Beta-turn formation by a six-residue linear peptide in solution.

A model peptide AAGDYY-NH2 (B1), which is found to adopt a beta-turn conformation in the TEM-1 beta-lactamase inhibitor protein (BLIP) in the TEM-1/BLIP co-crystal, was synthesized to elucidate the mechanism of its beta-turn formation and stability. Its structural preferences in solution were comprehensively characterized using CD, FT-IR and 1H NMR spectroscopy, respectively. The set of observed diagnostic NOEs, the restrained molecular dynamics simulation, CD and FT-IR spectroscopy confirmed the formation of a beta-turn in solution by the model peptide. The dihedral angles [(phi3, phi3) (phi4, phi4)] of [(-52 degrees, -32 degrees ) (-38 degrees, -44 degrees )] of Gly-Asp fragment in the model peptide are consistent with those of a type III beta-turn. In a conclusion, the conformational preference of the linear hexapeptide B1 in solution was determined, and it would provide a simple template to study the mechanism of beta-turn formation and stability.     

Formyl peptide chemotactic receptor. Evidence for an active proteolytic fragment

We have developed a radioiodinated photoaffinity label, N-formyl-Nle-Leu-Phe-Nle-125I-Tyr-Lys-N-6-(4'-azido-2'-nitrophenylamino) hexanoate (where Nle represents norleucine) (125I-PAL), which forms a covalent complex with the formyl peptide chemotactic receptor of living human neutrophils. Labeling was 12 to 16% efficient and did not alter cell viability. The receptor on live neutrophils and neutrophil membranes has an apparent molecular weight of 50,000 to 70,000 by sodium dodecyl sulfate-polyacrylamide electrophoresis. The receptor on intact cells possesses one predominant papain cleavage site, yielding a 35,000-Da fragment. This receptor fragment retains an affinity for N-formyl-Nle-Leu-Phe-Nle-125I-Tyr-Lys indistinguishable from the receptor on control cells (KD = 1.9 and 1.8 nM, respectively). The 35,000-Da papain fragment was biologically active as evidenced by an unchanged dose-response curve for peptide-stimulated beta-glucuronidase release and fluorescent peptide uptake. Papain treatment of 125I-PAL-labeled neutrophil membranes or of digitonin-soluble 125I-PAL-labeled receptors produced a predominant 28,000-Da fragment without evidence of the 35,000-Da fragment seen with whole cells. Pronase, which did not cleave the receptor on intact cells, produced multiple receptor fragments when used to treat 125I-PAL-labeled membranes.     

Solid state and solution conformations of a helical peptide with a central gly-gly segment

The influence of amino acids with contrasting conformational tendencies on the stereochemistry of oligopeptides has been investigated using an octapeptide Boc-Leu-Aib-Val-Gly-Gly-Leu-Aib-Val-OMe, which contains two helix-promoting Aib residues and a central helix-destabilizing Gly-Gly segment. Single crystal x-ray diffraction studies reveal that a 3 ₁₀-helix is formed up to the penultimate Aib residue, at which point there is a helix reversal in the backbone, reminiscent of a C-terminal 6 → I hydrogen bond. The curious feature in the crystal is the solvation of the possible 6 → 1 bond by a CH₃OH molecule, where the OH is inserted between O(3) and N(8) and participates in hydrogen bonds with both. The cell parameters are as follows: space group P2₁2₁2₁, a = 10.649(4) Å, b = 15.694(5) Å, c = 30.181(8) Å, R = 6.7% for 3427 data (| F₀| > 3σF) observed to 0.9 Å. Nuclear magnetic resonance studies in CDCl₃ using NH group solvent accessibility and nuclear Overhauser effects as probes are consistent with a 3 ₁₀-helical conformation. In contrast, in (CD₃)₂SO, unfolding of the central segment results in a multiple β-turn structure, with β-turn conformations populated at residues 1–2, 3–4, and 6–7. CD studies in methanol-2,2,2-trifluoroethanol (TFE) mixtures also provide evidence for a solvent-dependent structural transition. Helical conformations are populated in TFE, while type II β-turn structures are favored in methanol. © 1996 John Wiley & Sons, Inc.     

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].     

Solid and solution state conformation of 1L-1-O-acetyl-2,3:5,6-di-O-isopropylidene-chiro-inositol

The X-ray crystal structure of 1L-1-O-acetyl-2,3:5,6-di-O-isopropylidene-chiro-inositol is described. The inositol ring deviates considerably from the ideal chair conformation to a flattened chair. A comparison of its conformation in solution with that in solid was made by the use of 1H NMR. This conformational analysis revealed that the title compound adopts similar conformations in solid state and in solution states irrespective of solvent polarity.     

Synthesis and properties of chemotactic peptide analogs. I. Crystal structure and molecular conformation of HCO-Met-leu-Ain-OMe

HCO-Met-Leu-Ain-OMe (2), an analog of the chemotactic peptide HCO-Met-Leu-Phe-OH, containing the conformationally blocked residue of the 2-aminoindane-2-carboxylic acid (Ain) has been synthesized and its crystal and molecular conformation has been determined. Crystals of 2 are monoclinic, space group P2(1), with a = 15.059(7), b = 18.548(7), c = 9.600(4) A; beta = 85.04(3) degrees. The structure has been solved by direct methods and refined to R = 0.069 for 2813 independent reflections with I greater than 2.5 sigma (I). Two independent molecules A and B have been found in the asymmetric unit of the crystal of 2. Their conformation can be described as extended at the Met and Leu residues, but folded at the C-terminal Ain residue. The helical folding is left- and right-handed in the A and B molecule, respectively. The crystal packing is characterized by ribbons of intermolecular hydrogen bonded molecules extended along the c direction. The constrained analog 2 is highly active in the superoxide production, thus indicating that a stabilization of a helical folding at the C-terminal region of chemotactic tripeptides maintains the activity. The orientation of the aromatic ring, with respect to its adjacent backbone atoms, does not seem critical for the activity.     

A 1H-NMR study of the solution conformation of Icaria chemotactic peptide and its [Lys7] analog: effects on the physiological activity of a substitution of proline to lysine at position 7

Solution conformations of Icaria chemotactic peptide isolated from the Ropalidian wasp venom and its [Lys7] analog have been analyzed by the use of two-dimensional proton nuclear resonance spectroscopy. It has been shown that Icaria chemotactic peptide takes alpha helical conformation in the C-terminal 8-13 segment with the N-terminal 1-7 segment disordered. By contrast, the [Lys7] analog takes alpha helical conformation over a wider range from residues 3 to 13. The present results indicate that the substitution of proline to lysine at position 7 results in a drastic change in solution conformation, providing the Icaria chemotactic peptide with new physiological functions.     

N-formyl peptide receptors cluster in an active raft-associated state prior to phosphorylation

In response to ligand binding, G protein-coupled receptors undergo phosphorylation and activate cellular internalization machinery. An important component of this process is the concentration of receptors into clusters on the plasma membrane. Aside from organizing the receptor in anticipation of internalization, little is known of the function of ligand-mediated G protein-coupled receptor clustering, which has traditionally been thought of as being a phosphorylation-dependent event prior to receptor internalization. We now report that following receptor activation, the N-formyl peptide receptor (FPR) forms distinct membrane clusters prior to its association with arrestin. To determine whether this clustering is dependent upon receptor phosphorylation, we used a mutant form of the FPR, DeltaST-FPR, which lacks all phosphorylation sites in the carboxyl-terminal domain. We found that activation of the signaling-competent DeltaST-FPR resulted in rapid receptor clustering on the plasma membrane independent of Gi protein activation. This clustering required receptor activation since the D71A mutant receptor, which binds ligand but is incapable of transitioning to an active state, failed to induce receptor clustering. Furthermore we demonstrated that FPR-mediated clustering and signaling were cholesterol-dependent processes, suggesting that translocation of the active receptor to lipid rafts may be required for maximal signaling activity. Finally we showed that FPR stimulation in the absence of receptor phosphorylation resulted in translocation of FPR to GM1-rich clusters. Our results demonstrate for the first time that formation of a clustered activated receptor state precedes receptor phosphorylation, arrestin binding, and internalization.     

Conformation and dynamics of the chemotactic peptide formyl-L-methionyl-L-leucyl-L-phenylalanine in solution

Several conformational and dynamic features of the chemotactic peptide formyl-L-methionyl-L-leucyl-L-phenylalanine in solution have been delineated by investigations of NMR and IR spectroscopic parameters. Both 1D and 2D NMR experiments have been performed for detection of scalar and dipolar proton-proton connectivities, whereas 13C and 1H relaxation parameters have been interpreted in terms of molecular dynamics. The main conformation appeared to be unfolded with the three hydrophobic side chains extending in divergent directions with respect to the backbone. The existence of relatively weak intermolecular hydrogen bonds was demonstrated, involving the formamide end group, with increase in the hydrophobicity of the external surface.     

Solid state and solution conformations of a hybrid alphagammaalphaalphagammaalpha hexapeptide. Characterization of a backbone expanded analog of the alpha-polypeptide 3(10)-helix

The stereochemically constrained gamma amino acid residue gabapentin (1-(aminomethyl)cyclohexaneacetic acid, Gpn) has been incorporated into a host alpha-peptide sequence. The structure of a hybrid alphagammaalphaalphagammaalpha peptide, Boc-Leu-Gpn-Aib-Leu-Gpn-Aib-OMe in crystals reveals a continuous helical conformation stabilized by three intramolecular 4 --> 1 C(12) hydrogen bonds across the alphagamma/alphagamma segments and one C(10) hydrogen bond across the central alphaalpha segment. This conformation corresponds to an expanded analog of the canonical all-alpha polypeptide 3(10)-helix, with insertion of two additional backbone atoms at each gamma residue. Solvent dependence of NH chemical shifts in CDCl(3) solution are consistent with conformation in which the NH groups of Aib (3), Leu (4), Gpn (5), and Aib (6) are hydrogen bonded, a feature observed in the solid state. The nonsequential NOEs between Gpn (2) NH <--> Leu (4) NH and Gpn (2) NH <--> Gpn (5) NH support the presence of additional conformations in solution. Temperature-dependent line broadening of NH resonances confirms the occurrence of rapid exchange between multiple conformations at room temperature. Two conformational models which rationalize the observed nonsequential NOEs are presented, both of which contain three hydrogen bonds and are consistent with the known stereochemical preferences of the Gpn residue.     

Fluorescence study on the conformation of a cyclic enkephalin analog in aqueous solution

Certain patients with ovarian germ cell tumors develop a specific antibody reacting with glycoprotein-bound large carbohydrates of murine teratocarcinoma cells. The antigenic determinant was found to involve an alpha-galactosyl residue, since alpha-galactosidase from coffee bean, but not other glycosidases abolished the antigenic activity of the large glycan isolated from F9 and OTT6050 cells. Several evidences excluded the possibility that the antigen is blood group B or P1 antigen. These results indicate tumor-associated expression of an unusual alpha-galactosyl residue in human ovarian germ cell tumors.     

Structural and membrane modifying properties of suzukacillin, a peptide antibiotic related to alamethicin. Part A. Sequence and conformation.

The primary structure and conformation of the polypeptide antibiotic suzukacillin A are investigated. Suzukacillin A is isolated from the Trichoderma viride strain 1037 and exhibits membrane modifying and lysing properties similar to those of alamethicin. A combined gas chromatographic mass spectrometric analysis of the trifluoroacetylated peptide methyl esters of partial hydrolysates revealed a tentative sequence of 23 residues including 10 2-methylalanines and one phenylalaninol, which shows many fragments known from alamethicin: Ac-Aib-Pro-Val-Aib-Val-Ala-Aib-Ala-Aib-Aib-Gln-Aib-Leu-Aib-Gly-Leu-Aib-Pro-Val-Aib-Aib-Glu(Pheol)-Gln-OH. All chiral amino acids and phenylalainol have L-configuration. Ultraviolet and infrared spectroscopy, circular dichroism in various solvents and in particular 13C nuclear magnetic resonance have been used for a comparative study of suzukacillin with alamethicin. Suzukacillin has a partially alpha-helical structure and the helix content increases largely from polar to lipophilic solvents. Suzukacillin aggregates more strongly than alamethicin in aqueous medis due to a longer alpha-helical part and higher number of aliphatic residues. A part of the alpha-helix is exceptionally stabilized due to 2-methylalanine residues shielding the peptide bonds from interactions with polar solvents. In lipophilic solvents and lecithin vesicles particularly large temperature induced reductions of the high alpha-helix content are found for alamethicin. Suzukacillin shows similar temperature coefficients in lipophilic media, however, in contrast to alamethicin a more linear change in intensity of the Cotton effects is observed.     

Primary sequence and solution conformation of ferrocytochrome c-552 from Nitrosomonas europaea.

Cytochrome c-552 from Nitrosomonas europaea is a 9.1-kDa monoheme protein that is a member of the bacterial cytochrome c-551 family. The gene encoding for c-552 has been cloned and sequenced and the primary sequence of the product deduced. Proton resonance assignments were made for all main-chain and most side-chain protons in the diamagnetic, reduced form by two-dimensional NMR techniques. Distance constraints (1056) were determined from nuclear Overhauser enhancements, and torsion angle constraints (88) were determined from scalar coupling estimates. Solution conformations for the protein were computed by the hybrid distance geometry-simulated annealing approach. For 20 computed structures, the root mean squared deviation from the average position of equivalent atoms was 0.84 A (sigma = 0.12) for backbone atoms over all residues. Analysis by residue revealed there were three regions clearly less well defined than the rest of the protein: the first two residues at the N-terminus, the last two at the C-terminus, and a loop region from residues 34 to 40. Omitting these regions from the comparison, the root mean squared deviation was 0.61 A (sigma = 0.13) for backbone atoms, 0.86 A (sigma = 0.12) for all associated heavy atoms, and 0. 43 A (sigma = 0.17) for the heme group. The global folding of the protein is consistent with others in the c-551 family. A deletion at the N-terminus relative to other family members had no impact on the global folding, whereas an insertion at residue 65 did affect the way the polypeptide packs against the methionine-ligated side of the heme. The effects of specific substitutions will be discussed. The structure of c-552 serves to delineate essential features of the c-551 family.     

Crystal structure, conformation, and potential energy calculations of the chemotactic peptide N-formyl-L-Met-L-Leu-L-Phe-OMe

The tripeptide N-formyl-L-Met-L-Leu-L-Phe-OMe (FMLP-OMe) crystallizes in the orthorhombic system, space group P 2(1)2(1)2(1), with the following unit-cell parameters: a = 21.727, b = 21.836, c = 5.133 A, Z = 4. The structure has been solved and refined to a final R of 0.068 for 1838 independent reflexions with I greater than 2 omega (I). The peptide backbone is folded at the Leu residue (phi L = -67.7, psi L = -49.1 degrees) without intramolecular hydrogen bonds. Considering each peptide plane, the Leu side-chain is oriented on the same side of that of the Phe residue and on the opposite side of that of the Met residue, respectively. The crystal conformation differs from all the other conformations proposed for FMLP-OMe and the anionic form of N-formyl-L-Met-L-Leu-L-Phe-OH (FMLP) in solution accounts for the amphiphilic character of the peptide, giving rise, through intermolecular hydrogen bonds, to a stacking of molecules which could be maintained in the aggregation states experimentally observed in solvents of low polarity. Intramolecular potential energy calculations have been carried out in order to compare the energies of the various backbone conformers.     

Synthesis,conformation, and activity of HCO-Met-ΔZ Leu-Phe-Ome,an active analogue of chemotactic N-formyltripeptides

In order to induce a β-turn conformation into the chemotactic linear tripeptide N-formyl-L -methionyl-L -leucyl-L -phenylalanine (fMLP), the new analogue N-formyl-L -methionyl-Δ^Zleucyl-L -phenylalanine methyl ester [ Δ^ZLeu]²f MLP-OMe ( 1 ) has been synthesized. The conformational and biochemical consequences of this chemical modification have been determined. Analogue 1 has been synthesized by using N-carboxy-(Z)-α,β-didehydroleucine anhydride as key compound to introduce the unsaturated residue at the central position of the tripeptide 1 . The x-ray analysis shows that 1 adopts in the crystal a type II β-turn conformation in which the new residue occupies the (i + 2) position, and an intramolecular H bond is formed between the formylic oxygen and the Phe NH. ¹H-nmr analysis based on nuclear Overhauser effect measurements suggests that the same folded conformation is preferred in CDCl₃ solution; this finding is also supported by molecular dynamics simulation. The biological activity of 1 has been determined on human neutrophils (polymorphonuclear leukocytes) and compared to that shown by f MLP-OMe. Chemotactic activity, granule enzyme release, and superoxide anion production have been determined. Analogue 1 is practically inactive as chemoattractant, highly active in the superoxide generation, and similar to the parent in the lysozyme release. The conformational restriction imposed on the backbone by the presence of the unsaturated residue is discussed in relation with the observed bioselectivity. © 1993 John Wiley & Sons, Inc.     

Structural study of an active analog of EX-4 in solution and micelle associated states

Of many drug candidates designed for treatment of type II diabetes, an exendin-4 (EX-4) analog from the substitutions of both beta-Asp for Glu3 and Tyrfor Glnl3 of EX-4 was found to have a prolongation in biological half life, an increase in cell proliferation and a remarkable improvement in reducing blood glucose with respect to EX-4. In this study, we applied CD and NMR approaches to characterize the structures of this active EX-4 analog in water, trifluoroethanol (TFE) aqueous solution, and dodecylphosphocholine (DPC) micelles and compared the results of the EX-4 analog with those of EX-4. Both EX-4 peptides adopt alpha-helix structures with the N-termini disordered and the C-terminal parts folded as hydrophobic clusters in these media. However, the analog has a longer helical extension in the N-terminal part than EX-4. The increasing helical turns may favor affinity for extracellular domain of glucagon-like peptide-1 receptor and accurate positioning of the crucial N-terminal residues in the transmembrane domains of the receptor. The analog has a stronger propensity to aggregate than the native EX-4, which is attributed to more coiled-coil interaction in the analog than in its native type. We also probed the association of EX-4 and its analog to DPC micelles and observed micelle-induced insertion of both peptides with their N- and C-termini as well as the central parts embedded in micelles and the residues near Asp9 and the residues around Trp25-Ser32 more water exposed. A single-step ligand-receptor binding model was suggested based on the analysis of these results.     

Solid phase synthesis and conformation of sequential glycosylated polytripeptide sequences related to antifreeze glycoproteins

Sequential glycopeptides [Thr(beta-D-galactose)-Ala-Ala]n, with n ranging from 2 to 7, as models of natural antifreeze glycoproteins were synthesized by the continuous flow, solid phase procedure. The conformational properties of these materials in solution were investigated by c.d. and 1H-n.m.r. spectroscopy. In aqueous solution the c.d. pattern is practically independent of chain length and is very similar to that of natural antifreeze glycoproteins. The results are interpreted in terms of random coil structure. The absence of ordered structures is further confirmed by n.m.r. data. A small amount of ordered conformation can be induced either by increasing the temperature of the aqueous solution or by addition of TFE. The c.d. pattern of all glycopeptides in water at temperatures higher than 50 degrees C are compatible with the presence of a small amount of alpha-helix or 3(10) helix. Since the glyco-hexapeptide is too short to form an alpha-helix, the hypothesis is made that in the glycopeptides in water at high temperature a small amount of 3(10) helix is formed. The same is observed for the 21-residue glycopeptide in presence of 85% (v/v) TFE. In this medium, the c.d. data on the glyco-hexapeptide are more compatible with the presence of a small amount of beta-structure.     

Prediction of Xaa-Pro peptide bond conformation from sequence and chemical shifts

We present a program, named Promega, to predict the Xaa-Pro peptide bond conformation on the basis of backbone chemical shifts and the amino acid sequence. Using a chemical shift database of proteins of known structure together with the PDB-extracted amino acid preference of cis Xaa-Pro peptide bonds, a cis/trans probability score is calculated from the backbone and ¹³C^β chemical shifts of the proline and its neighboring residues. For an arbitrary number of input chemical shifts, which may include Pro-¹³C^γ, Promega calculates the statistical probability that a Xaa-Pro peptide bond is cis. Besides its potential as a validation tool, Promega is particularly useful for studies of larger proteins where Pro-¹³C^γ assignments can be challenging, and for on-going efforts to determine protein structures exclusively on the basis of backbone and ¹³C^β chemical shifts.