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.     

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.     

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.     

Membrane-induced structure of the mammalian tachykinin neuropeptide gamma

Neuropeptide gamma (NPgamma) is a neurokinin-2 (NK-2) receptor selective agonist, which plays an important role in mediation of asthma and elicits a wide range of biological responses like bronchoconstriction, vasodepression and regulation of endocrine functions. The structure determination of this peptide agonist is important in understanding the molecular basis of peptide ligand recognition by the receptor and for rational drug design. In the present study we report the solution structure of NPgamma characterized by circular dichroism (CD) spectropolarimetry and 2D (1)H NMR spectroscopy in both aqueous and membrane mimetic solvents. Effect of calcium ions on the conformation of NPgamma was also studied using CD spectropolarimetry. Sequence-specific 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 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 NPgamma prefers to be in an extended chain conformation whereas a helical conformation is induced in the central core and the C-terminal region of the peptide (K13-M21) in the presence of perdeuterated dodecylphosphocholine micelles, a membrane model system. A type II' beta turn from H9 to R11 precedes the helical core in the C-terminus of NPgamma. N-terminus of NPgamma also displays some degree of order and a possible turn structure. Conformation adopted by NPgamma in presence of lipid micelles represents a structural motif typical of NK-2 selective agonists and is similar to that observed for Neurokinin A in hydrophobic environment. The observed conformational features have been correlated to the binding ability and biological activity of NPgamma.     

Micelle-bound conformations of a bombesin/gastrin releasing peptide receptor agonist and an antagonist by two-dimensional NMR and restrained molecular dynamics.

Two nonapeptide analogs of the carboxyl termini of bombesin (Bn) and gastrin releasing peptide (GRP) have been synthesized. Despite the small difference in chemical composition between these peptides, one was a potent agonist and the other a potent antagonist of the Bn/GRP receptor in murine pancreas. All protons of both peptides, in dodecylphosphocholine micelles, were assigned by two-dimensional nuclear magnetic resonance spectroscopy. Interproton distance were derived from cross-peak volumes in nuclear Overhauser enhancement spectra. Conformations of both peptides were derived by distance-restrained molecular dynamics simulations using the interproton distances as constrains. The agonist conformation resembled a relaxed helix formed by three connected turns. The two N-terminal turns were similar for both peptides. The third turn of the agonist, at the carboxyl terminus, was absent in the antagonist. One interproton distance at the carboxyl terminus of the antagonist indicates that the chemical group connecting the last two residues of this peptide mimics a cis peptide bond geometry.     

Physicochemical studies of the protein-lipid interactions in melittin-containing micelles

Complexes of melittin with detergents and phospholipids have been characterized by fluorescence, circular dichroism, ultracentrifugation, quasi-elastic light scattering and 1H nuclear magnetic resonance (NMR) experiments. By ultracentrifugation and quasi-elastic light-scattering measurements it is shown that melittin forms stoichiometrically well-defined complexes with dodecylphosphocholine micelles consisting of one melittin molecule and approximately forty detergent molecules. Evidence from fluorescence, circular dichroism and 1H nuclear magnetic resonance experiments indicates that the conformation of melittin bound to micelles of various detergents or of diheptanoyl phosphatidylcholine is largely independent of the type of lipid and furthermore appears to be quite closely related to the conformation of melittin bound to phosphatidylcholine bilayers. 1H NMR is used to investigate the conformation of micelle-bound melittin in more detail and to compare certain aspects of the melittin conformation in the micelles with the spatial structures of monomeric and self-aggregated tetrameric melittin in aqueous solution. The experience gained with this system demonstrates that high resolution NMR of complexes of membrane proteins with micelles provides a viable method for conformational studies of membrane proteins.     

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.     

Three-dimensional structure of bradykinin in SDS micelles. Study using nuclear magnetic resonance, distance geometry, and restrained molecular mechanics and dynamics

The conformational properties of bradykinin in five molar excess sodium dodecyl sulfate (SDS) micelles have been examined by two-dimensional nuclear magnetic resonance (NMR) techniques at 500 MHz. Detailed structural information for bradykinin in SDS was obtained from quantitative 2-D nuclear Overhauser enhancement (n.O.e.) analyses, distance geometry, and restrained molecular mechanics and dynamics calculations. The conformation of bradykinin in SDS micelles, as determined by these methods, is characterized by a beta-turn-like structure at residues 6-9. A detailed comparison of the structures derived from distance geometry and restrained molecular mechanics and dynamics is also presented.     

Nuclear magnetic resonance investigation of the conformation of delta-haemolysin bound to dodecylphosphocholine micelles

delta-Haemolysin in mixed micelles with perdeuterated dodecylphosphocholine was investigated with two-dimensional proton nuclear magnetic resonance experiments at 500 MHz. A single set of resonance lines was observed for the micelle-bound polypeptide, indicating that delta-haemolysin adopts a single conformation in this environment. Nearly complete, sequence-specific assignments were obtained for the segment 5-23 of this 26-residue polypeptide chain. From the sequential connectivities and numerous medium-range nuclear Overhauser effects this central portion of the molecule was found to form an extended helix with pronounced amphipathic distribution of polar and nonpolar amino acid side-chains.     

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.     

Solution structures and model membrane interactions of Ctriporin,an anti‐methicillin‐resistant Staphylococcus aureus Peptide from Scorpion Venom

Ctriporin peptide (Ctr), a novel antimicrobial peptide isolated from the venom of the scorpion Chaerilus tricostatus, shows a broad‐spectrum of antimicrobial activity and is able to inhibit antibiotic resistant pathogens, including Methicillin resistant Staphylococcus aureus, Methicillin Resistant Coagulase‐negative Staphylococcus, and Penicillin Resistant Staphylococcus epidermidis strains. To understand the active conformation of the Ctr peptide in membranes, we have investigated the interaction of Ctr with the negatively charged and zwitterionic membrane‐mimetic micelles such as sodium dodecyl sulphate (SDS) and n‐dodecylphosphocholine (DPC), respectively. The interactions were studied using fluorescence and circular dichroism (CD) spectroscopy. Fluorescence experiments revealed that the N‐terminus tryptophan residue of Ctr interacted with the hydrophobic core of the membrane mimicking micelles. The CD results suggest that interactions with membrane‐mimetic micelles induce an α‐helix conformation in Ctr. Moreover, we have determined the solution structures of Ctr in SDS and DPC micelles using nuclear magnetic resonance (NMR) spectroscopy. The structural comparison of Ctr in the presence of SDS and DPC micelles showed significant conformational changes. The observed structural differences of Ctr in anionic versus zwitterionic membrane‐mimetic micelles suggest that the mode of interaction of this peptide may be different in two environments which may account for its ability to differentiate bacterial and eukaryotic cell membrane. © 2014 Wiley Periodicals, Inc. Biopolymers 101: 1143–1153, 2014.     

Nuclear magnetic resonance investigation of the conformation of δ-haemolysin bound to dodecylphosphocholine micelles

δ-Haemolysin in mixed micelles with perdeuterated dodecylphosphocholine was investigated with two-dimensional proton nuclear magnetic resonance experiments at 500 MHz. A single set of resonance lines was observed for the micelle-bound polypeptide, indicating that δ-haemolysin adopts a single conformation in this environment. Nearly complete, sequence-specific assignments were obtained for the segment 5–23 of this 26 residue polypeptide chain. From the sequential connectivities and numerous medium-range nuclear Overhauser effects this central portion of the molecule was found to form an extended helix with pronounced amphipathic distribution of polar and nonpolar amino acid side-chains.     

Physicochemical characterization of dodecylphosphocholine/palmitoyllysophosphatidic acid/myelin basic protein complexes.

The stoichiometry of dodecylphosphocholine/palmitoyllysophosphatidic acid/myelin basic protein complexes and the location of the protein in the micelles have been investigated by electron paramagnetic resonance, ultracentrifugation, small-angle X-ray scattering, 31P, 13C, and 1H nuclear magnetic resonance spectroscopy, and electron microscopy. Ultracentrifugation measurements indicated that well-defined complexes are formed by association of one protein molecule with approximately 133 detergent molecules. The spin-labels 5-, 12-, and 16-doxylstearate have been incorporated into detergent/protein aggregates. Electron paramagnetic resonance spectral parameters and 13C and 1H nuclear magnetic resonance relaxation times showed that the addition of myelin basic protein does not affect the environment and location of the labels or the organization of the micelles. Previous results suggesting that the protein lies primarily near the surface of the micelles have been confirmed by comparing 13C spectra of the detergents with and without protein with spectra of detergent/protein aggregates containing the spin labels. Electron micrographs of the complexes taken by using the freeze-fracture technique revealed the presence of particles with an estimated radius about three times the radius of the micelles measured by small-angle X-ray scattering. The structural integrity of the complexes appears to be based on intramolecular protein interactions as well as protein-detergent interactions.     

Structural Characterization of Neurokinin-3 Receptor Selective Peptide Agonist Scyliorhinin II Bound to DPC Micelles

Abstract

Scyliorhinin II, a cyclic Tachykinin peptide, is a potent NK3 receptor agonist. The pharmacology of NK3 receptor is least characterized out of the three tachykinin receptor subtypes cloned and characterized for Tachykinins. To understand the structural basis of peptide-receptor interaction, the three-dimensional structure of the Scyliorhinin II in aqueous and micellar environments has been studied by two-dimensional proton nuclear magnetic resonance (2D ¹H-NMR spectroscopy) and distance geometry calculations. Proton NMR assignments have been carried out with the aid of correlation spectroscopy (gradient-COSY and TOCSY) and nuclear Overhauser effect spectroscopy (NOESY and ROESY) experiments. The inter proton 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, Scyliorhinin II lacks a definite secondary structure. The structure is well-defined in presence of dodecyl phosphocholine micelles. The global fold of Scyliorhinin II bound to DPC micelles consists of a well-defined helix in the C-terminal region from residue 12–18 and a series of turns towards N-terminus. The structure is further stabilized by disulfide bond between Cys7 and Cys13. The conformational range of the peptide revealed by NMR and CD studies has been analyzed in terms of characteristic secondary features. Observed conformational features have been compared with those of Substance P, Neurokinin A and Neurokinin B, potent NK1, NK2, and NK3 agonists, respectively.     

Structural characterization of neurokinin-3 receptor selective peptide agonist scyliorhinin II bound to DPC micelles

Scyliorhinin II, a cyclic Tachykinin peptide, is a potent NK3 receptor agonist. The pharmacology of NK3 receptor is least characterized out of the three tachykinin receptor subtypes cloned and characterized for Tachykinins. To understand the structural basis of peptide-receptor interaction, the three-dimensional structure of the Scyliorhinin II in aqueous and micellar environments has been studied by two-dimensional proton nuclear magnetic resonance (2D 1H-NMR spectroscopy) and distance geometry calculations. Proton NMR assignments have been carried out with the aid of correlation spectroscopy (gradient-COSY and TOCSY) and nuclear Overhauser effect spectroscopy (NOESY and ROESY) experiments. The inter proton 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, Scyliorhinin II lacks a definite secondary structure. The structure is well-defined in presence of dodecyl phosphocholine micelles. The global fold of Scyliorhinin II bound to DPC micelles consists of a well-defined helix in the C-terminal region from residue 12-18 and a series of turns towards N-terminus. The structure is further stabilized by disulfide bond between Cys7 and Cys13. The conformational range of the peptide revealed by NMR and CD studies has been analyzed in terms of characteristic secondary features. Observed conformational features have been compared with those of Substance P, Neurokinin A and Neurokinin B, potent NK1, NK2, and NK3 agonists, respectively.     

Unique helical conformation of the fourth cytoplasmic loop of the CB1 cannabinoid receptor in a negatively charged environment

The proximal portion of the C-terminus of the CB(1) cannabinoid receptor is a primary determinant for G-protein activation. A 17 residue proximal C-terminal peptide (rodent CB1 401-417), the intracellular loop 4 (IL4) peptide, mimicked the receptor's G-protein activation domain. Because of the importance of the cationic amino acids to G-protein activation, the three-dimensional structure of the IL4 peptide in a negatively charged sodium dodecyl sulfate (SDS) micellar environment has been studied by two-dimensional proton nuclear magnetic resonance (2D (1)H NMR) spectroscopy and distance geometry calculations. Unambiguous proton NMR assignments were carried out with the aid of correlation spectroscopy (DQF-COSY and TOCSY) and nuclear Overhauser effect spectroscopy (NOESY and ROESY) experiments. The distance constraints were used in torsion angle dynamics algorithm for NMR applications (DYANA) to generate a family of structures which were refined using restrained energy minimization and dynamics. In water, the IL4 peptide prefers an extended conformation, whereas in SDS micelles, 3(10)-helical conformation is induced. The predominance of 3(10)-helical domain structure in SDS represents a unique difference compared with structure in alternative environments, which can significantly impact global electrostatic surface potential on the cytoplasmic surface of the CB(1) receptor and might influence the signal to the G-proteins.     

Pulmonary surfactant-associated polypeptide C in a mixed organic solvent transforms from a monomeric alpha-helical state into insoluble beta-sheet aggregates.

In the 35-residue pulmonary surfactant-associated lipopolypeptide C (SP-C), the stability of the valyl-rich alpha-helix comprising residues 9-34 has been monitored by circular dichroism, nuclear magnetic resonance, and Fourier transform infrared spectroscopy in both a mixed organic solvent and in phospholipid micelles. The alpha-helical form of SP-C observed in freshly prepared solutions in a mixed solvent of CHCl3/CH3OH/0.1 M HCl 32:64:5 (v/v/v) at 10 degrees C undergoes within a few days an irreversible transformation to an insoluble aggregate that contains beta-sheet secondary structure. Hydrogen exchange experiments revealed that this conformational transition proceeds through a transition state with an Eyring free activation enthalpy of about 100 kJ mol(-1), in which the polypeptide segment 9-27 largely retains a helical conformation. In dodecylphosphocholine micelles, the helical form of SP-C was maintained after seven weeks at 50 degrees C. The alpha-helical form of SP-C thus seems to be the thermodynamically most stable state in this micellar environment, whereas its presence in freshly prepared samples in the aforementioned mixed solvent is due to a high kinetic barrier for unfolding. These observations support a previously proposed pathway for in vivo synthesis of SP-C through proteolytic processing from a 21-kDa precursor protein.     

Combined use of proton-proton Overhauser enhancements and a distance geometry algorithm for determination of polypeptide conformations. Application to micelle-bound glucagon

In a new approach for the determination of polypeptide conformation, experimental data on intramolecular distances between pairs of hydrogen atoms obtained from nuclear Overhauser enhancement studies are used as input for a distance geometry algorithm. The algorithm determines the limits of the conformation space occupied by the polypeptide chain. The experimental data are used in such a way that the real conformation should in all cases be within these limits. Two important features of the method are that the results do not depend critically on the accuracy of the distance measurements by nuclear Overhauser enhancement studies and that internal mobility of the polypeptide conformation is explicitly taken into consideration. The use of this new procedure is illustrated with a structural study of the region 19-27 of glucagon bound to perdeuterated dodecylphosphocholine micelles.     

The interactions of the HIV gp41 fusion peptides with zwitterionic membrane mimics determined by NMR spectroscopy

The wild-type (wt) N-terminal 23-residue fusion peptide (FP) of the human immunodeficiency virus (HIV) fusion protein gp41 and its V2E mutant have been studied by nuclear magnetic resonance (NMR) spectroscopy in dodecylphosphocholine (DPC) micelles as membrane mimics. A number of NMR techniques have been used. Pulsed field-gradient diffusion measurements in DPC and in 4:1 DPC/sodium dodecylsulfate mixed micelles showed that there is no major difference between the partition coefficients of the fusogenic wt peptide and the V2E mutant in these micelles, indicating that there is no correlation between the activity of the fusion peptides and their membrane affinities. The nuclear Overhauser enhancement (NOE) patterns and the chemical shift index for these two peptides indicated that both FP are in an alpha helical conformation between the Ile4 to Leu12 or to Ala15 region. Simulated annealing showed that the helical region extends from Ile4 to Met19. The two FPs share similar conformational characteristics, indicating that the conformation of the FP is not an important factor determining its activity. The spin-label studies, utilizing spin labels 5- and 16-doxystearic acids in the DPC micelles, provided clear indication that the wt FP inserts its N-terminus into the micelles while the V2E mutant does not insert into the micelles. The conclusion from the spin-label results is corroborated by deuterium amide proton exchange experiments. The correlation between the oblique insertion of the FP and its fusogenic activity is in excellent agreement with results from our molecular dynamics simulation and from other previous studies.