Identification of neutral and anionic 8 alpha-substituted flavin semiquinones in flavoproteins by electron spin resonance spectroscopy

Circular dichroic spectra of metmyoglobin and apomyoglobin were measured in neutral and acidic solution. Addition of sodium dodecyl sulfate (NaDodSO₄) slightly reduces the helicity (based on the circular dichroic magnitude) of both proteins probably because of the loss of long-range interactions among helical segments. Lowering the pH of the protein-surfactant solution to 3 slightly enhances the helical conformation of myoglobin due to the protonation of acidic side groups and thereby the reduction of coulombic repulsion among negative charges. For BrCN-digested fragments the COOH-terminal peptide (22 residues) loses its helicity which can be restored by addition of NaDodSO₄. The middle fragment (76 residues) retains a considerable amount of helicity in water alone, which further increases in the presence of NaDodSO₄. The NH₂-terminal fragment (55 residues) also has some helical conformation in water, which is enhanced by the addition of NaDodSO₄. The circular dichroic spectrum of an equimolar mixture of the three peptides in NaDodSO₄ solution is the same as that calculated from the spectra of isolated peptides under similar conditions.     

Helical conformations of three crystalline pentapeptide fragments of suzukacillin,a membrane channel forming polypeptide

The crystal structures of three pentapeptide fragments of suzukacillin-A have been determined. Boc-Aib-Pro-Val-Aib-Val-OMe (peptide 1–5) adopts a distorted helical conformation, stabilized by three intramolecular hydrogen bonds (two 5→1, one 4→1). Boc-Ala-Aib-Ala-Aib-Aib-OMe (peptide 6–10) and Boc-Leu-Aib-Pro-Val-Aib-OMe (peptide 16–20) adopt 3₁₀ helical structures stabilized by three and two 4→1 intramolecular hydrogen bonds, respectively. These structures provide substantial support for a largely helical conformation for the suzukacillin membrane channel.     

Helical formation of a 13-residue C-peptide analogue of ribonuclease A in sodium dodecyl sulfate solution

The conformation of a 13-residue C-peptide analogue of ribonuclease A——in surfactant solutions was studied by CD. The CD spectrum of the peptide in excess NaDodSO₄ solution was typical for a helical conformation; the spectrum appeared to be virtually independent of pH (2.5–6) and temperature (3–25°C). Analysis of the CD data indicated a helicity of about 65–70% with no α-sheet and β-turn; this corresponded to 8 or 9 residues in the helical form or slightly more than two turns of α-helix. This compares with an average of about one turn of α-helix for the C-peptide analogue in water at pH 4.7 and 7°C. The conformation of the peptide in cationic surfactant, dodecyl ammonium chloride, and nonionic surfactant, dodecyl heptaoxyethylene ether, solution resembled that in water. We concluded that the C-peptide analogue can develop a maximum helicity close to the corresponding segment in ribonuclease A in hydrophobic environment provided by the clustering of NaDodSO₄ molecules to the cationic side groups of the peptide, except that the end effects may destabilize two or three residues each at both ends of the helix. Thus, in the interior of a protein molecule this hydrophobic effect may overshadow the charged-group effect than can be explained by the helix dipole model for the helical segments on the exterior of the protein molecule.     

Conformational properties of the isolated 1–23 fragment of human hemoglobin α-chain

With the purpose of establishing whether, as a general rule, regions of a protein chain that are helical in the native structure maintain, at least partially, the same helical structure when isolated in solution, we have prepared the 1–23 fragment of human hemoglobin α-chain, and studied its conformational properties in aqueous solution by CD and¹H-NMR. From the analysis of CD and NMR spectral changes with temperature, salt and addition of trifluoroethanol (TFE) it can be concluded that the 1–23 peptide forms a measurable population (18% at 22°C (pH 5.6) TFE/H₂O, 30:70 (v/v)) of an α-helix structure that spans the same residues that are helical in the native protein (namely, 6 to 17). These results, taken together with similar ones obtained previously in the 1–19, 21–42 and 50–61 RNAase fragments, support the idea that no helices other than the native ones are actually formed in solution by protein fragments. This implies that the final helical structure of a protein is present from the very beginning of the folding process, and also that such elements of secondary structure can act as primary nucleation centers.     

Stabilisation of a short α‐helical VIP fragment by side chain to side chain cyclisation: a comparison of common cyclisation motifs by circular dichroism

A model octapeptide segment derived from vasoactive intestinal peptide (VIP) was utilised to investigate the effect of several conventional cyclisation methods on the α‐helical conformation in short peptide fragments. Three of the classical macrocyclisation techniques (i.e. lactamisation, ring‐closing metathesis and Huisgen cycloaddition) were applied, and the conformations of the resulting cyclic peptides, as well as their linear precursors, were compared by CD analysis. The visibly higher folding propensity of the triazole‐tethered peptide after azide‐alkyne CuAAC macrocyclisation illustrates that the secondary structure of a short peptide fragment can differ significantly depending on the chemical strategy used to covalently cross‐link side chain residues in a ‘helical’ fragment. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Preferred side‐chain conformation of arginine residues in a triple‐helical structure

The crystal structure of the triple‐helical peptide (Pro‐Hyp‐Gly)₃‐Pro‐Arg‐Gly‐(Pro‐Hyp‐Gly)₄ (POG3‐PRG‐POG4) was determined at 1.45 Å resolution. POG3‐PRG‐POG4 was designed to permit investigation of the side‐chain conformation of the Arg residues in a triple‐helical structure. Because of the alternative structure of one of three Arg residues, four side‐chain conformations were observed in an asymmetric unit. Among them, three adopt a ttg^?t conformation and the other adopts a tg^?g^?t conformation. A statistical analysis of 80 Arg residues in various triple‐helical peptides showed that, unlike those in globular proteins, they preferentially adopt a tt conformation for χ₁ and χ₂, as observed in POG3‐PRG‐POG4. This conformation permits van der Waals contacts between the side‐chain atoms of Arg and the main‐chain atoms of the adjacent strand in the same molecule. Unlike many other host–guest peptides, in which there is a significant difference between the helical twists in the guest and the host peptides, POG3‐PRG‐POG4 shows a marked difference between the helical twists in the N‐terminal peptide and those in the C‐terminal peptide, separated near the Arg residue. This suggested that the unique side‐chain conformation of the Arg residue affects not only the conformation of the guest peptide, but also the conformation of the peptide away from the Arg residue. © 2014 Wiley Periodicals, Inc. Biopolymers 101: 1000–1009, 2014.     

Secondary structure mapping of DnaK-bound protein fragments: chain helicity and local helix unwinding at the binding site

Little is known about polypeptide conformation and folding in the presence of molecular chaperones participating in protein biosynthesis. In vitro studies on chaperone-substrate complexes have been mostly carried out with small peptide ligands. However, the technical challenges associated with either competing aggregation or spectroscopically unfavorable size and exchange rates have typically prevented analysis of larger substrates. Here, we report the high-resolution secondary structure of relatively large N-terminal protein fragments bound to the substrate-binding domain of the cotranslationally active chaperone DnaK. The all-alpha-helical protein apomyoglobin (apoMb), bearing the ubiquitous globin fold, has been chosen as a model substrate. On the basis of NMR secondary chemical shift analysis, we identify, for the first time, weak helical content (similar to that found in the chemically unfolded full-length protein) for the assigned residues of the chaperone-bound chain away from the chaperone binding sites. In contrast, we found that the residues corresponding to the strongest specific binding site for DnaK, examined via a short 13-mer apoMb peptide fragment matching the binding site sequence, display highly reduced helical content in their chaperone-bound form. Given that the free state of the peptide is weakly helical in isolation, we conclude that the substrate residues corresponding to the chaperone binding site undergo helix unwinding upon chaperone binding.     

Conformational properties of gastrin fragments of increasing chain length

The conformational properties of a series of biologically active gastrin peptides of increasing chain length have been investigated in TEE solution by spectroscopic techniques. It was found that elongation of the glutamic acid sequence from 1 to 5 residues at the N-terminal portion of the molecules causes a cooperative change of the conformation of the peptide backbone. The environment of the biologically important C-terminal sequence-Trp-Nle-Asp-Phe-NH₂ monitored by the near-uv chiroptical propertical properties is alos affected by chain elongation. However, the change of the structure of the C-terminal portion does not parallel the conformational change of the peptide backbone. In fact, the final folded structure at the C-terminus is almost reached in the fragment with a sequence of 4 glutamic acid residues, while an additiona, relevent conformational change of the backbne is observed on further elongation of the chain to minigastrin and little gastrin. The ability of the fragments to fold into an ordered conformation on chain elongation parallels the increase of biolocical potency tested in vivo, reported in the literature, and suggests a correlation between these two facts. Ionization of the carboxyl side chains is without effect on the structure of the fragments with 2, 3, and 4 glutamic acid residues, while an effect is observed in minigastrin and little gastrin. From analysis of the CD properties and from their dependence upon side-chain ionization a structural model is proposed for the hormones minigastrin and little gastrin. This tentative model includes a β-bend located in the sequence Ala-Tyr-Gly-Trp- and a short helical section at the N-terminal portion of the hormones.     

Conformations of isolated fragments of pancreatic polypeptide

In spite of its short polypeptide chain, the pancreatic polypeptide molecule consists of a polyproline II type helix and alpha-helix. To understand the stability and formation of the alpha-helical region, we prepared some peptide fragments including the helical segment of chicken pancreatic polypeptide and studied their conformations by circular dichroism (CD). PP7-36 (a peptide fragment corresponding to residues 7-36 of chicken pancreatic polypeptide) showed a CD spectrum characteristic of the helix at pH 4.6 and at peptide concentrations as low as 1 microM. PP11-36 was able to form a helical conformation only at high peptide concentrations and not at concentrations lower than 10 microM. However, acetyl PP11-36 (in which the alpha-amino group is acetylated so that no positive charge exists at the N terminus) was able to form the helical conformation at pH 4.6 and at the peptide concentrations where PP11-36 could not. Succinyl PP12-36 (in which the alpha-amino group is succinylated to introduce a negative charge) was also able to form the helical conformation. The CD spectra of PP12-36 and PP13-36 were not characteristic of the helical conformation at all the pH values and peptide concentrations studied. Acetyl PP13-36, which has no charge at the N terminus, did not form the helix. On the other hand, succinyl PP13-36, which has a negative charge at the N-terminal end, did form the helix at pH 4.6. These findings indicate that the presence of the negative charge of carboxylate at the N-terminal region of a peptide fragment is important for helix formation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Nicked apomyoglobin: a noncovalent complex of two polypeptide fragments comprising the entire protein chain

Limited proteolysis of the 153-residue chain of horse apomyoglobin (apoMb) by thermolysin results in the selective cleavage of the peptide bond Pro88-Leu89. The N-terminal (residues 1-88) and C-terminal (residues 89-153) fragments of apoMb were isolated to homogeneity and their conformational and association properties investigated in detail. Far-UV circular dichroism (CD) measurements revealed that both fragments in isolation acquire a high content of helical secondary structure, while near-UV CD indicated the absence of tertiary structure. A 1:1 mixture of the fragments leads to a tight noncovalent protein complex (1-88/89-153, nicked apoMb), characterized by secondary and tertiary structures similar to those of intact apoMb. The apoMb complex binds heme in a nativelike manner, as given by CD measurements in the Soret region. Second-derivative absorption spectra in the 250-300 nm region provided evidence that the degree of exposure of Tyr residues in the nicked species is similar to that of the intact protein at neutral pH. Also, the microenvironment of Trp residues, located in positions 7 and 14 of the 153-residue chain of the protein, is similar in both protein species, as given by fluorescence emission data. Moreover, in analogy to intact apoMb, the nicked protein binds the hydrophobic dye 1-anilinonaphthalene-8-sulfonate (ANS). Taken together, our results indicate that the two proteolytic fragments 1-88 and 89-153 of apoMb adopt partly folded states characterized by sufficiently nativelike conformational features that promote their specific association and mutual stabilization into a nicked protein species much resembling in its structural features intact apoMb. It is suggested that the formation of a noncovalent complex upon fragment complementation can mimic the protein folding process of the entire protein chain, with the difference that the folding of the complementary fragments is an intermolecular process. In particular, this study emphasizes the importance of interactions between marginally stable elements of secondary structure in promoting the tertiary contacts of a native protein. Considering that apoMb has been extensively used as a paradigm in protein folding studies for the past few decades, the novel fragment complementing system of apoMb here described appears to be very useful for investigating the initial as well as late events in protein folding.     

Conformational Analysis of the β-amyloid Peptide Fragment, β(12–28)

Abstract

NMR and CD spectroscopy have been used to examine the conformation of the peptide, β(12–28), (VHHQKLVFFAEDVGSNK) in aqueous and 60% TFE/40% H₂0 solution at pH 2.4. In 60% TFE solution, the peptide is helical as confirmed by the CD spectrum and by the pattern of the NOE cross peaks detected in the NOESY spectrum of the peptide. In aqueous solution, the peptide adopts a more extended and flexible conformation. Broadening of resonances at low temperature, temperature-dependent changes in the chemical shifts of several of the CH_α resonances and the observation of a number of NOE contacts between the hydrophobic side-chain protons of the peptide are indicative of aggregation in aqueous solution. The behavior of β(12–28) in 60% TFE and in aqueous solution are consistent with the overall conformation and aggregation behavior reported for the larger peptide fragment, β(1–28) and the parent β-amyloid peptide.     

High-resolution structural profile of hylaseptin-4: Aggregation,membrane topology and pH dependence of overall membrane binding process

Hylaseptin-4 (HSP-4, GIGDILKNLAKAAGKAALHAVGESL-NH₂) is an antimicrobial peptide originally isolated from Hypsiboas punctatus tree frog. The peptide has been chemically synthetized for structural investigations by CD and NMR spectroscopies. CD experiments reveal the high helical content of HSP-4 in biomimetic media. Interestingly, the aggregation process seems to occur at high peptide concentrations either in aqueous solution or in presence of biomimetic membranes, indicating an increase in the propensity of the peptide for adopting a helical conformation. High-resolution NMR structures determined in presence of DPC-d₃₈ micelles show a highly ordered α-helix from amino acid residues I2 to S24 and a smooth bend near G14. A large separation between hydrophobic and hydrophilic residues occurs up to the A16 residue, from which a shift in the amphipathicity is noticed. Oriented solid-state NMR spectroscopy show a roughly parallel orientation of the helical structure along the POPC lipid bilayer surface, with an insertion of the hydrophobic N-terminus into the bilayer core. Moreover, a noticeable pH dependence of the aggregation process in both aqueous and in biomimetic membrane environments is attributed to a single histidine residue (H19). The protonation degree of the imidazole side-chain might help in modulating the peptide-peptide or peptide-lipid interactions. Finally, molecular dynamics simulations confirm the orientation and preferential helical conformation and in addition, show that HSP-4 tends to self-aggregate in order to stabilize its active conformation in aqueous or phospholipid bilayer environments.     

The conformation of an alamethicin in methanol by multinuclear NMR spetroscopy and distance geometry/simulated annealing

The solution conformation of the antibiotic peptide alamethicin was investigated using multi-nuclear spectroscopy and the distance geometry/simulated annealing algorithms from the program DSPACE. ¹H-, ¹³C-, and ¹⁵N-nmr chemical shifts and homonuclear ¹H coupling constants suggest that the molecule is flexible in the vicinity of Gly-11 and Leu-12. The temperature dependence of the amide proton chemical shifts indicates that there is flexibility in the middle of the 20 residue peptide and provides evidence that, at the very N-terminus, the molecule adopts a 3₁₀-helical conformation. The large differences in the ¹³C chemical shifts of the pro-R and pro-S methyls of the α-aminoisobutyric acid residues were used to constrain those residues to the right-handed helical conformation in the distance geometry/simulated annealing algorithms. A family of 24 structures was generated but did not converge to a common conformation when superimposed over the entire polypeptide sequence. The molecules did converge to a helical conformation over residues 1–10 and residues 13–18. The lack of convergence when the entire lengths of the molecules are superimposed is explained by the flexibility of the peptide near Gly-11/Leu-12. The results suggest that the protein consists of two helices connected by a flexible “hinge.” The flexibility of the molecule is discussed with respect to the macrodipole model of voltage gating. © 1995 John Wiley & Sons, Inc.     

Use of a combination of the RDC method and NOESY NMR spectroscopy to determine the structure of Alzheimer’s amyloid Aβ10–35 peptide in solution and in SDS micelles

The spatial structure of Alzheimer’s amyloid Aβ_10–35-NH₂ peptide in aqueous solution at pH 7.3 and in SDS micelles was investigated by use of a combination of the residual dipolar coupling method and two-dimensional NMR spectroscopy (TOCSY, NOESY). At pH 7.3 Aβ_10–35-NH₂ adopts a compact random-coil conformation whereas in SDS micellar solutions two helical regions (residues 13–23 and 30–35) of Aβ_10–35-NH₂ were observed. By use of experimental data, the structure of “peptide–micelle” complex was determined; it was found that Aβ_10–35-NH₂ peptide binds to the micelle surface at two regions (residues 17–20 and 29–35).     

β‐Hairpin stabilization in a 28‐residue peptide derived from the β‐subunit sequence of human chorionic gonadotropin hormone

The β‐subunit of the human chorionic gonadotropin (hCG) hormone, which is believed to be related to certain types of cancer, contains three hairpin‐like fragments. To investigate the role of β‐hairpin formation in the early stages of the hCGβ folding, a 28‐residue peptide with the sequence RDVRFESIRLPGSPRGVNPVVSYAVALS, corresponding to the H3‐β hairpin fragment (residues 60–87) of the hCGβ subunit, was studied under various conditions using three optical spectroscopic methods: Fourier transform ir spectroscopy, electronic CD, and vibrational CD. Environmental conditions are critical factors for formation of secondary structure in this peptide. TFE : H₂O mixed solvents induced helical formation. Formation of β‐structure in this peptide, which may be related to the native β‐hairpin formation in the intact hormone, was found to be induced only under conditions such as high concentration, high temperature, and the presence of nonmicellar sodium dodecyl sulfate concentrations. These findings support a protein folding mechanism for the hCGβ subunit in which an initial hydrophobic collapse, which increases intermolecular interactions in hCGβ, is needed to induce the H3‐β hairpin formation. © 1999 John Wiley & Sons, Inc. Biopoly 50: 413–423, 1999     

Synthesis and FTIR Conformational Studies of Peptides From the Basic Region of c-Jun: a Critical Analysis on the Basis of CD and NMR Data

Abstract

The peptide (35 residues) corresponding to the basic subdomain (bSD) of c-Jun (residues 252–281) and its fragments NP (N-terminal peptide, 1–19) and CP (C-terminal peptide, 1635) were synthesized in stepwise solid-phase using the tert-butyloxycarbonyl/benzyl strategy. In a previous paper, we have shown that during its binding to the DNA site CRE (cAMP- responsive element) the bSD structure was converted into α-helix from an initial random coil conformation [Krebs, D., Dahmani, B., El Antri, S., Monnot, M., Convert, O., Mauffret, O., Troalen, F. & Fermandjian, S. Eur. J. Biochem. 231, 370–380 (1995)]. Our results suggested both a high flexibility and a helical potential in bSD, these two properties seeming crucial for the accommodation of the basic subdomain of c-Jun to its specific DNA targets. In this work, we assessed the conformational variability of bSD through the study of the secondary structures of its NP and CP fragments in trifluoroethanol (TFE)/²H₂O mixtures, using Fourier transform infrared (FTIR) spectroscopy. The IR results were critically analyzed in light of our previously reported circular dichroism (CD) and NMR data [Krebs, D., Dahmani, B., Monnot, M., Mauffret, O., Troalen, F. & Fermandjian, S. Eur. J. Biochem. 235, 699–712 (1996)]. Upon addition of TFE, the relative areas of the seven components of the amide I band (1700–1620 cm^?1) reflected the conversion of a large amount of random coil conformation into α-helix for the two fragments and bSD. This effect was accompanied by more subtle variations of the less populated structures, in agreement with the results of CD and NMR experiments. The IR results stipulated the conservation of the parent bSD secondary structures in both fragments; however, NP and CP peptides did not display similar random-to-α-helix stabilization pattern upon additions of TFE to aqueous solutions. The profile from CD signal at 222 nm was found sigmoidal for NP and almost linear for CP, while that corresponding to the parent peptide bSD was just in between those of its fragments. Thus, the present study confirms the high flexibility and helix propensity of the c-Jun basic subdomain and suggests that the N- and C-terminal parts of the peptide do not follow the same random-to-helix conversion profile during their complexation with DNA.     

Conformational studies of a short linear peptide corresponding to a major conserved neutralizing epitope of human respiratory syncytial virus fusion glycoprotein

The conformational properties of a 21-residue peptide, corresponding to amino acids 255 to 275 (F255-275) of the human respiratory syncytial virus fusion (F) glycoprotein, have been studied by CD and nmr spectroscopy. This peptide includes residues 262, 268, and 272 of the F polypeptide that are essential for integrity of most epitopes that mapped into a major antigenic site of the F molecule. CD data indicate that F255-275 adopts a random coil conformation in aqueous solution at low peptide concentrations. However, as the concentration of peptide is increased, a higher percentage of peptide molecules adopts an organized structure. This effect can be more easily observed when trifluoroethanol (30%) is added to peptide solutions, giving rise to CD spectra that resemble those of α-helix structures. These conformational changes were confirmed by nmr spectroscopy. The nuclear Overhauser effects observed in 30% trifluoroethanol/water together with the conformational H_α chemical shift data allowed us to propose a structural model of helix-loop-helix for the peptide in solution. In addition, these helical regions contain the amino acid residues essential for epitope integrity in the native F molecule. These results give new insights into the antigenic structure of the respiratory syncytial virus F glycoprotein. © 1996 John Wiley & Sons, Inc.     

Nonnative helical motif in a chaperone-bound protein fragment

The effect of cotranslationally active chaperones on the conformation of incomplete protein chains is poorly understood. The secondary structure of a 77-residue chaperone-bound N-terminal protein fragment corresponding to the first five helices (A-E) of apomyoglobin (apoMb_1-77) is investigated here at the residue-specific level by multidimensional NMR. The substrate-binding domain of DnaK, DnaK-β, is employed as a chaperone model. By taking advantage of the improved spectral quality resulting from chaperone deuteration, we find that DnaK-β-bound apoMb_1-77 displays a region of nonnative helicity at residues away from the main chaperone binding site. The nonnative structural motif comprises portions of the native D and E helices and has similar characteristics to the reported nonnative DE helical region of acid-unfolded full-length apoMb. Upon incorporation of the missing C-terminal amino acids, a structural kink develops between residues 56 and 57, and two separate native D and E helices are generated. This work highlights, for the first time to our knowledge, the presence of a nonnative helical motif in a large chaperone-bound protein fragment under physiologically relevant solution conditions.     

Peptide design: Crystal structure of a helical peptide module attached to a potentially nonhelical amino terminal segment

The peptide Boc-Gly-Dpg-Gly-Val-Ala-Leu-Aib-Val-Ala-Leu-OMe has been designed to examine the structural consequences of placing a short segment with a low helix propensity at the amino terminus of a helical heptapeptide module. The Gly-Dpg-Gly segment is a potential connecting element in the synthetic construction of a helix-linker-helix motif. Crystal parameters for the peptide are P2₁, a = 8.651(3) Å, b = 46.826(13) Å, c = 16.245 Å, β = 90.13(3)*, Z = 4; 2 independent molecules/asymmetric unit. The structure reveals almost identical conformations for the two independent molecules. The backbone is completely helical for residues 2–9, with one 4 → 1 hydrogen bond and six 5 → 1 hydrogen bonds. The α,α-di-n-propylglycine residue adopts a helical conformation. Gly(1) adopts an extended conformation resulting in a nonhelical N-terminus, with the Boc group swinging away from the helix. The lateral association of helices in the b axis direction is unusual in that the helix axes are directed up or down (parallel or antiparallel) by pairs: ↓↓↑↑↓↓, etc. © 1996 John Wiley & Sons, Inc.     

Helix formation by the phospholipase A2 38–59 fragment: Influence of chain shortening and dimerization monitored by nmr chemical shifts

The solution structure of a peptide fragment corresponding to the 38–59 region of porcine phospholipase A₂ has been investigated using CD, nmr chemical shifts, and nuclear over-hauser effects (NOEs). This isolated fragment of phospholipase forms an α-helix spanning residues 38–55, very similar to the one found in the native protein, except for residues 56–58, which were helical in the crystal but found random in solution. Addition of triflouro-ethanol (TFE) merely increased helix population but it did not redefine helix limits. To investigate how the folding information, in particular that concerning eventual helix start and stop signals, was coded in this particular amino acid sequence, the helices formed by synthetic peptides reproducing sections of this phospholipase 38–59 fragment, namely 40–59, 42–59, 38–50, and 45–57, were characterized using NOEs and helix populations quantitatively evaluated on different peptide chain segments using nmr chemical shifts in two solvents (H₂O and 30% TFE/H₂O). A set of nmr spectra was also recorded and assigned under denaturing conditions (6Murea) to obtain reliable values for the chemical shifts of each peptide in the random state. Based on chemical shift data, it was concluded that the helix formed by the phospholipase 38–59 fragment was not abruptly, but progressively, destabilized all along its length by successive elimination of residues at the N end, while the removal of residues at the C end affected helix stability more locally and to a lesser extent. These results are consistent with the idea that there are not single residues responsible for helix initiation or helix stability, and they also evidence an asymmetry for contributions to helix stability by residues located at the two chain ends. The restriction of molecular mobility caused by linking with a disulphide bridge at Cys 51 two identical 38–59 peptide chains did not increase helix stability. The helix formed by the covalently formed homodimer was very similar in length and population to that formed by the monomer. © 1994 John Wiley & Sons, Inc.