ultraviolet circular dichroism measurements, in aqueous solution under neutral conditions the fragment maintains a relative amount of helical structure which is comparable to that exhibited by the corresponding region in native thermolysin. The secondary structure attained by the fragment appears remarkably stable to the denaturing action of heat. By measuring the temperature dependence of the dichroic signal at 220 nm a Tm near 74d? was obtained. Immunodiffusion analyses indicated that the fragment recognizes and precipitates antibodies raised in rabbits using native thermolysin as immunogen. The overall conformational and immunochemical data indicate that fragment 121–316 of thermolysin is able to refold into a stable structure of native-like characteristics independently of the rest of the molecule. The results of this study complement those previously reported for fragment 206–316 (Vita, C., Fontana, A., Seeman, J.R. & Chaiken, I.M. (1979) Biochemistry 18 , 3023–3031).     

Conformational and biological properties of partial sequences of salmon calcitonin

According to the Chou-Fasman rules for predicting the secondary structures of proteins, the 12–20 portion of salmon calcitonin should adopt an alpha helical conformation. These residues would form an amphipathic helix and contribute to the solubilization of certain phospholipids by the peptide. Circular dichroism was used to assess the extent that peptide segments of salmon calcitonin fold into structures of higher helical content in the presence of dimyristoylphospha-tidylglycerol, lysolecithin or sodium dodecyl sulfate. All of the segments studied are carboxyl terminal amides as is the native, intact, salmon calcitonin. Salmon calcitonin segments 11–23 or 12–23 form no more helical structure in the presence of lipids or detergents than does a segment comprising the hydro-philic carboxyl terminal residues 22–32 which is not predicted to adopt a helical conformation. Even a larger segment containing residues 12–32 does not exhibit a great increase in helical content in the presence of lipids or detergents, and it causes only a small broadening of the phase transition of dimyristoyl-phosphatidylglycerol. In contrast, a preparation with an equivalent molar ratio of dimyristoylphosphatidylglycerol to the salmon calcitonin segment 1–23 exhibits a very marked broadening of the phase transition, similar to what is found with the 32 amino acid native hormone. This amino terminal segment also adopts a conformation of higher helical content than even the intact hormone. This 1–23 segment is the only one studied that showed significant interaction with lipids, and it is also the only one which exhibited any hypocalcemic activity. The association constant for the binding of the 1–23 segment to dimyristoylphosphatidylglycerol is 2.4 × 10⁴ M^-1 compared with 1.0 × 10⁵ M^-1 for salmon calcitonin. The reduction in the affinity for lipid which occurs by removing residues 24–32 is much less than the reduction in biological activity. These and previous results indicate that the properties which give rise to lipid binding contribute to receptor affinity but are not the sole determining factor.     

Conformational analysis by NMR and distance geometry techniques of a peptide mimetic of the third helix of the Antennapedia homeodomain*

Abstract: The Antennapedia homeodomain structure consists of four helices. The helices II and III are connected by a tripeptide that forms a turn, and constitute the well‐known helix‐turn‐helix motif. The recognition helix penetrates the DNA major groove, gives specific protein–DNA contacts and forms direct, or water‐mediated, intermolecular hydrogen bonds. It was suggested that helix III (and perhaps also helix IV) might represent the recognition helix of Antennapedia homeodomain, which makes contact with the surface of the major groove of the DNA. In an attempt to clarify the helix III capabilities of assuming an helical conformation when separated from the rest of the protein, we carried out the structural determination of the recognition helix III in different solvent media. The conformational study of fragments 42–53, where residues W48 and F49, not involved in the protein–DNA interaction, were substituted by two alanines, was conducted in sodium dodecyl sulfate (SDS), trifluoroethanol (TFE) and TFE/water, using circular dichroism, nuclear magnetic resonance (NMR) and distance geometry (DG) techniques. The fragment assumes a well‐defined secondary structure in TFE and in TFE/water (90/10, v/v) with an α‐helix encompassing residues 4–9, while in TFE/water (70/30, v/v) a less regular structure was found. The DG results in the micellar system evidence the presence of a distorted α‐helical conformation involving residues 4–8. Our results reveal that the isolated Antennapedia recognition helix III tend to preserve in solution the α‐helical conformation even if separated from the rest of the molecule.     

Evidence for helicity in insect diuretic peptide hormones: computational analysis,spectroscopic studies,and biological assays

The conformation of four insect diuretic hormones has been analyzed computationally using secondary structure prediction routines and comparison with structures in the Brookhaven Protein Databank. Based on this analysis, a common seven-residue peptide fragment (DVLRQRL) had a high probability of forming an α-helix. Circular dichroism (CD) studies found that addition of trifluoroethanol (TFE) to an aqueous solution of the seven-residue fragment induces a change from random coil to helix. Subsequent NMR studies in water-TFE (1 : 1) produced nOe values and ³J_αNH coupling constants confirming a helical conformation: ³J_αNH coupling constants for the first five residues (D1 to Q5) were all ≤ 6.0 Hz and two medium-range nOe values (d_{αN (i,i+3)}) were observed between V2 and Q5, and R4 and L7. The longer fragments PLDVLRQRL in water-TFE and Lom-DH 1–26 in water alone, both containing the DVLRQRL sequence of the locust (Locusta migratoria) diuretic hormone, maintained the helicity as determined by CD analysis. However, the remaining 20 residues of the locust diuretic hormone did not maintain the same amount of helicity in water and all of the truncated fragments were not biologically active.     

Stereochemical analysis of the antigenic tip of the V3 loop peptide of HIV-1 gp120

A novel multiple turn conformation has been observed for a segment GPGRAFY in the crystal structure of a complex of HIV-1 gp120 V3 loop peptide with the Fab fragment of a neutralizing antibody [Ghiara et al. (1994) Science 264 , 82–85]. A structural motif has been defined for the peptide segment, employing idealized backbone conformations characterized by ranges of virtual C^α torsion angles and bond angles. A search of 122 high-resolution protein crystal structures has permitted identification of 24 examples of similar structural motifs. Two major conformational families have been identified, which differ primarily in the conformation at residue 3. The observed conformation at residue 3 in family 1 is left-handed helical (α_L) and that in family 2 is right-handed helical (α_R). Of the 10 examples in family 1, 9 examples have Gly residues at position 3. Of the 12 examples in family 2, 7 examples have Asn/Asp at position 3. Computer modeling of the V3 loop tip sequence using the two backbone conformational families as starting points leads to minimum-energy conformations in which antigenically important side-chains occupy similar spatial arrangements. This stereo-chemical analysis of the V3 loop tip sequence suggests a rational basis for the design of synthetic analog peptides for use as viral antagonists or synthetic antigens. © Munksgaard 1995.     

Fluoroalcohols as structure modifiers in peptides and proteins: hexafluoroacetone hydrate stabilizes a helical conformation of melittin at low pH

Abstract: The effect of hexafluoroacetone hydrate (HFA) on the structure of the honey bee venom peptide melittin has been investigated. In aqueous solution at low pH melittin is predominantly unstructured. Addition of HFA at pH ≈ 2.0 induces a structural transition from the unstructured state to a predominantly helical conformation as suggested by intense diagnostic far UV CD bands. The structural transition is highly cooperative and complete at 3.6 m (50% v/v) HFA. A similar structural transition is also observed in 2,2,2 trifluoroethanol which is complete only at a cosolvent concentration of ≈ 8 m . Temperature dependent CD experiments support a ‘cold denaturation’ of melittin at low concentrations of HFA, suggesting that selective solvation of peptide by HFA is mediated by hydrophobic interactions. NMR studies in 3.6 m HFA establish a well-defined helical structure of melittin at low pH, as suggested by the presence of strong NH_i/NH_i+1 NOEs throughout the sequence, along with many medium range helical NOEs. Structure calculations using NOE-driven distance constraints reveal a well-ordered helical fold with a relatively flexible segment around residues T10–G11–T12. The helical structure of melittin obtained at 3.6 m HFA at low pH is similar to those determined in methanolic solution and perdeuterated dodecylphosphocholine micelles. HFA as a cosolvent facilitates helix formation even in the highly charged C-terminal segment.     

Conformational studies of a potent Leu11, D-Trp12-containing lactam-bridged parathyroid hormone-related protein-derived antagonist

Human parathyroid hormone-related protein (PTHrP) is expressed in various tissues where it acts as an endocrine/paracrine factor involved in cellular growth, differentiation and development of fetal skeleton. As for parathyroid hormone (PTH), which is the hormone responsible for regulation of extracellular calcium homeostasis, the N-terminal 1-34 fragment can reproduce the full spectrum of calciotropic activities inherent in full-length PTH. Truncation of six amino acid residues from the N-terminus of both hormone sequences generates 7-34 fragments which act as weak antagonists. Although PTH(7-34) is a pure antagonist, PTHrP(7-34) acts as partial agonist against the receptor shared by both hormones, the PTH/PTHrP receptor. In the current study, we analyzed the conformation of [Leu¹¹,d -Trp¹²,Lys²⁶,Asp³⁰]PTHrP(7-34)NH₂ (hybrid-lactam) in a 1:1 mixture of H₂O/TFE-d₃ at pH 4 by circular dichroism, nuclear magnetic resonance and distance geometry calculations. This weak antagonist (K_b= 650 nm ) combines two modifications: Leu¹¹,d -Trp¹² (K_b= 5.1 nm ), reported to eliminate partial agonism and enhance potency, and Lys²⁶-Asp³⁰ lactamization (K_b= 31 nm ), aimed to stabilize the helical structure of the principal binding domain attributed to residues 25-34. The helical content in 30% trifluoroethanol is 88%, i.e., higher than the corresponding linear analog, and comprises the d -Trp¹²-Thr³³ segment. This hybrid lactam contains a rigid helical segment spanning the 14-18 sequence followed by a hinge motif around Arg^19-20, but the sequence 14-18 forms a stable helix. In all potent lactam-containing, PTHrP-derived agonists and antagonists studied so far, the dominant structural motif consists of two helical domains at the two ends of the sequence and of two hinge regions centered around Gly¹²-Lys¹³ and Arg¹⁹. The weakly active agonists and antagonists do not exhibit the “hinge” around position 19. These findings suggest that the presence and location of discrete hinge regions that connect the N- and C-terminal helices are essential for generating the bioactive conformation of ligands for the PTH/ PTHrP receptor.     

NMR and CD conformational studies of the C‐terminal 16‐peptides of Pseudomonas aeruginosa c551 and Hydrogenobacter thermophilus c552 cytochromes

Abstract: The 16‐amino acid sequences of the C‐terminal helices of the homologous bacterial cytochromes c₅₅₁ from Pseudomonas aeruginosa and c₅₅₂ from Hydrogenobacter thermophilus were synthesized and their solution structure studied. Circular dichroism and NMR experiments in aqueous solution have shown the presence of α‐helices and 3₁₀‐helices. The populations of helical structures in phosphate buffer, pH 3.5, 293 K, were 21% for c₅₅₁ and 20% for c₅₅₂, but increased to 56.7 and 48%, respectively, in 50% aqueous 2,2,2‐trifluoroethanol. An isodichroic point was observed at 203 nm in CD spectra for the helix/coil transition in mixtures of water/2,2,2‐trifluoroethanol. NMR spectra in phosphate buffer show the presence of both α‐ and 3₁₀‐helical structures. In water/2,2,2‐trifluoroethanol (50 : 50) α‐helices are predominant. CD temperature‐dependency studies indicate that both peptides exhibit the same cooperativity for the transition in water/2,2,2‐trifluoroethanol (50 : 50). The experimental data show that the amino acid substitutions do not favor heat resistance of the secondary structure of the c₅₅₂ C‐terminal helix at the local level. Instead, they optimize nonlocal contacts of the polypeptide chain, which stabilize the tertiary structure in the native protein.     

Structural characterization of peptide fragments from hCD81–LEL

Abstract: The solution conformation of two peptides [ 1 : PSGSNIISNLFKED;  2 : GSSTLTALTTSVLKNNL] from human CD81 (hCD81) large extra‐cellular loop (LEL) with known importance in the hepatitis C virus glycoprotein E2 (HCV‐E2) binding interaction was characterized using circular dichroism spectroscopy. In addition, the solution structure of peptide 1 that contains a phenylalanine residue (F186 in hCD81) known to be critical in the binding interaction with HCV‐E2 was determined using 1D and 2D ¹H NMR spectroscopy. Both peptides are unstructured in water but begin forming significant helical conformation following the addition of 20% or more trifluoroethanol (v/v), a result consistent with their α‐helical conformation found in the native protein. The CD data recorded as a function of pH and NaCl concentration are consistent with stabilization of the helical structure from electrostatic forces for both peptides. Peptide 1 is able to block the binding interaction of recombinant HCV‐E2 (rHCV‐E2) to hCD81 expressed on Molt‐4 T cells at high concentrations (3.5 mm ), a low affinity that we attributed to the random coil structure in water.     

Conformation of a water-soluble β-sheet model peptide. A circular dichroism and Fourier-transform infrared spectroscopie study of double D-amino acid replacements

Among peptide secondary structures β-sheet domains have been much less intensively studied than α-helical conformations, mainly because of the lack of well characterized model peptides. In the present paper the secondary structure of a water-soluble de novo peptide consisting of 26 amino acids (DPKGDPKGVTVTVTVTVTGKGDPKPD-NH₂) and the corresponding double D-amino acid replacement set have been studied by circular dichroism and Fourier-transform infrared spectroscopy. The model peptide was found to be unstructured in aqueous solution at peptide concentrations < 10^?3 mol/L but to adopt a predominantly β-sheet structure in the presence of 15 mM sodium dodecyl sulfate or at apolar/water interfaces. Although the peptide is composed of amino acids with low helical propensity, it formed a single-stranded helical structure in aqueous trifluoroethanol. The D-amino acid replacement set was synthesized in order to study the conformational stability of the model peptide selectively in distinct regions. The data show that both the α-helix present in 50% trifluoroethanol as well as the β-sheet domain formed in the presence of sodium dodecyl sulfate or at apolar/water interfaces, are located in the region between Val⁹ and Thr¹⁸. Pairwise substitution of adjacent amino acids by their corresponding D-amino acids provides a pronounced β-sheet disturbance. These findings demonstrate that double D-amino acid replacements may be used to locate β-sheet domains in peptides.     

Synthetic and binding studies on the calcium binding site I of bovine brain calmodulin

The synthesis of the cyclic 20–31 sequence of bovine brain calmodulin corresponding to the loop of the hypothetical calcium binding site I of the protein has been accomplished by classical solution methods. The interaction of the synthetic cyclic fragment with calcium ions has been investigated by CD spectroscopy in water and in 98% trifluoroethanol solution. Calcium ions have no effect on the dichroic absorption of aqueous solution of the cyclic dodeca-peptide in the wavelength range 200–250 nm. In 98% trifluoroethanol the CD spectrum of the cyclic compound in the absence of calcium ions is almost identical to that of the linear dodecapeptide in the presence of saturation concentrations of calcium. This result supports our previous hypothesis of a folding of the linear sequence upon interaction with the metal ion. The cyclic peptide also interacts with calcium ions in 98% trifluoroethanol forming a 1:1 complex.     

CONFORMATIONAL ANALYSIS OF A CHAIN REVERSAL IN α-CHYMOTRYPSIN

A complete conformational analysis of the fold (Asp-Lys-Thr-Gly) (residues 35–38), and additional adjacent residues of α-chymotrypsin has been performed. A comparison of these findings with those of Lewis et al. (1) is made, and a discussion of the implications of protein-fold models is discussed. This particular residue sequence prefers to bend over maintaining a helical conformation. However, the bend conformation of the tetramer is different from that of the native bend. The native bend conformation is nearly realized when an additional residue of the native primary structure is added to each side of the tetramer. Early and late folding-sequence studies suggests that while the native fold is of low energy, there are fold-points along the primary structure which are more stable. The structural implications of this finding are discussed.     

Relationship between the tertiary structures of mastoparan B and its analogs and their lytic activities studied by NMR spectroscopy

Abstract: Mastoparan B (MP‐B), an antimicrobial cationic tetradecapeptide amide isolated from the venom of the hornet Vespa basalis, is an amphiphilic α‐helical peptide. MP‐B possesses a variety of biological activities, such as mast cells degradation histamine release, erythrocyte lysis and inhibition of the growth of Gram‐positive and Gram‐negative bacteria. In order to study the relationship between the structure and the biological activity of MP‐B, we used four analogs by replacing amino acids with alanine. Tertiary structures of MP‐B and its analogs in 2,2,2‐trifluoroethanol (TFE)‐containing aqueous solution have been determined by NMR spectroscopy and molecular modeling. The results indicate that [Ala⁴]MP‐B and [Ala¹²]MP‐B with higher hydrophobicity adopt a higher content of amphiphilic helical structures, and have better antimicrobial and hemolytic activities than MP‐B. However, [Ala³]MP‐B and [Ala⁹]MP‐B with lower hydrophobicity have disordered structures. [Ala³]MP‐B and [Ala⁹]MP‐B have low antimicrobial activity and much less hemolytic activity relative to MP‐B. It is likely that tryptophan residue in MP‐B and appropriate hydrophobicity of MP‐B to induce α‐helical structure is essential for the antibacterial and hemolytic activity of MP‐B. This study can aid understanding of the structure–activity relationship of MP‐B and to design peptides to possess lytic activity.     

Conformational behaviour of a synthetic peptide of the C-terminus of villin that interacts with actin: an NMR,CD and simulated annealing study

The solution structure of a synthetic 22-amino acid peptide (P1) corresponding to the extreme C-terminal end and one of the F-actin binding sites of villin has been determined by ¹H NMR and CD spectroscopy. The structure of this peptide was compared to that of a peptide in which lysine to glutamic acid substitutions were introduced at positions 17 and 19 (P11), abolishing F-actin binding. Both peptides are largely unstructured in aqueous solution. Changes observed in the NMR and CD spectra of both peptides are consistent with α-helix formation in trifluoroethanol/water mixtures. A set of 189 interproton distances derived from nuclear Overhauser enhancement (NOE) measurements, 17 φ-angle constraints obtained from ³J_nhα coupling constants, as well as about 10 N···O distance restraints deduced from amide proton exchange kinetics with deuterium, were used for the structure determination. The three-dimensional structure of P1 and P11 is characterized by two helical regions, one extending from residues 2 to 5 and a second covering residues 7 to 17. The central fragment, ranging from Leu-7 to Leu-15, is more stable. The C-terminal residues are less structured, particularly within peptide P11. The significance of these structural results is discussed in relation to the biological activity of villin. © Munksgaard 1995.     

Solution structure of the hydrophilic region of HIV-1 encoded virus protein U (Vpu) by CD and lH NMR spectroscopy

The HIV-1 specific Vpu is a class I oligomeric membrane phosphoprotein of unknown structure and mechanism. The first experimental evidence for the position of secondary structural elements present in the hydrophilic C-terminal region of Vpu under various solution regimes is reported. CD data for nine overlapping 15 amino-acid fragments and 3 longer fragments indicate the presence of only transitory amounts of stable structure in aqueous solution alone, while with increasing trifluoroethanol content limiting structures were found indicating two helical segments in the hydrophilic region of Vpu. These limiting structures were more precisely defined from a detailed study of Vpu^63–81, Vpu^52–74 and Vpu^63–81, by a combination of 2D ²H NMR spectroscopy, distance geometry, and restrained molecular dynamics and energy minimization calculations. Sets of low-energy conformations compatible with the quantitative NOE data indicate that Vpu^41–58 has an α-helix from residues 42 to 50 while a second helix is found for VPU^52–74 from residues 57 to 69. Vpu^63–81 shows only the presence of a single reverse turn at residues 74 to 77, without any evidence of helix, under the same conditions. From CD measurements the first helix extends back to residue 30 and is connected to the N-terminal anchor of Vpu. Thus the hydrophilic region of Vpu consists of two α-helices joined by a flexible region of 6 or 7 residues, which contains the phosphoacceptor sites of Vpu at positions 52 and 56. The second helix is followed by a single reverse turn and a flexible C-terminus. © Munksgaard 1995.     

Synthesis and conformational study in solution and in solid state of oligopeptides containing l-leucine and glycine

The synthesis and conformational studies of the oligopeptide N-tert.-butyloxycarbonyl-l -Leu-(l -Leu-Gly)_n-OBzl (n = 1, 3, 5) and N-tert.-butyloxycarbonyl-(l -Leu-Gly)₂-OBzl are described. The peptides were synthesized by stepwise and fragment condensation techniques using dicyclohexylcarbodiimide as the condensing agent in solution. The conformational study of the oligopeptides was carried out using CD, u.v. and i.r. spectra. The conformation in solution was examined in trifluoroethanol, hexafluoroisopropanol. hexafluoroacetone trihydrate, and methanol. CD spectra in trifluoroethanol exhibited a gradual variation with increasing peptide chain length. This can be interpreted as a formation of an ordered structure which is already present in the heptapeptide and, to a greater extent, in the undecapeptide. The results obtained from the CD profiles and i.r. spectra showed the presence of β structure with antiparallel chains in the heptapeptide and undecapeptide. Finally, CD spectra revealed in trifluoroethanol-water solution the binding of Ca²⁺ to heptapeptide and undecapeptide together with a contemporaneous conformational change. This change is probably due to the formation of β-turns. No change in the CD profiles was obtained by using Mg²⁺, K⁺, Na⁺, and Li⁺ ions instead of Ca²⁺.     

A peptide of nine amino acid residues from α-sarcin cytotoxin is a membrane-perturbing structure

A water-soluble synthetic peptide with only nine amino acid residues, comprising the 131–139 sequence region of the cytotoxic protein α-sarcin (secreted by the mold Aspergillus giganteus), interacts with large unilamellar vesicles composed of acid phospholipids. It promotes lipid mixing between bilayers and leakage of vesicle aqueous contents, and it also abolishes the phospholipid phase transition. Other larger peptides containing such an amino acid sequence also produce these effects. These peptides acquire α-helical conformation in the presence of trifluoroethanol, but display β-strand conformation in the presence of sodium dodecyl sulfate. The interaction of these peptides with the lipid vesicles also results in β-structure. The obtained data are discussed in terms of the involvement of the 131–139 stretch of α-sarcin in its interaction with lipid membranes.     

Antibacterial activities and conformations of synthetic α‐defensin HNP‐1 and analogs with one,two and three disulfide bridges

Abstract: Structure and biological activities of synthetic peptides corresponding to human α‐defensin HNP‐1, AC¹YC²RIPAC³IAGERRYGTC⁴IYQGRLWAFC⁵C⁶ with the S–S connectivities: C¹–C⁶, C²–C⁴, C³–C⁵, and its variants with one, two and three disulfide bridges were investigated. Oxidation of synthetic, reduced HNP‐1 yielded a peptide with S–S connectivities C¹–C³, C²–C⁴ and C⁵–C⁶, and not with the S–S linkages as in naturally occurring HNP‐1. Selective protection of cysteine sulfhydryls was necessary for the formation of S–S bridges as in native HNP‐1. Likewise, oxidation of peptide encompassing the segment from C² to C⁵, resulted in the S–S linkages C²–C³ and C⁴–C⁵ instead of the expected linkage C²–C⁴ and C³–C⁵. Antibacterial activities were observed for all peptides, irrespective of how the S–S bridges were linked. Linear peptides without S–S bridges were inactive. Circular dichroism (CD) spectra suggest that peptides constrained by one and two S–S bridges do not form rigid β‐sheet structures in an aqueous environment. The spectrum of HNP‐1 in an aqueous environment suggests the presence of a β‐hairpin conformation. In the presence of lipid vesicles, the S–S constrained peptides tend to adopt a β‐structure. Although the S–S connectivities observed in HNP‐1 may be necessary for other physiological activities, such as chemotaxis, they are clearly not essential for antibacterial activity.