Secondary structure and position of the cell-penetrating peptide transportan in SDS micelles as determined by NMR

Transportan is a 27-residue peptide (GWTLN SAGYL LGKIN LKALA ALAKK IL-amide) which has the ability to penetrate into living cells carrying a hydrophilic load. Transportan is a chimeric peptide constructed from the 12 N-terminal residues of galanin in the N-terminus with the 14-residue sequence of mastoparan in the C-terminus and a connecting lysine. Circular dichroism studies of transportan and mastoparan show that both peptides have close to random coil secondary structure in water. Sodium dodecyl sulfate (SDS) micelles induce 60% helix in transportan and 75% helix in mastoparan. The 600 MHz (1)H NMR studies of secondary structure in SDS micelles confirm the helix in mastoparan and show that in transportan the helix is localized to the mastoparan part. The less structured N-terminus of transportan has a secondary structure similar to that of the same sequence in galanin [Ohman, A., et al. (1998) Biochemistry 37, 9169-9178]. The position of mastoparan and transportan relative to the SDS micelle surface was studied by adding spin-labeled 5-doxyl- or 12-doxyl-stearic acid or Mn2+ to the peptide/micelle system. The combined results show that the peptides are for the most part buried in the SDS micelles. Only the C-terminal parts of both peptides and the central segment connecting the two parts of transportan are clearly surface exposed. For mastoparan, the secondary chemical shifts of the amide protons were found to vary periodically and display a pattern almost identical to those reported for mastoparan in phospholipid bicelles [Vold, R., et al. (1997) J. Biomol. NMR 9, 329-335], indicating similar structures and interactions in the two membrane-mimicking environments.     

Structural characterization of the interaction of the delta and alpha subunits of the Escherichia coli F1F0-ATP synthase by NMR spectroscopy

A critical point of interaction between F(1) and F(0) in the bacterial F(1)F(0)-ATP synthase is formed by the alpha and delta subunits. Previous work has shown that the N-terminal domain (residues 3-105) of the delta subunit forms a 6 alpha-helix bundle [Wilkens, S., Dunn, S. D., Chandler, J., Dahlquist, F. W., and Capaldi, R. A. (1997) Nat. Struct. Biol. 4, 198-201] and that the majority of the binding energy between delta and F(1) is provided by the interaction between the N-terminal 22 residues of the alpha- and N-terminal domain of the delta subunit [Weber, J., Muharemagic, A., Wilke-Mounts, S., and Senior, A. E. (2003) J. Biol. Chem. 278, 13623-13626]. We have now analyzed a 1:1 complex of the delta-subunit N-terminal domain and a peptide comprising the N-terminal 22 residues of the alpha subunit by heteronuclear protein NMR spectroscopy. A comparison of the chemical-shift values of delta-subunit residues with and without alpha N-terminal peptide bound indicates that the binding interface on the N-terminal domain of the delta subunit is formed by alpha helices I and V. NOE cross-peak patterns in 2D (12)C/(12)C-filtered NOESY spectra of the (13)C-labeled delta-subunit N-terminal domain in complex with unlabeled peptide verify that residues 8-18 in the alpha-subunit N-terminal peptide are folded as an alpha helix when bound to delta N-terminal domain. On the basis of intermolecular contacts observed in (12)C/(13)C-filtered NOESY experiments, we describe structural details of the interaction of the delta-subunit N-terminal domain with the alpha-subunit N-terminal alpha helix.     

The micelle-bound structure of an antimicrobial peptide derived from the alpha-chain of bovine hemoglobin isolated from the tick Boophilus microplus

Hemoglobin is known to be a source of peptides involved in several functions. The peptide FLSFPTTKTYFPHFDLSHGSAQVKGHGAK (Hb33-61) is a proteolytic product of the bovine hemoglobin alpha-chain found in the gut content of the cattle tick, Boophilus microplus, and it possesses antimicrobial activity. Since in the past we showed that the amidated form of Hb33-61, Hb33-61a, is active against a few Gram-positive bacteria and fungi strains at micromolar concentration [Fogaca et al. (1999) J. Biol. Chem. 274, 25330-25334], we have been prompted to shed more light on its functional and structural features. Here we show that the peptide is able to disrupt the bacterial membrane ofMicrococcus luteus A270. As for its structure, it has a random conformation in water, and it does not interact with zwitterionic micelles. On the other hand, it binds to negatively charged micelles acquiring a finite structural organization. The 3D structure of Hb33-61a bound to SDS micelles exhibits a nonconventional conformation for an antimicrobial peptide. The backbone is characterized by the presence of a beta-turn in the N-terminus and by a beta-turn followed by a alpha-helical stretch in the C-terminus. A hinge, whose spatial organization is stabilized by side-chain-side-chain interactions, joins these two regions. Interestingly, it preserves structural features present in the corresponding segment of the bovine hemoglobin alpha-chain. Hb33-61a does not possess a well-defined amphipathic nature, and H/D exchange experiments show that while the C-terminal region is embedded in the SDS micelle, one face of the N-terminal half is partly exposed to the solvent.     

NMR structures of the C-terminal segment of surfactant protein B in detergent micelles and hexafluoro-2-propanol

Although the membrane-associated surfactant protein B (SP-B) is an essential component of lung surfactant, which is itself essential for life, the molecular basis for its activity is not understood. SP-B's biophysical functions can be partially mimicked by subfragments of the protein, including the C-terminus. We have used NMR to determine the structure of a C-terminal fragment of human SP-B that includes residues 63-78. Structure determination was performed both in the fluorinated alcohol hexafluoro-2-propanol (HFIP) and in sodium dodecyl sulfate (SDS) micelles. In both solvents, residues 68-78 take on an amphipathic helical structure, in agreement with predictions made by comparison to homologous saposin family proteins. In HFIP, the five N-terminal residues of the peptide are largely unstructured, while in SDS micelles, these residues take on a well-defined compact conformation. Differences in helical residue side chain positioning between the two solvents were also found, with better agreement between the structures for the hydrophobic face than the hydrophilic face. A paramagnetic probe was used to investigate the position of the peptide within the SDS micelles and indicated that the peptide is located at the water interface with the hydrophobic face of the helix oriented inward, the hydrophilic face of the helix oriented outward, and the N-terminal residues even farther from the micelle center than those on the hydrophilic face of the alpha-helix. Interactions of basic residues of SP-B with anionic lipid headgroups are known to have an impact on function, and these studies demonstrate structural ramifications of such interactions via the differences observed between the peptide structures determined in HFIP and SDS.     

Structural analysis of synthetic peptide fragments from EmrE,a multidrug resistance protein,in a membrane-mimetic environment

EmrE, a multidrug resistance protein from Escherichia coli, renders the bacterium resistant to a variety of cytotoxic drugs by active translocation out of the cell. The 110-residue sequence of EmrE limits the number of structural possibilities that can be envisioned for this membrane protein. Four helix bundle models have been considered [Yerushalmi, H., Lebendiker, M., and Schuldiner, S. (1996) J. Biol. Chem. 271, 31044-31048]. The validity of EmrE structural models has been probed experimentally by investigations on overlapping peptides (ranging in length from 19 to 27 residues), derived from the sequence of EmrE. The choice of peptides was made to provide sequences of two complete, predicted transmembrane helices (peptides H1 and H3) and two helix-loop-helix motifs (peptides A and B). Peptide (B) also corresponds to a putative hairpin in a speculative beta-barrel model, with the "Pro-Thr-Gly" segment forming a turn. Structure determination in SDS micelles using NMR indicates peptide H1 to be predominantly helical, with helix boundaries in the micellar environment corroborating predicted helical limits. Peptide A adopts a helix-loop-helix structure in SDS micelles, and peptide B was also largely helical in micellar environments. An analogue peptide, C, in which the central "Pro-Thr-Gly" was replaced by "(D)Pro-Gly" displays local turn conformation at the (D)Pro-Gly segment, but neither a continuous helical stretch nor beta-hairpin formation was observed. This study implies that the constraints of membrane and micellar environments largely direct the structure of transmembrane peptides and proteins and study of judiciously selected peptide fragments can prove useful in the structural elucidation of membrane proteins.     

The NMR-derived conformation of orexin-A: an orphan G-protein coupled receptor agonist involved in appetite regulation and sleep

The conformation of orexin-A, an orphan G-protein coupled receptor agonist has been determined when bound to sodium dodecylsulphate-d(25) (SDS) micelles by (1)H and (13)C NMR and molecular modeling. Orexin-A has been implicated in sleep-wakefulness and feeding regulation. The conformational preference of orexin-A consists of a short helical section, involving Asp(5) to Gln(9) that makes up helix I, followed by a bend from Lys(10) to Ser(13). Residues Leu(16) to Gly(22) make up helix II. The conformation of orexin-A can now be used to explain the results of earlier Ala substitution mutagenesis experiments (J. G. Darker et al., Bioorg. Med. Chem. Lett. 11, 737-740 (2001); S. Ammoun, et al., J. Pharmacol. Expt. Ther. 305, 507-514 (2003)). Darker et al., working with orexin-A (15-33) amide, observed a significant drop in functional potency at the OX(1)R receptor when Leu(16), Leu(19), Leu(20), His(26), Gly(29), Ile(30), Leu(31), Thr(32), and Leu(33) were replaced by Ala. Ammoun et al. identified three areas of interest, which were the same for OX(1)R and OX(2)R receptors, as amino acids 15-17, 20 and 25-26 with the most marked reduction in activity being produced by the replacement of Leu(20) by Ala. We suggest that Leu(16), Leu(19), and Leu(20), which are in helix II, are likely responsible for binding orexin-A to the surface of the micelle.     

Key motif to gain selectivity at the neuropeptide Y5-receptor: structure and dynamics of micelle-bound [Ala31, Pro32]-NPY

The structure of [Ala(31), Pro(32)]-NPY, a neuropeptide Y mutant with selectivity for the NPY Y(5)-receptor (Cabrele, C., Wieland, H. A., Stidsen, C., Beck-Sickinger, A. G., (2002) Biochemistry XX, XXXX-XXXX (companion paper)), has been characterized in the presence of the membrane mimetic dodecylphosphocholine (DPC) micelles using high-resolution NMR techniques. The overall topology closely resembles the fold of the previously described Y(5)-receptor-selective agonist [Ala(31), Aib(32)]-NPY (Cabrele, C., Langer, M., Bader, R., Wieland, H. A., Doods, H. N., Zerbe, O., and Beck-Sickinger, A. G. (2000) J. Biol. Chem 275, 36043-36048). Similar to wild-type neuropeptide Y (NPY) and [Ala(31), Aib(32)]-NPY, the N-terminal residues Tyr(1)-Asp(16) are disordered in solution. Starting from residue Leu(17), an alpha helix extends toward the C-terminus. The decreased density of medium-range NOEs for the C-terminal residues resulting in larger RMSD values for the backbone atoms of Ala(31)-Tyr(36) indicates that the alpha helix has become interrupted through the [Ala(31), Pro(32)] mutation. This finding is further supported by (15)N-relaxation data through which we can demonstrate that the well-defined alpha helix is restricted to residues 17-31, with the C-terminal tetrapeptide displaying increased flexibility as compared to NPY. Surprisingly, increased generalized order parameter as well as decreased (3)J(HN)(alpha) scalar coupling constants reveal that the central helix is stabilized in comparison to wild-type NPY. Micelle-integrating spin labels were used to probe the mode of association of the helix with the membrane mimetic. The Y(5)-receptor-selective mutant and NPY share a similar orientation, which is parallel to the lipid surface. However, signal reductions due to efficient electron, nuclear spin relaxation were much less pronounced for the surface-averted residues in [Ala(31), Pro(32)]-NPY when compared to wild-type DPC-bound NPY. Only the signals of residues Asn(29) and Leu(30) were significantly more reduced in the mutant. The postulation of a different membrane binding mode of [Ala(31), Pro(32)]-NPY is further supported by the faster H/D exchange at the C-terminal amide protons. We conclude that arginine residues 33 and 35, which are believed to be directly involved in forming contacts to acidic receptor residues at the membrane-water interface, are no longer fixed in a well-defined conformation close to the membrane surface in [Ala(31), Pro(32)]-NPY.     

2D NMR and structural model for a mitochondrial signal peptide bound to a micelle

The 19 amino acid signal peptide of rat liver aldehyde dehydrogenase, possessing a lysine substitution for an arginine and containing 3 extra amino acid residues at the C terminus, was studied by two-dimensional NMR in a dodecylphosphocholine micelle. In this membrane-like environment, the peptide contains two alpha-helical regions, both of which are amphiphilic, separated by a hinge region. The helix located closer to the C terminus is more stable than is the helix located near the N terminus. This suggests that the hydrophobic face of the C-terminal helix is buried within the hydrophobic region of the micelle. On the basis of these results a general model for protein translocation is presented in which the C-terminal amphiphilic helix of the signal region in the preprotein first binds to the mitochondrial membrane and then diffuses to the translocation receptor. The receptor then recognizes the N-terminal helix of the signal region, which is not anchored to the membrane. To explain how this signal peptide was imported into isolated mitochondria in the absence of energy or receptor protein [Pak, Y. K., & Weiner, H. (1990) J. Biol. Chem. 265, 14298-14307], a model for signal peptide translocation across a membrane barrier without the need for auxiliary membrane proteins is proposed. In this model the faces of the two helices fold upon each other, resulting in the mutual shielding of positively charged residues by the complementary hydrophilic face of the other amphiphilic helix.     

A minor beta-structured conformation is the active state of a fusion peptide of vesicular stomatitis virus glycoprotein.

Entry of enveloped animal viruses into their host cells always depends on a step of membrane fusion triggered by conformational changes in viral envelope glycoproteins. Vesicular stomatitis virus (VSV) infection is mediated by virus spike glycoprotein G, which induces membrane fusion at the acidic environment of the endosomal compartment. In a previous work, we identified a specific sequence in the VSV G protein, comprising the residues 145-164, directly involved in membrane interaction and fusion. In the present work we studied the interaction of pep[145-164] with membranes using NMR to solve the structure of the peptide in two membrane-mimetic systems: SDS micelles and liposomes composed of phosphatidylcholine and phosphatidylserine (PC:PS vesicles). The presence of medium-range NOEs showed that the peptide has a tendency to form N- and C-terminal helical segments in the presence of SDS micelles. Analysis of the chemical shift index indicated helix-coil equilibrium for the C-terminal helix under all conditions studied. At pH 7.0, the N-terminal helix also displayed a helix-coil equilibrium when pep[145-164] was free in solution or in the presence of PC:PS. Remarkably, at the fusogenic pH, the region of the N-terminal helix in the presence of SDS or PC:PS presented a third conformational species that was in equilibrium with the helix and random coil. The N-terminal helix content decreases pH and the minor beta-structured conformation becomes more prevalent at the fusogenic pH. These data point to a beta-conformation as the fusogenic active structure-which is in agreement with the X-ray structure, which shows a beta-hairpin for the region corresponding to pep[145-164].     

The nociceptin pharmacophore site for opioid receptor binding derived from the NMR structure and bioactivity relationships

Nociceptin, a 17 amino acid opioid-like peptide that has an inhibitory effect on synaptic transmission in the nervous system, is involved in learning, memory, attention, and emotion and is also implicated in the perception of pain and visual, auditory, and olfactory functions. In this study, we investigated the NMR solution structure of nociceptin in membrane-like environments (trifluoroethanol and SDS micelles) and found it to have a relatively stable helix conformation from residues 4-17 with functionally important N-terminal residues being folded aperidoically on top of the helix. In functional assays for receptor binding and calcium flux, alanine-scanning variants of nociceptin indicated that functionally important residues generally followed helix periodicity, consistent with the NMR structural model. Structure-activity relationships allowed identification of pharmacophore sites that were used in small molecule data base searches, affording hits with demonstrated nociceptin receptor binding affinities.     

The glycine residue in cyclic lactam analogues of galanin(1-16)-NH2 is important for stabilizing an N-terminal helix

The neuropeptide galanin is a 29- or 30-residue peptide whose physiological functions are mediated by G-protein-coupled receptors. Galanin's agonist activity has been shown to be associated with the N-terminal sequence, galanin(1-16). Conformational investigations previously carried out on full-length galanin have, furthermore, indicated the presence of a helical conformation in the neuropeptide's N-terminal domain. Several cyclic lactam analogues of galanin(1-16)-NH2 were prepared in an attempt to stabilize an N-terminal helix in the peptide. Here we describe and compare the solution conformational properties of these analogues in the presence of SDS micelles as determined by NMR, CD, and fluorescence spectroscopy. Differences in CD spectral profiles were observed among the compounds that were studied. Both c[D4, K8]Gal(1-16)-NH2 and c[D4,K8]Gal(1-12)-NH2 adopted stable helical conformations in the micelle solution. On the basis of the analyses of their respective alpha H chemical shifts and NOE patterns, this helix was localized to the first 10 residues. The distance between the aromatic rings of Trp2 and Tyr9 in c[D4, K8]Gal(1-16)-NH2 was determined to be 10.8 +/- 3 A from fluorescence resonance energy transfer measurements. This interchromophore spacing was found to be more consistent with a helical structure than an extended one. Removal of the Gly1 residue in compounds c[D4,K8]Gal(1-16)-NH2 and c[D4, K8]Gal(1-12)-NH2 resulted in a loss of helical conformation and a concomitant reduction in binding potency at the GalR1 receptor but not at the GalR2 receptor. The nuclear Overhauser enhancements obtained for the Gly1 deficient analogues did, however, reveal the presence of nascent helical structures within the N-terminal sequence. Decreasing the ring structure size in c[D4, K8]Gal(1-16)-NH2 by replacing Lys8 with an ornithine residue or by changing the position of the single lysine residue from eight to seven was accompanied by a complete loss of helical structure and dramatically reduced receptor affinity. It is concluded from the data obtained for the series of cyclic galanin(1-16)-NH2 analogues that both the ring structure size and the presence of an N-terminal glycine residue are important for stabilizing an N-terminal helix in these compounds. However, although an N-terminal helix constitutes a predominant portion of the conformational ensemble for compounds c[D4,K8]Gal(1-16)-NH2 and c[D4, K8]Gal(1-12)-NH2, these peptides nevertheless are able to adopt other conformations in solution. Consequently, the correlation between the ability of the cyclic galanin analogues to adopt an N-terminal helix and bind to the GalR1 receptor may be considered as a working hypothesis.     

NMR studies of the structure and dynamics of peptide E, an endogenous opioid peptide that binds with high affinity to multiple opioid receptor subtypes

Structural and dynamic properties of opioid peptide E have been examined in an sodium dodecyl sulfate (SDS) micelle. Structural and dynamic studies both indicate that this peptide exhibits greater segmental mobility than typical structured proteins. An nmr structural analysis of adrenal peptide E in SDS micelles indicated the presence of two well-defined beta-turns, one at the N-terminus encompassing residues 3 to 6, and the second in the region between residues 15 and 18. Certain side chain dihedral angles were also remarkably well defined, such as the chi 1 angle of F4, which exhibited a trans configuration. These calculated structures were based on a set of 9.5 restraints per residue. The backbone dynamics of peptide E in SDS micelles were examined through an analysis of 15N-relaxation parameters. An extended model-free analysis was used to interpret the relaxation data. The overall rotational correlation time is 19.7 ns. the average order parameter S2 is 0.66 +/- 0.15. The N-terminal loop region residues including G3 to R6 have an average order parameter of 0.70 +/- 0.23. The average order parameter lies somewhere between that observed for a random coil (e.g., S2 = 0.3) and that of a well-defined tertiary fold (e.g., S2 = 0.86). This suggests that peptide E in SDS micelles adopts a restricted range of conformations rather than a random coil. Based on the helical structure recently obtained for the highly homologous kappa-agonist dynorphin-A(1-17) and the beta-turn in the same region of peptide E, it is reasonable to assume that these two elements of secondary structure reflect different receptor subtype binding geometries. The intermediate order parameters observed for peptide E in an SDS micelle suggest a degree of dynamic mobility that may enable facile interconversion between helical and beta-turn geometries in the N-terminal agonist domain.     

Pituitary adenylate cyclase activating polypeptide (PACAP) with 27 residues. Conformation determined by 1H NMR and CD spectroscopies and distance geometry in 25% methanol solution.

The conformation of pituitary adenylate cyclase activating polypeptide with 27 residues (PACAP27) has been determined by two-dimensional NMR and CD spectroscopies and distance geometry in 25% methanol. Residues 9-20 and 22-25 have well-defined conformations but other residues do not show ordered conformations. The conformation of residues 9-20 is composed of three distinct regions of beta turn-like conformation (residues 9-12), alpha helix (residues 12-14) and the looser helical conformation (residues 15-20), while residues 22-24 form alpha helix. PACAP27 has a 2 helices separated by a disordered region similar to a VIP analog reported by Fry et al. but is distinct from the VIP analog in the position of the first helix, which is shifted by 2 residues toward the C-terminus, and in the form of the second helix [Fry, D.C., Madison, V.S., Bolin, D.R., Greeley, D.N., Toome, V. and Wegrzynski, B.B. (1989) Biochemistry 28, 2399-2409].     

Comparison of the conformation and electrostatic surface properties of magainin peptides bound to sodium dodecyl sulfate and dodecylphosphocholine micelles

The role played by noncovalent interactions in inducing a stable secondary structure onto the sodium dodecyl sulfate (SDS) and dodecylphosphocholine (DPC) micelle-bound conformations of (Ala(8,13,18))magainin 2 amide and the DPC micelle bound conformation of magainin 1 were determined. Two-dimensional NMR and molecular modeling investigations indicated that (Ala(8,13,18))magainin 2 amide bound to DPC micelles adopts a alpha-helical secondary structure involving residues 2-16. The four C-terminal residues converge to a lose beta-turn structure. (Ala(8,13,18))magainin 2 amide bound to SDS miscelles adopts a alpha-helical secondary structure involving residues 7-18. The C- and N-terminal residues exhibited a great deal of conformational flexibility. Magainin 1 bound to DPC micelles adopts a alpha-helical secondary structure involving residues 4-19. The C-terminal residues converge to a lose beta-turn structure. The results of this investigation indicate hydrophobic interactions are the major contributors to stabilizing the induced helical structure of the micelle-bound peptides. Electrostatic interactions between the polar head groups of the micelle and the cationic side chains of the peptides define the positions along the peptide backbone where the helical structures begin and end.     

Signaling domain of the aspartate receptor is a helical hairpin with a localized kinase docking surface: cysteine and disulfide scanning studies.

Cysteine and disulfide scanning has been employed to probe the signaling domain, a highly conserved motif found in the cytoplasmic region of the aspartate receptor of bacterial chemotaxis and related members of the taxis receptor family. Previous work has characterized the N-terminal section of the signaling domain [Bass, R. B., and Falke, J. J. (1998) J. Biol. Chem. 273, 25006-25014], while the present study focuses on the C-terminal section and the interactions between these two regions. Engineered cysteine residues are incorporated at positions Gly388 through Ile419 in the signaling domain, thereby generating a library of receptors each containing a single cysteine per receptor subunit. The solvent exposure of each cysteine is ascertained by chemical reactivity measurements, revealing a periodic pattern of buried hydrophobic and exposed polar residues characteristic of an amphipathic alpha-helix, denoted helix alpha8. The helix begins between positions R392 and Val401, then continues through the last residue scanned, Ile419. Activity assays carried out both in vivo and in vitro indicate that both the buried and exposed faces of this amphipathic helix are critical for proper receptor function and the buried surface is especially important for kinase downregulation. Patterns of disulfide bond formation suggest that helix alpha8, together with the immediately N-terminal helix alpha7, forms a helical hairpin that associates with a symmetric hairpin from the other subunit of the homodimer, generating an antiparallel four helix bundle containing helices alpha7, alpha7', alpha8, and alpha8'. Finally, the protein-interactions-by-cysteine-modification (PICM) method suggests that the loop between helices alpha7 and alpha8 interacts with the kinase CheA and/or the coupling protein CheW, expanding the receptor surface implicated in kinase docking.     

Solution structure of neuromedin B by (1)H nuclear magnetic resonance spectroscopy.

The solution structure of neuromedin B (NMB) was investigated using two-dimensional nuclear magnetic resonance (NMR) spectroscopy in membrane-mimicking environments. NMB adopts a relaxed helical conformation from Trp(4) to Met(10) in 50% aqueous 2,2, 2-trifluoroethanol (TFE) solution and in 150 mM SDS micelles. Sidechain atoms of the three residues, Trp(4), His(8) and Phe(9) orient toward the same direction and these residues might play a key role on interacting with hydrophobic acyl chains of the phospholipids in the membrane. NOESY experiments performed on NMB in non-deuterated SDS micelle show that aromatic ring protons of Trp(4) and Phe(9) residues are in close contact with methylene protons of SDS micelles. In addition, proton longitudinal relaxation data proved that the interactions between NMB with SDS micelle are characterized as extrinsic interaction. Trp(4) and Phe(9) seem to be important in interaction with receptor and this agrees with the previous studies of structure-activity relationship (Howell, D.C. et al. (1996) Int. J. Pept. Protein Res. 48, 522-531). These conformational features might be helpful in understanding the molecular mechanism of the function of NMB and developing the efficient drugs.     

Helical structure of dermaseptin B2 in a membrane-mimetic environment

Dermaseptins are antimicrobial peptides from frog skin that have high membrane-lytic activity against a broad spectrum of microorganisms. The structure of dermaseptin B2 in aqueous solution, in TFE/water mixtures, and in micellar and nonmicellar SDS was analyzed by CD, FTIR, fluorescence, and NMR spectroscopy combined with molecular dynamics calculations. Dermaseptin B2 is unstructured in water, but helical conformations, mostly in segment 3-18, are stabilized by addition of TFE. SDS titration showed that dermaseptin B2 assumes nonhelical structures at SDS concentrations far below the critical micellar concentration and helical structures at micellar concentrations. Dermaseptin B2 bound to SDS micelles (0.4 mM peptide, 80 mM SDS) adopts a well-defined amphipathic helix between residues 11-31 connected to a more flexible helical segment spanning residues 1-8 by a flexible hinge region around Val9 and Gly10. Experiments using paramagnetic probes showed that dermaseptin B2 lies near the surface of SDS micelles and that residue Trp3 is buried in the SDS micelle, but close to the surface. A slow exchange equilibrium occurs at higher peptide/SDS ratios (2 mM peptide, 80 mM SDS) between forms having distinct sets of resonances in the N-terminal 1-11 segment. This equilibrium could reflect different oligomeric states of dermaseptin B2 interacting with SDS micelles. Structure-activity studies on dermaseptin B2 analogues showed that the N-terminal 1-11 segment is an absolute requirement for antibacterial activity, while the C-terminal 10-33 region is also important for full antibiotic activity.     

Structure and dynamics of micelle-bound neuropeptide Y: comparison with unligated NPY and implications for receptor selection

The biological importance of the neuropeptide Y (NPY) has steered a number of investigations about its solution structure over the last 20 years. Here, we focus on the comparison of the structure and dynamics of NPY free in solution to when bound to a membrane mimetic, dodecylphosphocholine (DPC) micelles, as studied by 2D (1)H NMR spectroscopy. Both, free in solution and in the micelle-bound form, the N-terminal segment (Tyr1-Glu15) is shown to extend like a flexible tail in solution. This is not compatible with the PP-fold model for NPY that postulates backfolding of the flexible N terminus onto the C-terminal helix. The correlation time (tau(c)) of NPY in aqueous solution, 5.5 (+/-1.0) ns at 32 degrees C, is only consistent with its existence in a dimeric form. Exchange contributions especially enhancing transverse relaxation rates (R(2)) of residues located on one side of the C-terminal helix of the molecule are supposed to originate from dimerization of the NPY molecule. The dimerization interface was directly probed by looking at (15)N-labeled NPY/spin-labeled [TOAC34]-[(14)N]-NPY heterodimers and revealed both parallel and anti-parallel alignment of the helices. The NMR-derived three-dimensional structure of micelle-bound NPY at 37 degrees C and pH 6.0 is similar but not identical to that free in solution. The final set of 17 lowest-energy DYANA structures is particularly well defined in the region of residues 21-31, with a mean pairwise RMSD of 0.23 A for the backbone heavy atoms and 0.85 A for all heavy atoms. The combination of NMR relaxation data and CD measurements clearly demonstrates that the alpha-helical region Ala18-Thr32 is more stable, and the C-terminal tetrapeptide becomes structured only in the presence of the phosphocholine micelles. The position of NPY relative to the DPC micelle surface was probed by adding micelle integrating spin labels. Together with information from (1)H,(2)H exchange rates, we conclude that the interaction of NPY with the micelle is promoted by the amphiphilic alpha-helical segment of residues Tyr21-Thr32. NPY is located at the lipid-water interface with its C-terminal helix parallel to the membrane surface and penetrates the hydrophobic interior only via insertions of a few long aliphatic or aromatic side-chains. From these data we can demonstrate that the dimer interface of neuropeptide Y is similar to the interface of the monomer binding to DPC-micelles. We speculate that binding of the NPY monomer to the membrane is an essential key step preceeding receptor binding, thereby pre-orientating the C-terminal tetrapeptide and possibly inducing the bio-active conformation.     

Conformational studies of human [15-2-aminohexanoic acid]little gastrin in sodium dodecyl sulfate micelles by 1H NMR

Human little gastrin is a 17 amino acid peptide that adopts a random conformation in water and an ordered structure in sodium dodecyl sulfate (SDS) micelles as well as in trifluoroethanol (TFE). The circular dichroism spectra in these two media have the same shape, indicative of a similar preferred conformation [Mammi, S., Mammi, N. J., Foffani, M. T., Peggion, E., Moroder, L., & Wünsch, E. (1987) Biopolymers 26, S1-S10]. We describe here the assignment of the proton NMR resonances and the conformational analysis of [Ahx15]little gastrin in SDS micelles. Two-dimensional correlation techniques form the basis for the assignment. The conformational analysis utilized NOE's, NH to C alpha H coupling constants, and the temperature coefficients of the amide chemical shifts. The NMR data indicate a helical structure in the N-terminal portion of the peptide. These results are compared with the conformation that we recently proposed for a minigastrin analogue (fragment 5-17 of [Ahx15]little gastrin) in TFE.     

Solution structure of chi-conopeptide MrIA, a modulator of the human norepinephrine transporter

The chi-conopeptides MrIA and MrIB are 13-residue peptides with two disulfide bonds that inhibit human and rat norepinephrine transporter systems and are of significant interest for the design of novel drugs involved in pain treatment. In the current study we have determined the solution structure of MrIA using NMR spectroscopy. The major element of secondary structure is a beta-hairpin with the two strands connected by an inverse gamma-turn. The residues primarily involved in activity have previously been shown to be located in the turn region (Sharpe, I. A.; Palant, E.; Schroder, C. I.; Kaye, D. M.; Adams, D. J.; Alewood, P. F.; Lewis, R. J. J Biol Chem 2003, 278, 40317-40323), which appears to be more flexible than the beta-strands based on disorder in the ensemble of calculated structures. Analogues of MrIA with N-terminal truncations indicate that the N-terminal residues play a role in defining a stable conformation and the native disulfide connectivity. In particular, noncovalent interactions between Val3 and Hyp12 are likely to be involved in maintaining a stable conformation. The N-terminus also affects activity, as a single N-terminal deletion introduced additional pharmacology at rat vas deferens, while deleting the first two amino acids reduced chi-conopeptide potency.