Structural comparison in solution of a native and retro peptide derived from the third helix of Staphylococcus aureus protein A,domain B: retro peptides,a useful tool for the discrimination of helix stabilization factors dependent on the peptide chain orientation

A peptide fragment corresponding to the third helix of Staphylococcus Aureus protein A, domain B, was chosen to study the effect of the main‒chain direction upon secondary structure formation and stability, applying the retro‒enantio concept. For this purpose, two peptides consisting of the native (Ln) and reversed (Lr) sequences were synthesized and their conformational preferences analysed by CD and NMR spectroscopy. A combination of CD and NMR data, such as molar ellipcitity, NOE connectivities, Hα and NH chemical shifts, ³J_αN coupling constants and amide temperature coefficients indicated the presence of nascent helices for both Ln and Lr in water, stabilized upon addition of the fluorinated solvents TFE and HFIP. Helix formation and stabilization appeared to be very similar in both normal and retro peptides, despite the unfavourable charge–macrodipole interactions and bad N-capping in the retro peptide. Thus, these helix stabilization factors are not a secondary structure as determined for this specific peptide. In general, the synthesis and confirmational analysis of peptide pairs with opposite main‒chain directions, normal and retro peptides, could be useful in the determination of secondary structure stabilization factors dependent on the direction. © 1997 European Peptide Society and John Wiley & Sons, Ltd.     

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.     

CD and NMR investigations on trifluoroethanol-induced step-wise folding of helical segment from scorpion neurotoxin.

A 14 amino acid residue peptide from the helical region of Scorpion neurotoxin has been structurally characterized using CD and NMR spectroscopy in different solvent conditions. 2,2,2-Trifluoroethanol (TFE) titration has been carried out in 11 steps from 0 to 90% TFE and the gradual stabilization of the conformation to form predominantly alpha-helix covering all of the 14 residues has been studied by 1H and 13C NMR spectroscopy. Detailed information such as coupling constants, chemical shift indices, NOESY peak intensities and amide proton temperature coefficients at each TFE concentration has been extracted and analysed to derive the step-wise preferential stabilization of the helical segments along the length of the peptide. It was found that there is a finite amount of the helical conformation in the middle residues 5-11 even at low TFE concentrations. It was also observed that > 75% TFE (v/v) is required for the propagation of the helix to the N and C termini and for correct packing of the side chains of all of the residues. These observations are significant to understanding the folding of this segment in the protein and may throw light on the inherent preferences and side chain interactions in the formation of the helix in the peptide.     

(φ,ψ)₂ motifs: a purely conformation-based fine-grained enumeration of protein parts at the two-residue level

A deep understanding of protein structure benefits from the use of a variety of classification strategies that enhance our ability to effectively describe local patterns of conformation. Here, we use a clustering algorithm to analyze 76,533 all-trans segments from protein structures solved at 1.2 Å resolution or better to create a purely φ,ψ-based comprehensive empirical categorization of common conformations adopted by two adjacent φ,ψ pairs (i.e., (φ,ψ)₂ motifs). The clustering algorithm works in an origin-shifted four-dimensional space based on the two φ,ψ pairs to yield a parameter-dependent list of (φ,ψ)₂ motifs, in order of their prominence. The results are remarkably distinct from and complementary to the standard hydrogen-bond-centered view of secondary structure. New insights include an unprecedented level of precision in describing the φ,ψ angles of both previously known and novel motifs, ordering of these motifs by their population density, a data-driven recommendation that the standard C^α_i…C^α_i + 3 < 7 Å criteria for defining turns be changed to 6.5 Å, identification of β-strand and turn capping motifs, and identification of conformational capping by residues in polypeptide II conformation. We further document that the conformational preferences of a residue are substantially influenced by the conformation of its neighbors, and we suggest that accounting for these dependencies will improve protein modeling accuracy. Although the CUEVAS-4D(r₁₀?₁₄) ‘parts list’ presented here is only an initial exploration of the complex (φ,ψ)₂ landscape of proteins, it shows that there is value to be had from this approach, and it opens the door to more in-depth characterizations at the (φ,ψ)₂ level and at higher dimensions.     

A conformational study of the tetrapeptide CH3CO-Ala-Asp-Gly-Lys-NHCH3 corresponding to a β-bend in staphylococcal nuclease

The tetrapeptide sequence Ala-Asp-Gly-Lys occurs as a type I′ β-bend at residues 94–97 in staphylococcal nuclease. We have synthesized theN-acetyl,N′-methylamide derivative of this tetrapeptide and studied its conformation in solution, using nuclear magnetic resonance (NMR) and circular dichroism (CD) spectroscopy. In the synthesis, special attention was paid to the possibility of cyclic aspartimide formation giving rise to mixtures of α- and β-Asp-Gly products. The presence of such a mixture was excluded by infrared, NMR, and other analytical procedures applied to the products and to models for α- and β-linked aspartyl residues. The CD spectra of the protected tetrapeptide in water, methanol, and trifluoroethanol show no evidence of preferred chain conformations. In dimethylsulfoxide-d ₆ , however, the NMR spectra are consistent with the presence of a population of conformers in which the Lys and C-terminal NHCH₃ amide protons are shielded from solvent. Taken together with the observed³J_NH-C ^α _H coupling constants for all residues, this permitted the construction and energetic evaluation of possible conformations in solution. Only one such conformation was fully compatible with the NMR data; this is a type II β-bend in which the Lys and C-terminal NHCH₃ amide protons are close to the Ala C=O group and may form bifurcated hydrogen bonds with it. This conformation can be converted into the conformation existing in staphylococcal nuclease by rotating the plane of the Ala-Asp peptide group by about 120° around a line connecting the Ala and Asp C_α atoms and by making small shifts in dihedral angles elsewhere in the peptide.     

Solution structure of the first and second transmembrane segments of the mitochondrial oxoglutarate carrier

The structures of the first and the second transmembrane segment of the bovine mitochondrial oxoglutarate carrier (OGC) were studied by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopies. Peptides 21–46 and 78–108 of its primary sequence were synthesized and structurally characterized in membrane-mimetic environments. CD data showed that at high concentrations of TFE (>50%) and SDS (>2%) both peptides assume α-helical structures, whereas in more hydrophilic environments only peptide 78–108 has a helical structure. ¹H-NMR spectra of the two peptides in TFE/water and SDS were fully assigned, and the secondary structures of the peptides were obtained from nuclear Overhauser effects, ³J_αH-NH coupling constants and αH chemical shifts. The three-dimensional solution structures of the peptides in TFE/water were generated by distance geometry calculations. A well-defined α-helix was found in the region K24-V39 of peptide 21–46 and in the region A86–F106 of peptide 78–108. We cannot exclude that in intact OGC the extension of these helices is longer. The helix of peptide 21–46 is essentially hydrophobic, whereas that of peptide 78–108 is predominantly hydrophilic.     

Solution structure of the fifth and sixth transmembrane segments of the mitochondrial oxoglutarate carrier

The structures of the fifth and sixth transmembrane segments of the bovine mitochondrial oxoglutarate carrier (OGC) and of the hydrophilic loop that connects them were studied by CD and NMR spectroscopies. Peptides F215-R246, W279-K305 and P257-L278 were synthesized and structurally characterized. CD data showed that at high concentrations of TFE and SDS all peptides assume α-helical structures. ¹H-NMR spectra of the three peptides in TFE/water were fully assigned and the secondary structures of the peptides were obtained from nuclear Overhauser effects, ³J_αH-NH coupling constants and αH chemical shifts. The three-dimensional solution structures of the peptides were generated by distance geometry calculations. A well-defined α–helix was found in the region L220-V243 of peptide F215-R246 (TMS-V), in the region P284-M303 of peptide W279-K305 (TMS-VI) and in the region N261-F275 of peptide P257-L278 (hydrophilic loop). The helix L220-V243 exhibited a sharp kink at P239, while a little bend around P291 was observed in the helical region P284-M303. Fluorescence studies performed on peptide W279-K305, alone and together with other transmembrane segments of OGC, showed that the W279 fluorescence was quenched upon addition of peptide F215-R246, but not of peptides K21-K46, R78-R108 and P117-A149 suggesting a specific interaction between TMS-V and TMS-VI of OGC.     

Conformation of malformin A: a proton magnetic resonance and a circular dichroism study

Malformin A is a cyclic pentapeptide with an intramolecular disulfide bridge. The conformation in solution of this molecule has been studied by NMR and CD. The 270 MHz Proton spectrum in dimethyl sulfoxide is well resolved and the peaks corresponding to the five residues have been assigned. From the temperature dependence of chemical shifts of the peptide protons and from the exchange rate of these protons, it is concluded that the NH proton of one Cys is shielded from the solvent. This observation and H? N? _αC? H angles, estimated from the corresponding coupling constants, a proposed conformation of the peptide backbone. From the H? _βC? _αC? H coupling constants, a P chirality for the disulfide bridge is proposed. Such a conformation is confirmed by the circular dichroism spectrum which shows a negative band at λ > 250 nm. It is concluded that the conformation of malformin A is rigid and that the disulfide bridge is exposed to interact with biological receptors.     

Solution conformation study of substance P methyl ester and [Nle10]-neurokinin A (4–10) by nmr spectroscopy

High-resolution proton spectra at 500 MHz of two tachykinin peptides, substance P methyl ester (SPOMe) and [Nle¹⁰]-neurokinin A (4–10), have been obtained in dimethylsulfoxide (DMSO), and for SPOMe, also in 2, 2, 2-trifluoroethanol (TFE)/water mixtures. Complete chemical shift assignments for these peptides were made based on two-dimensional (2D) nmr techniques, correlated spectroscopy and total COSY. J coupling measurement and nuclear Overhauser effect spectroscopy (NOESY) were then used to determine the conformation of these peptides in the various solvents. Based on the J coupling, NOE correlations, and temperature coefficients of the NH resonances, it is concluded that these two peptides exist in DMSO at room temperature as a mixture of conformers that are primarily extended. For SPOMe in TFE/water with high TFE content, however, helical structures are found to be present, and they become quite clear at temperatures between 270 and 280 K. The variation of the ¹³C chemical shifts of the C_α (the secondary shift) with TFE contents corroborates this conclusion. The NOE and C_α shifts show that the main helical region for SPOMe lies between ⁴P and ⁹G. The C-terminus segment L? M? NH₂ is found to be quite flexible, which appears to be quite common for neurokinin-1 selective peptides. © 1993 John Wiley & Sons, Inc.     

Solution structure of human calcitonin in membrane-mimetic environment: The role of the amphipathic helix

The 32 amino acid hormone human calcitonin was studied at pH 3.7 and 7.4 by multidimensional NMR spectroscopy in sodium dodecyl sulfate micelles at 310K. The secondary structure was obtained from nuclear Overhauser enhancement spectroscopy (NOESY), ³J_HNα coupling constants, and slowly exchanging amide data. Three-dimensional structures consistent with NMR data were generated by using distance geometry calculations. A set of 265 interproton distances derived from NOESY experiments, hydrogen-bond constraints obtained from amide exchange, and coupling constants were used. From the initial random conformations, 30 distance geometry structures with minimal violations were selected for further refinement with restrained energy minimization. In micelles, at both pHs, the hormone assumes an amphipathic α-helix from Leu9 to Phe16, followed by a type-I β-turn between residues Phe16 and Phe19. From His20 onward the molecule is extended and no interaction with the helix was observed. The relevance of the amphipathic helix for the structure–activity relationship, the possible mechanisms of interaction with the receptor, as well as the formation of fibrillar aggregates, is discussed. Proteins 32:314–323, 1998. © 1998 Wiley-Liss, Inc.     

Synthesis and conformational analysis of carbasugar bioisosteres of α-l-iduronic acid and its methyl glycoside

The synthesis of two novel carbasugar analogues of α-l-iduronic acid is described in which the ring-oxygen is replaced by a methylene group. In analogy with the conformational equilibrium described for α-l-IdopA, the conformation of the carbasugars was investigated by ¹H and ¹³C NMR spectroscopy. Hadamard transform NMR experiments were utilised for rapid acquisition of ¹H,¹³C-HSQC spectra and efficient measurements of heteronuclear long-range coupling constants. Analysis of ¹H NMR chemical shifts and J_H,H coupling constants extracted by a total-lineshape fitting procedure in conjunction with J_H,C coupling constants obtained by three different 2D NMR experiments, viz., ¹H,¹³C-HSQC-HECADE, J-HMBC and IPAP-HSQC-TOCSY-HT, as well as effective proton-proton distances from 1D ¹H,¹H T-ROE and NOE experiments showed that the conformational equilibrium ⁴C₁?²S_5a?¹C₄ is shifted towards ⁴C₁ as the predominant or exclusive conformation. These carbasugar bioisosteres of α-l-iduronic acid do not as monomers show the inherent flexibility that is anticipated to be necessary for biological activity.     

Conformational analysis of the type II and type III collagen α-1 chain N-telopeptides by 1H-NMR spectroscopy and restrained molecular mechanics calculations

The type II and type III collagen α-1 chain N-telopeptides are a nonadecamer with the sequence pEMAGGFDEKAGGAQLGVMQ-NH₂ and a tetradecamer with the sequence pEYEAYDVKSGVAGG-NH₂, respectively. Their conformations have been studied in CD₃OH/H₂O (60/40) solution by means of two-dimensional proton nmr spectroscopy. Based on double quantum filtered correlation spectroscopy, total correlation spectroscopy, rotating frame nuclear Overhauser enhancement (ROE) spectroscopy, and nuclear Over-hauser enhancement (NOE) spectroscopy experiments, all resonances were assigned and the conformational properties were analyzed in terms of vicinal NH-H_α coupling constants, sequential and medium-range NOEs (ROEs), and amide proton temperature coefficients. The NOE distance constraints as well as dihedral constraints based on the vicinal NH-H_α coupling constants were used as input parameters for restrained molecular mechanics, consisting of restrained molecular dynamics and restrained energy minimization calculations. The type II N-telopeptide's conformation is dominated by a fused βγ-turn between Phe⁶ and Ala¹⁰, stabilized by three hydrogen bonds and a salt bridge between the side-chain end groups of Glu⁸ and Lys⁹. The first 5 amino acids are extended with a much higher degree of conformational freedom. The 2 Gly residues following the turns were found to be highly flexible (hinge-like), leaving the spatial position of the second half of the molecule relative to the fused βγ-turn undefined. In the type III telopeptide, a series of sequential NH(i)-NH(i + 1) ROEs were observed between the amino acids Tyr² and Ser⁹, indicating that a fraction of the conformational space is helical. However, the absence of medium-range ROEs and the lack of regularity of the effects associated with α-helices suggest the presence of a nascent rather than a complete helix. © 1993 John Wiley & Sons, Inc.     

Conformational analysis of short polar side-chain amino-acids through umbrella sampling and DFT calculations

Molecular and quantum mechanics calculations were carried out in a series of tripeptides (GXG, where X?=?D, N and C) as models of the unfolded states of proteins. The selected central amino acids, especially aspartic acid (D) and asparagine (N) are known to present significant average conformations in partially allowed areas of the Ramachandran plot, which have been suggested to be important in unfolded protein regions. In this report, we present the calculation of the propensity values through an umbrella sampling procedure in combination with the calculation of the NMR J-coupling constants obtained by a DFT model. The experimental NMR observations can be reasonably explained in terms of a conformational distribution where PP_II and β basins sum up propensities above 0.9. The conformational analysis of the side chain dihedral angle (χ₁), along with the computation of ³J(H^αH^β), revealed a preference for the g _? and g ₊ rotamers. These may be connected with the presence of intermolecular H-bonding and carbonyl–carbonyl interactions sampled in the PP_II and β basins. Taking into account all those results, it can be established that these residues show a similar behavior to other amino acids in short peptides regarding backbone φ,ψ dihedral angle distribution, in agreement with some experimental analysis of capped dipeptides.     

Structural and positional studies of the antimicrobial peptide brevinin‐1BYa in membrane‐mimetic environments

Brevinin‐1BYa (FLPILASLAAKFGPKLFCLVTKKC), first isolated from skin secretions of the foothill yellow‐legged frog Rana boylii, shows broad‐spectrum activity, being particularly effective against opportunistic yeast pathogens. The structure of brevinin‐1BYa was investigated in various solution and membrane‐mimicking environments by proton nuclear magnetic resonance (¹H‐NMR) spectroscopy and molecular modelling. The peptide does not possess a secondary structure in aqueous solution. In a 33% 2,2,2‐trifluoroethanol (TFE‐d₃)‐H₂O solvent mixture, as well as in membrane‐mimicking sodium dodecyl sulfate and dodecylphosphocholine micelles, the peptide's structure is characterised by a flexible helix‐hinge‐helix motif, with the hinge located at the Gly¹³/Pro¹⁴ residues, and the two α‐helices extending from Pro³ to Phe¹² and from Pro¹⁴ to Thr²¹. Positional studies involving the peptide in sodium dodecyl sulfate and dodecylphosphocholine micelles using 5‐doxyl‐labelled stearic acid and manganese chloride paramagnetic probes show that the peptide's helical segments lie parallel to the micellar surface, with the residues on the hydrophobic face of the amphipathic helices facing towards the micelle core and the hydrophilic residues pointing outwards, suggesting that the peptide exerts its biological activity by a non–pore‐forming mechanism.     

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% H(2)0 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.     

Identification of initiation sites for T4 lysozyme folding using CD and NMR spectroscopy of peptide fragments

Using CD and 2D (1)H NMR spectroscopy, we have identified potential initiation sites for the folding of T4 lysozyme by examining the conformational preferences of peptide fragments corresponding to regions of secondary structure. CD spectropolarimetry showed most peptides were unstructured in water, but adopted partial helical conformations in TFE and SDS solution. This was also consistent with the (1)H NMR data which showed that the peptides were predominantly disordered in water, although in some cases, nascent or small populations of partially folded conformations could be detected. NOE patterns, coupling constants, and deviations from random coil Halpha chemical shift values complemented the CD data and confirmed that many of the peptides were helical in TFE and SDS micelles. In particular, the peptide corresponding to helix E in the native enzyme formed a well-defined helix in both TFE and SDS, indicating that helix E potentially forms an initiation site for T4 lysozyme folding. The data for the other peptides indicated that helices D, F, G, and H are dependent on tertiary interactions for their folding and/or stability. Overall, the results from this study, and those of our earlier studies, are in agreement with modeling and HD-deuterium exchange experiments, and support an hierarchical model of folding for T4 lysozyme.     

Heterochiral Ala/(αMe)Aze sequential oligopeptides: Synthesis and conformational study

α‐Amino acid residues with a ?,ψ constrained conformation are known to significantly bias the peptide backbone 3D structure. An intriguing member of this class of compounds is (αMe)Aze, characterized by an N^α‐alkylated four‐membered ring and C^α‐methylation. We have already reported that (S)‐(αMe)Aze, when followed by (S)‐Ala in the homochiral dipeptide sequential motif ‐(S)‐(αMe)Aze‐(S)‐Ala‐, tends to generate the unprecedented γ‐bend ribbon conformation, as formation of a regular, fully intramolecularly H‐bonded γ‐helix is precluded, due to the occurrence of a tertiary amide bond every two residues. In this work, we have expanded this study to the preparation and 3D structural analysis of the heterochiral (S)‐Ala/(R)‐(αMe)Aze sequential peptides from dimer to hexamer. Our conformational results show that members of this series may fold in type‐II β‐turns or in γ‐turns depending on the experimental conditions.     

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.     

A CD and an nmr study of multiple bradykinin conformations in aqueous trifluoroethanol solutions

CD and nmr studies have been carried out on aqueous trifluoroethanol (TFE) solutions of bradykinin (BK) and a bradykinin antagonist. The CD results exhibit a striking effect of TFE on the spectra of BK, with sequence Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg, and the BK antagonist, with sequence D -Arg-Arg-Pro-Hyp-Gly-Thi-D -Ser-D -Cpg-Cpg-Arg [where Hyp is 4-hydroxy-L -proline; Thi refers to β-(2-thienyl)-L -alanine and Cpg refers to α-cyclopentylglycine]. The effect of increasing concentration of TFE in water on the difference ellipticity at 222 nm was examined and showed that BK may be a mixture of at least two different conformers, one of which largely forms when the TFE concentration is increased beyond 80%. The linear extrapolation of 100% of the difference ellipticity of BK at low TFE concentrations yields a value in agreement with that shown by the BK antagonist, indicating that the conformation of BK at the lower TFE concentrations is similar to that of the BK antagonist. The conformational analysis was carried out using both one-dimensional and two-dimensional ¹H-nmr techniques. The total correlation spectroscopy (TOCSY) spectrum of BK in a 60/40% (v/v) TFE/H₂O solution at 10°C and a nuclear Overhauser effect spectroscopy (NOESY) spectrum that shows only sequential H_α(i) – NH(i + 1) or the H_α(i) – H_δδ′(i + 1) NOEs indicate that the majority of the molecules adopt an all-trans extended conformation. The TOCSY for BK in the 95/5% (v/v) TFE/H₂O solution shows that there are two major conformations in the solution with about equal population. The NOESY experiment shows two new important cross peaks for one conformation, namely Pro²(α)-Pro³ (α) and the Pro²(α)-Gly⁴(NH), indicating a cis Pro²-Pro³ bond and a type VI β-turn between residues Arg¹ and Gly⁴ involving cis proline at position 3, respectively. The low temperature coefficient of Gly⁴ for this conformation suggests the presence of an intramolecular hydrogen bond, therefore a type VIa β-turn is present. The other conformation is all trans and extended. The BK antafonist shows difference CD spectra in TFE solutions referred to H₂O that are superficially indicative of a β-bend. However, nmr speaks against this possibility, as only one set of peaks were observed in the TOCSY and NOESY experiments, indicating an all-trans extended confirmation over the range of TFE concentrations. The BK-antagonist CD data suggest that solvent perturbation of the CD of an extended confirmation perturbation of the optical activity of the thienyl moiety of the peptide since the CD spectrum of N-acetyl-β-thienyl-L -alanine N-methylamide is strongly perturbed by TFE. The present results again demonstrate the complementary relationship between CD and nmr. © 1994 John Wiley & Sons, Inc.