Peptide-lanthanide interactions. Crystal structure of a europium(III)-triglycine complex

Crystals of Eu-(Gly-Gly-Gly).(H2O)5.(ClO4)3 are triclinic, spacegroup P1BAR with a = 9.123 (2), b = 11.185 (5), c = 11.426 (2) angstrom; alpha = 90.79 (2), beta = 98.08 (1), gamma = 98.57 (2)-degrees; Z = 2. The europium cation is surrounded by four oxygens from three different peptide units and four oxygens from water molecules. The geometry around the metal is a distorted bi-capped trigonal prism. The peptide backbone conformation in this complex is compared with those in the free peptide and in various metal complexes. Considerable differences are observed between Eu(III) and Ca(II) complexes of triglycine. (C) Munksgaard 1994.

Hydration and distortion of peptide helices in crystals. α-Helical structure of a dodecapeptide, Boc-(Ala-Leu-Aib)4-OMe

The dodecapeptide Boc-(Ala-Leu-Aib)(4)-OMe crystallized with two independent helical molecules in a triclinic cell. The two molecules are very similar in conformation, with a 3(10)-helix turn at the N-terminus followed by an alpha-helix, except for an elongated N(7)...O(3) distance in both molecules. All the helices in the crystal pack in a parallel motif. Eleven water sites have been found in the head-to-tail region between the apolar helices that participate in peptide-water hydrogen bonds and a network of water-water hydrogen bonds. The crystal parameters are as follows: 2(C58H104N12O15)+ca. 10H(2)O, space group P1 with a = 12.946(2), b = 17.321(3), c = 20.465(4) Angstrom, alpha = 103.12(2), beta = 105.63(2), gamma = 107.50(2)degrees, Z = 2, R = 10.9% for 5152 data observed > 3 sigma(F), resolution 1.0 Angstrom. In contrast to the shorter sequences [Karle et al. (1988)Proc. Natl. Acad. Sci. USA 85, 299-303] and Boc-(Ala-Leu-Aib)(2)-OMe [Karle et al. (1989) Biopolymers 28, 773-781], no insertion of a water molecule into the helix is observed. However, the elongated N---O distance between Ala(7) NH and Aib(3) CO in both molecules (molecule A, 3.40 Angstrom; molecule B, 3.42 Angstrom) is indicative of an incipient break in the helices. (C) Munksgaard 1994.

Stereochemical analysis of the antigenic tip of the V3 loop peptide of HIV-1 gp120

A novel multiple turn conformation has been observed for a segment GPGRAFY in the crystal structure of a complex of HIV-1 gp120 V3 loop peptide with the Fab fragment of a neutralizing antibody [Ghiara ct al. (1994) Science 264, 82-85]. A structural motif has been defined for the peptide segment, employing idealized backbone conformations characterized by ranges of virtual C-alpha torsion angles and bond angles. A search of 122 high-resolution protein crystal structures has permitted identification of 24 examples of similar structural motifs. Two major conformational families have been identified, which differ primarily in the conformation at residue 3. The observed conformation at residue 3 in family 1 is left-handed helical (alpha(L)) and that in family 2 is right-handed helical (alpha(R)). Of the 10 examples in family 1, 9 examples have Gly residues at position 3. Of the 12 examples in family 2, 7 examples have Asn/Asp at position 3. Computer modeling of the V3 loop tip sequence using the two backbone conformational families as starting points leads to minimum-energy conformations in which antigenically important side-chains occupy similar spatial arrangements. This stereochemical analysis of the V3 loop tip sequence suggests a rational basis for the design of synthetic analog peptides for use as viral antagonists or synthetic antigens. (C) Munksgaard 1995.

Stereochemical Analysis of Higher α,α-Dialkylglycine Containing Peptides. Characterization of Local Helical Conformations at Dipropylglycine Residues and Observation of a Novel Hydrated Multiple β-Turn Structure in Crystals of a Glycine Rich Peptide

The peptide Boc-Gly-Dpg-Gly-Gly-Dpg-Gly-NHMe (1) has been synthesized to examine the conformational preferences of Dpg residues in the context of a poor helix promoting sequence. Single crystals of 1 were obtained in the space group P21/c with a = 13.716(2) Å, b = 12.960(2) Å, c = 22.266(4) Å, and β = 98.05(1)°; R = 6.3% for 3660 data with |Fo| > 4σ. The molecular conformation in crystals revealed that the Gly(1)-Dpg(2) segment adopts φ, ψ values distorted from those expected for an ideal type II‘ β-turn (φGly(1) = +72.0°, ψGly(1) = −166.0°; φDpg(2) = −54.0°, ψDpg(2) = −46.0°) with an inserted water molecule between Boc-CO and Gly(3)NH. The Gly(3)-Gly(4) segment adopts φ, ψ values which lie broadly in the right handed helical region (φGly(3) = −78.0°, ψGly(3) = −9.0°; φGly(4) = −80.0°, ψGly(4) = −18.0°). There is a chiral reversal at Dpg(5) which takes up φ, ψ values in the left handed helical region. The Dpg(5)-Gly(6) segment closely resembles an ideal type I‘ β-turn (φDpg(5) = +56.0°, ψDpg(5) = +32.0°; φGly(6) = +85.0°, ψGly(6) = −3.0°). Molecules of both chiral senses are found in the centrosymmetric crystal. The C-terminus forms a hydrated Schellman motif, with water insertion into the potential 6 → 1 hydrogen bond between Gly(1)CO and Gly(6)NH. NMR studies in CDCl3 suggest substantial retention of the multiple turn conformation observed in crystals. In solution the observed NOEs support local helical conformation at the two Dpg residues.

The role of polar and facial amphipathic character in determining lipopolysaccharide-binding properties in synthetic cationic peptides

Two series of peptides, designated K and NK were synthesized and tested for lipid A binding and neutralizing properties. K-2, which has an 11-residue amphiphilic core, and a branched N-terminus bearing two branched lysinyl residues does not bind lipid A, while NK2, also with an 11-residue amphiphilic core comprised entirely of non-ionizable residues, and a similarly branched, cationic N-terminus, binds lipid A very weakly. Both peptides do not inhibit lipopolysaccharide (LPS) activity in the Limulus assay, nor do they inhibit LPS-induced TNF-alpha and NO production in 5774 cells. These results are entirely unlike a homologous peptide with an exclusively hydrophobic core whose LPS-binding and neutralizing properties are very similar to that of polymyxin B [David SA, Awasthi SK, Wiese A et al. Characterization of the interactions of a polycationic, amphiphilic, terminally branched oligopeptide with lipid A and lipopolysaccharide from the deep rough mutant of Salmonella minnesota. J Endotoxin Res 1996; 3: 369-379]. These data suggest that a clear segregation of charged and apolar domains is crucial in molecules designed for purposes of LPS sequestration and that head-tail (polar) orientation of the cationic/hydrophobic regions is preferable to molecules with mixed or facial cationic/amphipathic character.

Hybrid peptide hairpins containing alpha- and omega-amino acids: Conformational analysis of decapeptides with unsubstituted beta-, gamma-, and delta-residues at positions 3 and 8

The effects of inserting unsubstituted omega-amino acids into the strand segments of model beta-hairpin peptides was investigated by using four synthetic decapeptides, Boc-Lcu-Val-Xxx-Val-D-Pro-Gly-Leu-Xxx-Val-Val- OMe: pepticle 1 (Xxx=Gly), pepticle 2 (Xxx=beta Gly=beta hGly=homoglycine, beta-glycine), pepticle 3 (Xxx=gamma Abu=gamma-aminobutyric acid), pepticle 4 (Xxx= delta Ava=delta-aminovaleric acid). H-1 NMR studies (500 MHz, methanol) reveal several critical cross-strand NOEs, providing evidence for P-hairpin conformations in peptides 2-4. In peptide 3, the NMR results support the formation of the nucleating turn, however, evidence for cross-strand registry is not detected. Single-crystal X-ray diffraction studies of peptide 3 reveal a beta-hairpin conformation for both molecules in the crystallographic asymmetric unit, stabilized by four cross-strand hydrogen bonds, with the gamma Abu residues accommodated within the strands. The D-Pro-Gly segment in both molecules (A,B) adopts a type II' beta-turn conformation. The circular dichroism spectrum for peptide 3 is characterized by a negative CD band at 229 rim, whereas for peptides 2 and 4, the negative band is centered at 225 nm, suggesting a correlation between the orientation of the amide units in the strand segments and the observed CD pattern.

Polypeptide Helices in Hybrid Peptide Sequences

A new class of polypeptide helices in hybrid sequences containing alpha-, beta-, and gamma-residues is described. The molecular conformations in crystals determined for the synthetic peptides Boc-Leu-Phe-Val-Aib-beta Phe-Leu-Phe-Val-OMe 1 (beta Phe: (S)-beta(3)-homophenylalanine) and Boc-Aib-Gpn-AibGpn-OM2(Gpn:1-(aminomethyl)cycl hexaneacetic acid) reveal expanded helical turns in the hybrid sequences (alpha alpha beta)(n) and (ay), In 1, a repetitive helical structure composed Of C-14 hydrogen-bonded units is observed, whereas 2 provides an example of a repetitive C-12 hydrogen-bonded structure. Using experimentally determined backbone torsion angles for the hydrogen-bonded units formed by hybrid sequences, we have generated energetically favorable hybrid helices. Conformational parameters are provided for C-11, C-12, C-13, C-14, and C-15 helices in hybrid sequences.

Characterization of Alkali Induced Formation of Lanthionine, Trisulfides, and Tetrasulfides from Peptide Disulfides using Negative Ion Mass Spectrometry

Peptide disulfides are unstable under alkaline conditions, resulting in the formation of products containing lanthionine and polysulfied linkages. Electrospray ionization mass spectrometry has been used to characterize major species obtained when cyclic and acyclic peptide disulfides are exposed to alkaline media. Studies on a model cyclic peptide disulfide (Boc - Cys - Pro - Leu - Cys - NHMe) and an acyclic peptide, oxidized glutathione, bis ((gamma)Glu Cys - Gly - COOH), are described. Disulfide cleavage reactions are initiated by the abstraction of (CH)-H-alpha or (CH)-H-beta protons of Cys residues, with Subsequent elimination of H2S or H2S2. The buildup of reactive thiol species which act on intermediates containing dehydroalanine residues, rationalizes the formation of lanthionine and polysulfide products. In the case of the cyclic peptide disulfide, the formation of cyclic products is facilitated by the intramolecular nature of the Michael addition reaction of thiols to the dehydroalanine residue. Mass spectral evidence for the intermediate species is presented by using alkylation of thiol groups as a trapping method. Mass spectral fragmentation in the negative ion mode of the peptides derived from trisulfides and tetrasulfides results in elimination of S-2. (J Am Soc Mass Spectrom 2009, 20, 783-791) (C) 2009 American Society for Mass Spectrometry.

Characterization of Alkali Induced Formation of Lanthionine, Trisulfides, and Tetrasulfides from Peptide Disulfides using Negative Ion Mass Spectrometry

Peptide disulfides are unstable under alkaline conditions, resulting in the formation of products containing lanthionine and polysulfied linkages. Electrospray ionization mass spectrometry has been used to characterize major species obtained when cyclic and acyclic peptide disulfides are exposed to alkaline media. Studies on a model cyclic peptide disulfide (Boc - Cys - Pro - Leu - Cys - NHMe) and an acyclic peptide, oxidized glutathione, bis ((gamma)Glu Cys - Gly - COOH), are described. Disulfide cleavage reactions are initiated by the abstraction of (CH)-H-alpha or (CH)-H-beta protons of Cys residues, with Subsequent elimination of H2S or H2S2. The buildup of reactive thiol species which act on intermediates containing dehydroalanine residues, rationalizes the formation of lanthionine and polysulfide products. In the case of the cyclic peptide disulfide, the formation of cyclic products is facilitated by the intramolecular nature of the Michael addition reaction of thiols to the dehydroalanine residue. Mass spectral evidence for the intermediate species is presented by using alkylation of thiol groups as a trapping method. Mass spectral fragmentation in the negative ion mode of the peptides derived from trisulfides and tetrasulfides results in elimination of S-2. (J Am Soc Mass Spectrom 2009, 20, 783-791) (C) 2009 American Society for Mass Spectrometry.

Tribology of steel/steel interaction in oil-in-water mulsion; a rationale for lubricity

Oil droplets are dispersed in water by an anionic urfactant to form an emulsion. The lubricity of this emulsion in steel/steel interaction is explored in a ball on flat nanotribometer. The droplet size and charge are measured using dynamic light scattering, while the substrate charge density is estimated using the pH titration method. These data are combined to calculate the DLVO forces for the droplets generated for a range of surfactant concentration and two oil to water volume ratios. The droplets have a clear bi-modal size distribution. The study shows that the smaller droplets which experience weak repulsion are situated (at the highest DLVO barrier) much closer to the substrate than thebigger droplets, which experience the same DLVO force, are. We suggest that the smaller droplets thus play a more important role in lubricity than what the bigger droplets do. The largest volume of such small droplets occurs in the 0.5 mM-1 mM range of surfactant concentration and 1% oil to water volume ratio, where the coefficient of friction is also observed to be the least. (C) 2010 Elsevier Inc. All rights reserved.

N-H center dot center dot center dot O and C-H center dot center dotcenter dot O interaction mimicry in the 1:1 molecular complexes of 5,5-diethylbarbituric acid with urea and acetamide

The analogy between N-H center dot center dot center dot O and C-H center dot center dot center dot O intermolecular interactions is studied with variable temperature (180-100 K) single crystal X-ray diffraction analysis.5,5-Diethylbarbituric acid (barbital) forms isostructural molecular complexes (co-crystals) with urea (1) and acetamide (2) that respectively contain these analogous interactions.The behaviour of these two interactions as a function of temperature is very similar. This indicates that the C-H center dot center dot center dot O bond in barbital acetamide plays a similar chemical and structural role as does the N-H center dot center dot center dot O bond in barbital urea. The close relationship between these interactions and their comparable nature is further adduced from the formation of a ternary solid solution (3) of barbital, urea and acetamide. The fact that the C-H center dot center dot center dot O interaction in barbital acetamide is weaker than the N-H center dot center dot center dot O interaction in barbital urea is shown by the fact that acetamide is under expressed and urea is over expressed with respect to the quantities of these substances present in solution prior to crystallization of these ternary crystals.

Crystal structure of dimeric FabZ of Plasmodium falciparum reveals conformational switching to active hexamers by peptide flips

The crystal structure of beta-hydroxyacyl acyl carrier protein dehydratase of Plasmodium falciparum (PfFabZ) has been determined at a resolution of 2.4 angstrom. PfFabZ has been found to exist as a homodimer (d-PfFabZ) in the crystals of the present study in contrast to the reported hexameric form (h-PfFabZ) which is a trimer of dimers crystallized in a different condition. The catalytic sites of this enzyme are located in deep narrow tunnel-shaped pockets formed at the dimer interface. A histidine residue from one subunit of the dimer and a glutamate residue from the other subunit lining the tunnel form the catalytic dyad in the reported crystal structures. While the position of glutamate remains unaltered in the crystal structure of d-PffabZ compared to that in b-PfFabZ, the histidine residue takes up an entirely different conformation and moves away from the tunnel leading to a His-Phe cis-trans peptide flip at the histidine residue. In addition, a loop in the vicinity has been observed to undergo a similar flip at a Tyr-Pro peptide bond. These alterations not only prevent the formation of a hexamer but also distort the active site geometry resulting in a dimeric form of FabZ that is incapable of substrate binding. The dimeric state and an altered catalytic site architecture make d-PfFabZ distinctly different from the FabZ structures described so far. Dynamic light scattering and size exclusion chromatographic studies clearly indicate a pH-related switching of the dimers to active hexamers. (c) 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserv.

Exploring the interaction energies for the binding of hydroxydiphenyl ethers to enoyl-acyl carrier protein reductases

It is now well established that the potent anti-microbial compound, triclosan, interrupts the type II fatty acid synthesis by inhibiting the enzyme enoyl-ACP reductase in a number of organisms. Existence of a high degree of similarity between the recently discovered enoyl-ACP reductase from R falciparum and B. napus enzyme permitted building of a satisfactory model for the former enzyme that explained some of the key aspects of the enzyme such as its specificity for binding to the cofactor and the inhibitor. We now report the interaction energies between triclosan and other hydroxydiphenyl ethers with the enzymes from B. napus, E. coli and R falciparum. Examination of the triclosan-enzyme interactions revealed that subtle differences exist in the ligand binding sites of the enzymes from different sources i.e., B. napus, E. coli and P falciparum. A comparison of their binding propensities thus determined should aid in the design of effective inhibitors for the respective enzymes.

Kinetics and the Mechanism of Interaction of the Endoplasmic Reticulum Chaperone, Calreticulin, with Monoglucosylated (Glc1Man9GlcNAc2) Substrate

Calreticulin is a lectin-like molecular chaperone of the endoplasmic reticulum in eukaryotes. Its interaction with N-glycosylated polypeptides is mediated by the glycan, Glc(1)Man(9)GlcNAc(2), present on the target glycoproteins. In this work, binding of monoglucosyl IgG (chicken) substrate to calreticulin has been studied using real time association kinetics of the interaction with the biosensor based on surface plasmon resonance (SPR). By SPR, accurate association and dissociation rate constants were determined, and these yielded a micromolar association constant. The nature of reaction was unaffected by immobilization of either of the reactants. The Scatchard analysis values for K-a agreed web crith the one obtained by the ratio k(1)/k(-1). The interaction was completely inhibited by free oligosaccharide, Glc(1)Man(9)GlcNAc(2), whereas Man(9)GlcNAc(2) did not bind to the calreticulin-substrate complex, attesting to the exquisite specificity of this interaction. The binding of calreticulin to IgG was used for the development of immunoassay and the relative affinity of the lectin-substrate association was indirectly measured. The values are in agreement with those obtained with SPR. Although the reactions are several orders of magnitude slower than the diffusion controlled processes, the data are qualitatively and quantitatively consistent with single-step bimolecular association and dissociation reaction. Analyses of the activation parameters indicate that reaction is enthalpically driven and does not involve a highly ordered transition state. Based on these data, the mechanism of its chaperone activity is briefly discussed.

Specificity for the Exchange of Phospholipids Through Polymyxin B Mediated Intermembrane Molecular Contacts

Structural specificity for the direct vesicle−vesicle exchange of phospholipids through stable molecular contacts formed by the antibiotic polymyxin B (PxB) is characterized by kinetic and spectroscopic methods. As shown elsewhere [Cajal, Y., Rogers, J., Berg, O. G., & Jain, M. K. (1996) Biochemistry 35, 299−308], intermembrane molecular contacts between anionic vesicles are formed by a small number of PxB molecules, which suggests that a stoichiometric complex may be responsible for the exchange of phospholipids. Larger clusters containing several vesicles are formed where each vesicle can make multiple contacts if sterically allowed. In this paper we show that the overall process can be dissected into three functional steps: binding of PxB to vesicles, formation of stable vesicle−vesicle contacts, and exchange of phospholipids. Polycationic PxB binds to anionic vesicles. Formation of molecular contacts and exchange of monoanionic phospholipids through PxB contacts does not depend on the chain length of the phospholipid. Only monoanionic phospholipids (with methanol, serine, glycol, butanol, or phosphatidylglycerol as the second phosphodiester substituent in the head group) exchange through these contacts, whereas dianionic phosphatidic acid does not. Selectivity for the exchange was also determined with covesicles of phosphatidylmethanol and other phospholipids. PxB does not bind to vesicles of zwitterionic phosphatidylcholine, and its exchange in covesicles is not mediated by PxB. Vesicles of dianionic phospholipids, like phosphatidic acid, bind PxB; however, this phospholipid does not exchange. The structural features of the contacts are characterized by the spectroscopic and chemical properties of PxB at the interface. PxB in intermembrane contacts is readily accessible from the aqueous phase to quenchers and reagents that modify amino groups. Results show that PxB at the interface can exist in two forms depending on the lipid/PxB ratio. Additional studies show that stable PxB-mediated vesicle−vesicle contacts may be structurally and functionally distinct from “stalks”, the putative transient intermediate for membrane fusion. The phenomenon of selective exchange of phospholipids through peptide-mediated contacts could serve as a prototype for intermembrane targeting and sorting of phospholipids during their biosynthesis and trafficking in different compartments of a cell. The protocols and results described here also extend the syllogistic foundations of interfacial equilibria and catalysis.