Three-dimensional structure of the mammalian tachykinin peptide neurokinin A bound to lipid micelles

The solution structure of NKA, a decapeptide of mammalian origin, has been characterized by CD spectropolarimetry and 2D proton nuclear magnetic resonance (2D 1H-NMR) spectroscopy in both aqueous and membrane mimetic solvents. Unambiguous NMR assignments of protons have been made with the aid of correlation spectroscopy (DQF-COSY and TOCSY) experiments and nuclear Overhauser effect spectroscopy (NOESY and ROESY) experiments. The distance constraints obtained from the NMR data have been utilized to generate a family of structures, which have been refined using restrained energy minimization and dynamics. These data show that in water NKA prefers to be in an extended chain conformation whereas a helical conformation is induced in the central core and the C-terminal region (D4-M10) of the peptide in the presence of perdeuterated dodecylphosphocholine (DPC) micelles, a membrane model system. Though less defined the N-terminus also displays some degree of order and a possible turn structure. The conformation adopted by NKA in the presence of DPC micelles represents a structural motif typical of neurokinin-2 selective agonists and is similar to that reported for eledoisin in hydrophobic environment.     

Three dimensional structure of mammalian tachykinin peptide neurokinin B bound to lipid micelles

Neurokinin B (NKB), a decapeptide of mammalian origin exhibits a variety of biological activities such as regulatory functions in reproduction, pre-eclampsia and neuroprotection in Alzheimer's disease. In order to gain insight into structure-function relationship, three-dimensional structure of NKB has been investigated using CD spectropolarimetry and two-dimensional proton nuclear magnetic resonance (2D 1H-NMR) spectroscopy in aqueous and membrane mimetic solvents. Unambiguous NMR assignments of resonances have been made with the aid of correlation spectroscopy (DQF-COSY and TOCSY) experiments and Nuclear Overhauser Effect Spectroscopy (NOESY) experiments. Distance constraints obtained from the NMR data have been used to generate a family of structures, which have been refined using restrained energy minimization and dynamics. Our data show that a helical structure is induced in NKB, in presence of perdeuterated dodecyl phosphocholine (DPC) micelles, a membrane model system. Further, the conformation adopted by NKB in presence of DPC micelles represents a structural motif typical of neurokinin-3 selective agonists.     

Conformational solution studies of neuropeptide gamma using CD and NMR spectroscopy.

Neuropeptide gamma is one of the largest members of the tachykinin family of peptides, exhibiting strong agonistic activity towards the NK-2 tachykinin receptor. This peptide was synthesized by the solid-phase method using the Fmoc chemistry. Circular-dichroism spectroscopy (CD) investigations of this peptide were performed in phosphate buffer, in the presence of sodium dodecylsulphate (SDS) micelles and trifluoroethanol (TFE) solutions and in DMSO-d6 using the 2D NMR technique in conjunction with two different theoretical approaches. The first assumes multiconformational equilibrium of the peptide studied characterized by the values of statistical weights of low-energy conformations. These calculations were performed using three different force fields ECEPP/3, AMBER4.1 and CHARMM (implemented in the X-PLOR program). The second method incorporates interproton distance and dihedral angle constraints into the starting conformation using the Simulated Annealing algorithm (X-PLOR program). The CD experiments revealed that although the peptide studied is flexible in polar solvents, a tendency to adopt a helical structure was observed in the hydrophobic environment. The NMR data (NOE effects) indicate a helical or reverse structure in the Ile7-His12 fragment of the peptide studied in DMSO-d6 solution. The results obtained cannot be interpreted in terms of a single conformation. Most of the conformations obtained with the ECEPP/3 force field possess a high content of a helical structure. None of the conformers, obtained with the AMBER4.1 and CHARMM force fields, can be considered as the dominant one. In all conformations several beta-turns were detected and in some cases gamma-turns were also found. But in fact, it is rather difficult to select the position of the secondary element(s) present in the structure of NPgamma in solution. All conformers calculated with the X-PLOR program (with using NMR derived distance and torsion angle constraints) are stabilized by several beta-turns. Common structural motives are a type IV beta-turn in the Gln6-His12 fragment. All conformations obtained using two approaches adopt very similar turn shapes in the middle region of molecule and a random structure on the N- and C-terminal fragments.     

Membrane-Induced Structure of Scyliorhinin I: A Dual NK1/NK2 Agonist

Scyliorhinin I, a linear decapeptide, is the only known tachykinin that shows high affinity for both NK-1 and NK-2 binding sites and low affinity for NK-3 binding sites. As a first step to understand the structure-activity relationship, we report the membrane-induced structure of scyliorhinin I with the aid of circular dichroism and 2D-(1)H NMR spectroscopy. Sequence specific resonance assignments of protons have been made from correlation spectroscopy (TOCSY, DQF-COSY) and NOESY spectroscopy. The interproton distance constraints and dihedral angle constraints have been utilized to generate a family of structures using DYANA. The superimposition of 20 final structures has been reported with backbone pairwise root mean-square deviation of 0.38 +/- 0.19 A. The results show that scyliorhinin I exists in a random coil state in aqueous environments, whereas helical conformation is induced toward the C-terminal region of the peptide (D4-M10) in the presence of dodecyl phosphocholine micelles. Analysis of NMR data is suggestive of the presence of a 3(10)-helix that is in equilibrium with an alpha-helix in this region from residue 4 to 10. An extended highly flexible N-terminus of scyliorhinin I displays some degree of order and a possible turn structure. Observed conformational features have been compared with respect to that of substance P and neurokinin A, which are endogenous agonists of NK-1 and NK-2 receptors, respectively.     

Three-dimensional structure of neuropeptide k bound to dodecylphosphocholine micelles

Neuropeptide K (NPK), an N-terminally extended form of neurokinin A (NKA), represents the most potent and longest lasting vasodepressor and cardiomodulatory tachykinin reported thus far. NPK has been shown to have high selectivity for the NK2 receptor. Because the micelle-associated structure may be relevant to the NPK-receptor interaction, the three-dimensional structure of the NPK in aqueous and micellar environments has been studied by two-dimensional proton nuclear magnetic resonance (2D (1)H NMR spectroscopy) and distance geometry calculations. Proton NMR assignments have been carried out with the aid of correlation spectroscopy (DQF-COSY and TOCSY) and nuclear Overhauser effect spectroscopy (NOESY and ROESY) experiments. The interproton distances and dihedral angle constraints obtained from the NMR data have been used in torsion angle dynamics algorithm for NMR applications (DYANA) to generate a family of structures, which have been refined using restrained energy minimization and dynamics. The results show that in an aqueous environment NPK lacks a definite secondary structure, although some turn-like elements are present in the N terminus. The structure is well-defined in the presence of dodecylphosphocholine micelles. The global fold of NPK bound to DPC micelles consists of two well-defined helices from residues 9 to 18 and residues 27 to 33 connected by a noncanonical beta turn. The N terminus of the peptide is characterized by a 3(10) helix or a series of dynamic beta turns. The conformational range of the peptide revealed by NMR and circular dichroism (CD) studies has been analyzed in terms of characteristic secondary features. The observed conformational features have been further compared to a NKA and neuropeptide gamma (NPgamma) potent endogenous agonist for the NK2 receptor.     

Three Dimensional Structure of Mammalian Tachykinin Peptide Neurokinin B Bound to Lipid Micelles

Abstract

Neurokinin B (NKB), a decapeptide of mammalian origin exhibits a variety of biological activities such as regulatory functions in reproduction, pre-eclampsia and neuroprotection in Alzheimer's disease. In order to gain insight into structure-function relationship, three- dimensional structure of NKB has been investigated using CD spectropolarimetry and two-dimensional proton nuclear magnetic resonance (2D ¹H-NMR) spectroscopy in aqueous and membrane mimetic solvents. Unambiguous NMR assignments of resonances have been made with the aid of correlation spectroscopy (DQF-COSY and TOCSY) experiments and Nuclear Overhauser Effect Spectroscopy (NOESY) experiments. Distance constraints obtained from the NMR data have been used to generate a family of structures, which have been refined using restrained energy minimization and dynamics. Our data show that a helical structure is induced in NKB, in presence of perdeuterated dodecyl phosphocholine (DPC) micelles, a membrane model system. Further, the conformation adopted by NKB in presence of DPC micelles represents a structural motif typical of neurokinin-3 selective agonists.     

Lipid induced conformation of the tachykinin peptide Kassinin

Both the aqueous and lipid-induced structure of Kassinin, a dodecapeptide of amphibian origin, has been studied by two-dimensional proton nuclear magnetic resonance (2D 1H-NMR) spectroscopy and distance geometry calculations. Unambiguous NMR assignments of protons have been made with the aid of correlation spectroscopy (DQF-COSY and TOCSY) experiments and nuclear Overhauser effect spectroscopy (NOESY and ROESY) experiments. The distance constraints obtained from the NMR data have been utilized in a distance geometry algorithm to generate a family of structures, which have been refined using restrained energy minimization and dynamics. These data show that, while in water Kassinin prefers to be in an extended chain conformation, in the presence of perdeuterated dodecylphosphocholine (DPC) micelles, a membrane model system, helical conformation is induced in the central core and C-terminal region (K4-M12) of the peptide. N-terminus though less defined also displays some degree of order and a possible turn structure. The conformation adopted by Kassinin in the presence of DPC micelles is consistent with the structural motif typical of neurokinin-1 selective agonists and with that reported for Eledoisin in hydrophobic environment.     

Protein Folding: A Few Random Thoughts

Abstract

Both the aqueous and lipid-induced structure of Kassinin, a dodecapeptide of amphibian origin, has been studied by two-dimensional proton nuclear magnetic resonance (2D ¹H-NMR) spectroscopy and distance geometry calculations. Unambiguous NMR assignments of protons have been made with the aid of correlation spectroscopy (DQF-COSY and TOCSY) experiments and nuclear Overhauser effect spectroscopy (NOESY and ROESY) experiments. The distance constraints obtained from the NMR data have been utilized in a distance geometry algorithm to generate a family of structures, which have been refined using restrained energy minimization and dynamics. These data show that, while in water Kassinin prefers to be in an extended chain conformation, in the presence of perdeuterated dodecylphosphocholine (DPC) micelles, a membrane model system, helical conformation is induced in the central core and C-terminal region (K4-M12) of the peptide. N-terminus though less defined also displays some degree of order and a possible turn structure. The conformation adopted by Kassinin in the presence of DPC micelles is consistent with the structural motif typical of neurokinin-1 selective agonists and with that reported for Eledoisin in hydrophobic environment.     

Solution structure of the tachykinin peptide eledoisin

Both the aqueous and the lipid-induced structure of eledoisin, an undecapeptide of mollusk origin, have been studied by two-dimensional proton nuclear magnetic resonance spectroscopy and distance geometry calculations. Unambiguous nuclear magnetic resonance assignments of protons have been made with the aid of correlation spectroscopy experiments and nuclear Overhauser effect spectroscopy experiments. The distance constraints obtained from the nuclear magnetic resonance data have been utilized in a distance geometry algorithm to generate a family of structures, which have been refined using restrained energy minimization and dynamics. These data show that, while in water and dimethyl sulfoxide, eledoisin prefers to be in an extended chain conformation, whereas in the presence of perdeuterated dodecylphosphocholine micelles, a membrane model system, helical conformation is induced in the central core and C-terminal region (K4-M11) of the peptide. N terminus, though less defined, also displays some degree of order and a possible turn structure. The conformation adopted by eledoisin in the presence of dodecylphosphocholine micelles is similar to the structural motif typical of neurokinin-2 selective agonists and with that reported for kassinin in hydrophobic environment.     

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.     

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.     

NMR studies of the low-density lipoprotein receptor-binding peptide of apolipoprotein E bound to dodecylphosphocholine micelles.

Circular dichroism and NMR spectroscopy have been used to determine the structure of the low-density lipoprotein (LDL) receptor-binding peptide, comprising residues 130-152, of the human apolipoprotein E. This peptide has little persistent three-dimensional structure in solution, but when bound to micelles of dodecylphosphocholine (DPC) it adopts a predominantly alpha-helical structure. The three-dimensional structure of the DPC-bound peptide has been determined by using 1H-NMR spectroscopy: the structure derived from NOE-based distance constraints and restrained molecular dynamics is largely helical. The derived phi and psi angle order parameters show that the helical structure is well defined but with some flexibility that causes the structures not to be superimposable over the full peptide length. Deuterium exchange experiments suggest that many peptide amide groups are readily accessible to the solvent, but those associated with hydrophobic residues exchange more slowly, and this helix is thus likely to be positioned on the surface of the DPC micelles. In this conformation the peptide has one hydrophobic face and two that are rich in basic amino acid side chains. The solvent-exposed face of the peptide contains residues previously shown to be involved in binding to the LDL receptor.     

Characterization of the bioactive conformation of the C-terminal tripeptide Gly-Leu-Met-NH2 of substance P using [3-prolinoleucine10]SP analogues.

Residue Leu10 of substance P (SP) is critical for NK-1 receptor recognition and agonist activity. In order to probe the bioactive conformation of this residue, cis- and trans-3-substituted prolinoleucines were introduced in position 10 of SP. The substituted SP analogues were tested for their affinity to human NK-1 receptor specific binding sites (NK-1M and NK-1m) and their potency to stimulate adenylate cyclase and phospholipase C in CHO cells transfected with the human NK-1 receptor. [trans-3-prolinoleucine10]SP retained affinity and potency similar to SP whereas [cis-3-prolinoleucine10]SP shows dramatic loss of affinity and potency. To analyze the structural implications of these biological results, the conformational preferences of the SP analogues were analyzed by NMR spectroscopy and minimum-energy conformers of Ac-cis-3-prolinoleucine-NHMe, Ac-trans-3-prolinoleucine-NHMe and model dipeptides were generated by molecular mechanics calculations. From NMR and modeling studies it can be proposed that residue Leu10 of SP adopts a gauche(+) conformation around the chi1 angle and a trans conformation around the chi2 angle in the bioactive conformation. Together with previously published results, our data indicate that the C-terminal SP tripeptide should preferentially adopt an extended conformation or a PPII helical structure when bound to the receptor.     

Circular dichroism studies on a synthetic peptide corresponding to the membrane-spanning region of vesicular stomatitis virus G protein and its fatty acyl derivative

The conformations of synthetic peptides Lys-Phe-Phe-Phe-Ile-Ile-Gly-Leu-Ile-Ile-Gly-Leu-Phe-OCH3 and Lys(epsilon-palmitoyl)-Phe-Phe-Phe-Ile-Ile-Gly-Leu-Ile-Ile-Gly-Leu-Phe-O CH3, which constitute a part of the membrane-spanning region of the vesicular stomatitis virus G protein, have been studied by circular dichroism (CD) spectroscopy. Secondary structural features are observed for both peptides in trifluoroethanol, methanol, aqueous mixtures of trifluoroethanol and methanol and in a micellar environment. In trifluoroethanol, the CD spectra indicate the presence of a helical conformation, whereas in aqueous mixtures of organic solvents, both helical and beta-conformations are observed. While fatty acid acylation does not directly modulate peptide conformation, it promotes self-association of the acylated peptide and association with micelles. In a micellar environment, the acylated peptide adopts an alpha-helical conformation.     

Synthesis,biological activity,and conformational analysis of [pGlu6, N-MePhe8, Aib9] substance P (6–11): A selective agonist for the NK-3 receptor

A highly potent and selective agonist to the tachykinin NK-3 receptor, [pGlu⁶, N-MePhe⁸, Aib⁹] substance P (6–11) ( I ), was synthesized via the solid phase method. The ED₅₀ of I was 4n M in the guinea pig ileum in the absence of atropine (NK-1 + NK-3 receptors) and this agonist was 5000-fold less potent in the presence of atropine (NK-1 receptor). The analogue was virtually inactive in the rat vas deferens (NK-2 receptor). A detailed analysis of the solution conformation of this analogue in DMSO-d₆ and in a DMSO-d₆ H₂O cryornixture was carried out by a combination of ¹H-nmr 2D techniques (DQF-COSY, TOCSY, NOESY and ROESY) and model building based on empirical energy calculations. Peptide I exists as a mixture of isomers containing cis and trans Phe-N-MePhe peptide bonds. The main isomer, containing a cis Phe-N-MePhe peptide bond, shows a preferred folded conformation characterized by a type VI β-turn with Phe and N-MePhe in the i + 1 and i + 2 positions. The turn is followed by a helical segment extending to the C-terminal. This conformation is compared to previously reported conformations of other selective tachykinin agonists and may be a promising lead for the design of novel NK-3 agonists with additional conformational constraints. © 1993 John Wiley & Sons, Inc.     

The application of DOSY NMR and molecular dynamics simulations to explore the mechanism(s) of micelle binding of antimicrobial peptides containing unnatural amino acids

Anionic and zwitterionic micelles are often used as simple models for the lipids found in bacterial and mammalian cell membranes to investigate antimicrobial peptide‐lipid interactions. In our laboratory we have employed a variety of 1D, 2D, and diffusion ordered (DOSY) NMR experiments to investigate the interactions of antimicrobial peptides containing unnatural amino acids with SDS and DPC micelles. Complete assignment of the proton spectra of these peptides is prohibited by the incorporation of a high percentage of unnatural amino acids which don't contain amide protons into the backbone. However preliminary assignment of the TOCSY spectra of compound 23 in the presence of both micelles indicated multiple conformers are present as a result of binding to these micelles. Chemical Shift Indexing agreed with previously collected CD spectra that indicated on binding to SDS micelles compound 23 adopts a mixture of α‐helical structures and on binding to DPC micelles this peptide adopts a mixture of helical and β‐turn/sheet like structures. DOSY NMR experiments also indicated that the total positive charge and the relative placement of that charge at the N‐terminus or C‐terminus are important in determining the mole fraction of the peptide that will bind to the different micelles. DOSY and ¹H‐NMR experiments indicated that the length of Spacer #1 plays a major role in defining the binding conformation of these analogs with SDS micelles. Results obtained from molecular simulations studies of the binding of compounds 23 and 36 with SDS micelles were consistent with the observed NMR results. © 2013 Wiley Periodicals, Inc. Biopolymers 99: 548–561, 2013.     

Solvent-induced beta-hairpin to helix conformational transition in a designed peptide

An octapeptide containing a central -Aib-Gly- segment capable of adopting beta-turn conformations compatible with both hairpin (beta(II') or beta(I')) and helical (beta(I)) structures has been designed. The effect of solvent on the conformation of the peptide Boc-Leu-Val-Val-Aib-Gly-Leu-Val-Val-OMe (VIII; Boc: t-butyloxycarbonyl; OMe: methyl ester) has been investigated by NMR and CD spectroscopy. Peptide VIII adopts a well-defined beta-hairpin conformation in solvents capable of hydrogen bonding like (CD(3))(2)SO and CD(3)OH. In solvents that have a lower tendency to interact with backbone peptide groups, like CDCl(3) and CD(3)CN, helical conformations predominate. Nuclear Overhauser effects between the backbone protons and solvent shielding of NH groups involved in cross-strand hydrogen bonding, backbone chemical shifts, and vicinal coupling constants provide further support for the conformational assignments in different solvents. Truncated peptides Boc-Val-Val-Aib-Gly-Leu-Val-Val-OMe (VII), Boc-Val-Val-Aib-Gly-Leu-Val-OMe (VI), and Boc-Val-Aib-Gly-Leu-OMe (IV) were studied in CDCl(3) and (CD(3))(2)SO by 500 MHz (1)H-NMR spectroscopy. Peptides IV and VI show no evidence for hairpin conformation in both the solvents. The three truncated peptides show a well-defined helical conformation in CDCl(3). In (CD(3))(2)SO, peptide VII adopts a beta-hairpin conformation. The results establish that peptides may be designed, which are poised to undergo a dramatic conformational transition.     

Conformational requirement of signal sequences functioning in yeast: circular dichroism and 1H nuclear magnetic resonance studies of synthetic peptides

Recently, we have designed a series of simplified artificial signal sequences and have shown that a proline residue in the signal sequence plays an important role in the secretion of human lysozyme in yeast, presumably by altering the conformation of the signal sequence [Yamamoto, Y., Taniyama, Y., & Kikuchi, M. (1989) Biochemistry 28, 2728-2732]. To elucidate the conformational requirement of the signal sequence in more detail, functional and nonfunctional signal sequences connected to the N-terminal five residues of mature human lysozyme were chemically synthesized and their conformations in a lipophilic environment [aqueous trifluoroethanol (TFE) or sodium dodecyl sulfate micelles] analyzed by circular dichroism (CD) and 1H nuclear magnetic resonance (NMR) spectroscopy. The helix content of the peptides, including functional (L8, CL10) and nonfunctional (L8PL, L8PG, L8PL2) signal sequences, was estimated from CD spectra to be 40-50% and 60-70%, respectively, indicating that the helical structure is more abundant in the nonfunctional signal sequences. Two-dimensional NMR analyses in 50% TFE/H2O revealed that each peptide adopted a helical conformation throughout the sequence except for a few residues at the N- and C-termini. Furthermore, H-D exchange experiments indicated that the helical structure of the C-terminal region of the functional signal sequences (L8 and CL10) was less stable than that of the nonfunctional signal sequences (L8PL and L8PL2). On the basis of these results, a model was developed in which the functional signal sequence is inserted in the membrane with a helical conformation and the C-terminal helix unraveled in an extended conformational form through an interaction with the signal peptidase.     

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

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 approximately 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 approximately 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/NHi+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.     

Solution structure of a novel ETB receptor selective agonist ET1-21 [Cys(Acm)1,15, Aib3,11, Leu7] by nuclear magnetic resonance spectroscopy and molecular modelling.

The solution structure of a biologically active modified linear endothelin-1 analogue, ET1-21[Cys(Acm)1,15, Aib3,11, Leu7], has been determined for the first time by two-dimensional nuclear magnetic resonance spectroscopy in a methanol-d3/water solvent mixture. Out of approximately one hundred linear peptide analogues tested by biological assay, this peptide, together with a dozen others, showed significant ETB selective agonist activity. Here we report the solution structure of an ETB selective agonist of a full-length, synthetic linear endothelin analogue. The calculated structures indicate that the peptide adopts an alpha-helical conformation between residues Ser5-His16, whilst both N- and C-termini show no preferred conformation. These results suggest that the disulphide bridges normally associated with endothelin and sarafotoxin peptides may not necessarily be important for either ETB receptor binding activity or the formation of a helical conformation in solution.