Inhibition of Escherichia coli growth and respiration by polymyxin B covalently attached to agarose beads.

Polymyxin B was attached to agarose beads by stable covalent bonds and the antimicrobial activity of the immobilized peptide was examined. Polymyxin-agarose inhibited the growth of Escherichia coli and Pseudomonas aeruginosa, but not Bacillus subtilis. In addition, the respiration of E. coli, E. coli spheroplasts, and B. subtilis protoplasts was inhibited by immobilized polymyxin, whereas the respiration of B. subtilis was unaffected by polymyxin-agarose. The activity of polymyxin-agarose was not due to the release of free peptide from the derivative. These data indicate that polymyxin can inhibit the growth and respiration of gram-negative bacteria by interacting with the outer surface of these cells. It is proposed that perturbation of outer membrane structure by polymyxin-agarose indirectly affected the selective permeability of the inner membrane and inhibited respiration. The results of this study emphasize the importance of outer membrane structural integrity for the normal functions of gram-negative bacteria.     

Deuterium and phosphorus nuclear magnetic resonance studies on the binding of polymyxin B to lipid bilayer-water interfaces

Deuterium and phosphorus NMR methods have been used to study the binding of polymyxin B to the surface of bilayers containing lipids that were deuterated at specific positions in the polar head-group region. The binding of polymyxin B to acidic dimyristoylphosphatidylglycerol (DMPG) membranes induces only small structural distortions of the glycerol head group. The deuterium spin-lattice relaxation times for the different carbon-deuterium bonds in the head group of the same phospholipid are greatly reduced on binding of polymyxin B, indicating a restriction of the motional rate of the glycerol head group. Only very weak interactions were detected between polymyxin B and bilayers of zwitterionic dimyristoylphosphatidylcholine (DMPC). In mixed bilayers of the two phospholipid types, in which either of the two phospholipids was deuterated, the presence of polymyxin B caused a lateral phase separation into DMPG-enriched phospholipid-peptide clusters and a DMPG-depleted phase. Complete phase separation did not occur: peptide-containing complexes with charged phosphatidylglycerol contained substantial amounts of zwitterionic phosphatidylcholine. Exchange of both phospholipid types between complexes and the bulk lipid matrix was shown to be fast on the NMR time scale, with a lifetime for phospholipid-peptide association of less than 1 ms.     

Structural flexibility of Aib-containing peptides: the N-terminal tripeptide of trichotoxin

The sequence Aib-Gly-Aib which corresponds to the N-terminus of the microheterogeneous peptide antibiotic trichotoxin has been studied crystallographically in the context of different protecting groups. Peptides Ac-Aib-Gly-Aib-OH (A) and Z-Aib-Gly-Aib-OH (B) form beta-turns. Both peptides show a remarkable conformational flexibility forming a large variety of beta-turns of different types.     

Designed beta-hairpin peptides with defined tight turn stereochemistry

The conformational analysis of two synthetic octapeptides, Boc-Leu-Val-Val-D-Pro-L-Ala-Leu-Val-Val-OMe (1) and Boc-Leu-Val-Val-D-Pro-D-Ala-Leu-Val-Val-OMe (2) has been carried out in order to investigate the effect of beta-turn stereochemistry on designed beta-hairpin structures. Five hundred megahertz (1)H NMR studies establish that both peptides 1 and 2 adopt predominantly beta-hairpin conformations in methanol solution. Specific nuclear Overhauser effects provide evidence for a type II' beta-turn conformation for the D-Pro-L-Ala segment in 1, while the NMR data suggest that the type I' D-Pro-D-Ala beta-turn conformation predominates in peptide 2. Evidence for a minor conformation in peptide 2, in slow exchange on the NMR time scale, is also presented. Interstrand registry is demonstrated in both peptides 1 and 2. The crystal structure of 1 reveals two independent molecules in the crystallographic asymmetric unit, both of which adopt beta-hairpin conformations nucleated by D-Pro-L-Ala type II' beta-turns and are stabilized by three cross-strand hydrogen bonds. CD spectra for peptides 1 and 2 show marked differences, presumably as a consequence of the superposition of spectral bands arising from both beta-turn and beta-strand conformations.     

The histamine-liberating action of polymyxin B and its analogs

Histamine-releasing effect of polymyxin B1 and its deacylated analogues has been studied on purified rat mast cells. The structure-activity analysis showed that cyclic peptide fragment and acyl residue of molecule of polymyxin plays an important role in histamine-releasing activity. Histamine release, induced by polymyxin B1 and its analogue was blocked by metabolic inhibitor antimycin A. Preincubation of polymyxin B1 with lipopolysaccharide inhibits in dose-dependent manner polymyxin-induced histamine secretion from rat mast cells.     

Design of peptides with alpha,beta-dehydro-residues: synthesis, and crystal and molecular structure of a 3(10)-helical tetrapeptide Boc-L-Val-deltaPhe-deltaPhe-L-Ile-OCH3.

The peptide Boc-L-Val-deltaPhe-deltaPhe-L-Ile-OCH3 was synthesized using the azlactone method in the solution phase, and its crystal and molecular structures were determined by X-ray diffraction. Single crystals were grown by slow evaporation from solution in methanol at 25 degrees C. The crystals belong to an orthorhombic space group P2(1)2(1)2(1) with a = 12.882(7) A, b = 15.430(5) A, c = 18.330(5) A and Z = 4. The structure was determined by direct methods and refined by a least-squares procedure to an R-value of 0.073. The peptide adopts a right-handed 3(10)-helical conformation with backbone torsion angles: phi1 = 56.0(6)degrees, psi1 = -38.0(6)degrees, phi2 = -53.8(6)degrees, psi2 = 23.6(6)degrees, phi3 = -82.9(6)degrees, psi3 = -10.6(7)degrees, phi4 = 124.9(5)degrees. All the peptide bonds are trans. The conformation is stabilized by intramolecular 4-->1 hydrogen bonds involving Boc carbonyl oxygen and NH of deltaPhe3 and CO of Val1 and NH of Ile4. It is noteworthy that the two other chemically very similar peptides: Boc-Val-deltaPhe-deltaPhe-Ala-OCH3 (i) and Boc-Val-deltaPhe-deltaPhe-Val-OCH3 (ii) with differences only at the fourth position have been found to adopt folded conformations with two overlapping beta-turns of types II and III', respectively, whereas the present peptide adopts two overlapping beta-turns of type III. Thus the introduction of Ile at fourth position in a sequence Val-deltaPhe-deltaPhe-X results in the formation of a 3(10)-helix. The crystal structure is stabilized by intermolecular hydrogen bonds involving NH of Val1 and carbonyl oxygen of a symmetry related (-x, y - 1/2, 1/2 + z) deltaPhe2 and NH of deltaPhe2 with carbonyl oxygen of a symmetry related (x, y1/2, 1/2 + z) Ile4. This gives rise to long columns of helical molecules linked head to tail running along [010] direction.     

Isolation and partial characterization of antagonistic peptides produced by Paenibacillus sp. strain B2 isolated from the sorghum mycorrhizosphere

Paenibacillus sp. strain B2, isolated from the mycorrhizosphere of sorghum colonized by Glomus mosseae, produces an antagonistic factor. This factor has a broad spectrum of activity against gram-positive and gram-negative bacteria and also against fungi. The antagonistic factor was isolated from the bacterial culture medium and purified by cation-exchange, reverse-phase, and size exclusion chromatography. The purified factor could be separated into three active compounds following characterization by amino acid analysis and by combined reverse-phase chromatography and mass spectrometry (liquid chromatography-mass spectrometry and mass spectrometry-mass spectrometry). The first compound had the same retention time as polymyxin B1, whereas the two other compounds were more hydrophobic. The molecular masses of the latter compounds are 1,184.7 and 1,202.7 Da, respectively, and their structure is similar to that of polymyxin B1, with a cyclic heptapeptide moiety attached to a tripeptide side chain and a fatty acyl residue. They both contain threonine, phenylalanine, leucine, and 2,4-diaminobutyric acid residues. The peptide with a molecular mass of 1,184.7 contains a 2,3-didehydrobutyrine residue with a molecular mass of 101 Da replacing a threonine at the A2 position of the polymyxin side chain. This modification could explain the broader range of antagonistic activity of this peptide compared to that of polymyxin B.     

Role of a two-residue spacer in an alpha,beta-Didehydrophenylalanine containing hexapeptide: crystal and solution structure of Boc-val-deltaPhe-Leu-Ala-deltaPhe-Ala-OMe.

The peptide Boc-Val1-deltaPhe2-Leu3-Ala4-deltaPhe5-Ala6-OMe has been examined for the structural consequence of placing a two-residue segment between the deltaPhe residues. The peptide is stabilized by four consecutive beta-turns. The overall conformation of the molecule is a right-handed 3(10)-helix, with average (phi, psi) values (-67.7 degrees, -22.7 degrees), unwound at the C-terminus. The 1H NMR results also suggest that the peptide maintains its 3(10)-helical structure in solution as observed in the crystal state. The crystal structure is stabilized through head-to-tail hydrogen bonds and a repertoire of aromatic interactions laterally directed between adjacent helices, which are antiparallel to each other. The aromatic ring of deltaPhe5 forms the hub of multicentred interactions, namely as a donor in aromatic C-H...pi and aromatic C-H...O=C interactions and as an acceptor in a CH3...pi interaction. The present structure uniquely illustrates the unusual capability of a deltaPhe ring to host such concerted interactions and suggests its exploitation in introducing long-range interactions in the folding of supersecondary structures.     

Molecular mechanics studies of dermorphin

Molecular mechanical simulations have been carried out on dermorphin. Presence of D-Ala2 at the N-terminus and L-Pro6 residue at the C-terminus indicated the probability of beta-turns. From the stereochemical considerations, three types- II', III' and V' - for the beta-turn at the N-terminus of the peptide and two types-I and III- for the C-terminus side of the peptide are possible. In our molecular mechanics calculations, we considered six folded and one extended conformations for dermorphin to asses the relative stabilities. Three of the six folded conformations are lower in energy and have the following general feature-similar in energy, three hydrogen bonds, semirigid beta-sheet segment and favorable Tyr1-Tyr5 interaction. The presence of beta-sheet structure might play a role in mu-receptor selective interaction of dermorphin.     

Effects of polymyxin B sulfate and polymyxin B nonapeptide on growth and permeability of the yeast Saccharomyces cerevisiae

Summary Polymyxin B, a toxic, membrane-affecting antibiotic, can be rendered harmless to yeast cells by enzymatic removal of its fatty acyl moiety. The remaining cyclic peptide portion, polymyxin B nonapeptide, has no significant effect on growth and viability but it drastically reduces mating efficiency. In addition, the cyclic peptide enhances sensitivity of cells to several drugs, presumably by increasing membrane permeability. Mutants resistant to polymyxin B are simultaneously less responsive to the combination of the nonapeptide and the drugs. This indicates that the peptide portion of polymyxin B is the moiety responsible for the permeability changes. The resistance is inherited as a simple recessive trait. The mutation has been mapped to chromosome XV of Saccharomyces cerevisiae.     

Combined use of COSY and double quantum two-dimensional NMR spectroscopy for elucidation of spin systems in polymyxin B

Two-dimensional single quantum correlation NMR spectroscopy (COSY) and two-dimensional double quantum NMR spectroscopy (2QT) are used to study spin systems in the 1H NMR spectrum of polymyxin B. Because of different frequency relationships, the two types of two-dimensional NMR experiments are found to be highly complementary. This is demonstrated by combined use of COSY and 2QT spectroscopy to obtain a complete analysis of the complicated spectral overlap which occurs in the 1H NMR spectrum of polymyxin B.     

gβ- and gγ-turns in proteins revisited: A new set of amino acid turn-type dependent positional preferences and potentials

The number of beta-turns in a representative set of 426 protein three-dimensional crystal structures selected from the recent Protein Data Bank has nearly doubled and the number of gamma-turns in a representative set of 320 proteins has increased over seven times since the previous analysis. Beta-turns (7153) and gamma-turns (911) extracted from these proteins were used to derive a revised set of type-dependent amino acid positional preferences and potentials. Compared with previous results, the preference for proline, methionine and tryptophan has increased and the preference for glutamine, valine, glutamic acid and alanine has decreased for beta-turns. Certain new amino acid preferences were observed for both turn types and individual amino acids showed turn-type dependent positional preferences. The rationale for new amino acid preferences are discussed in the light of hydrogen bonds and other interactions involving the turns. Where main-chain hydrogen bonds of the type NH(i + 3) --> CO(i) were not observed for some beta-turns, other main-chain hydrogen bonds or solvent interactions were observed that possibly stabilize such beta-turns. A number of unexpected isolated beta-turns with proline at i + 2 position were also observed. The NH(i + 2) --> CO(i) hydrogen bond was observed for almost all gamma-turns. Nearly 20% classic gamma-turns and 43% inverse gamma-turns are isolated turns.     

Polymyxin B-phosphatidylglycerol interactions. A monolayer (π, ΔV) study

Through a monolayer investigation (π, ΔV), it is shown that the cationic antibiotic polymyxin B (or E) strongly interacts with films of acidic lipids, namely the didodecanoyl- and dihexadecanoylphosphatidylglycerol. The zwitterionic dihexadecanoylphosphatidylcholine was an unsuitable substrate. Interactions occurred at and above a polymyxin B concentration in the subphase of 2.5 · 10^?7 M, bringing about a considerable increase of both π and ΔV. These interactions proceeded in two steps, as revealed by a biphasic change of ΔV with time. They were independent of the film molecular packing (fluid or gel states) and of the initial film pressure.Since it was possible to monitor the relative number of polymyxin B and didodecanoyl- or dihexadecanoylphosphatidylglycerol molecules in the monolayer, it is demonstrated that, at saturation, one polymyxin B molecule is bound to five phosphatidylglycerol molecules, a result which accounts for an exact neutralization of the charges.From competition experiments, it is shown that Na⁺ is ineffective in removing polymyxin B from the interface. Ca²⁺ appeared to be a stronger competitor but no complete antibiotic desorption was observed even at a Ca²⁺ concentration of 100 mM.As a working hypothesis, the antibiotic/lipid ($\text{1}\text{5}$) system was assumed to constitute by itself one molecular species. The mixing of the polymyxin B/didodecanoylphosphatidylglycerol ($\text{1}\text{5}$) system with an excess of lipid molecules in the monolayer was found to be ideal both in terms of π and ΔV. With dihexadecanoylphosphatidylglycerol, a small condensing effect could be detected only at intermediate surface pressures, in a region where the lipid phase transition occurred.The molecular area of polymyxin B interacting with didodecanoylphosphatidylglycerol can be calculated to be 1.23 ± 0.05 nm². It is proposed that the whole antibiotic molecule penetrates the film, the five bound lipid molecules being distributed around the peptide structure, at given positions imposed by the five 2,4-diaminobutyric acid residues.     

Structure characterization of the central repetitive domain of high molecular weight gluten proteins. II. Characterization in solution and in the dry state.

The structure of the central repetitive domain of high molecular weight HMW) wheat gluten proteins was characterized in solution and in the dry state using HMW proteins Bx6 and Bx7 and a subcloned, bacterially expressed part of the repetitive domain of HMW Dx5. Model studies of the HMW consensus peptides PGQGQQ and GYYPTSPQQ formed the basis for the data analysis (van Dijk AA et al., 1997, Protein Sci 6:637-648). In solution, the repetitive domain contained a continuous nonoverlapping series of both type I and type II II beta-turns at positions predicted from the model studies; type II beta-turns occurred at QPGQ and QQGY sequences and type I beta-turns at YPTS and SPQQ. The subcloned part of the HMW Dx5 repetitive domain sometimes migrated as two bands on SDS-PAGE; we present evidence that this may be caused by a single amino acid insertion that disturbs the regular structure of beta-turns. The type I beta-turns are lost when the protein is dried on a solid surface, probably by conversion to type II beta-turns. The homogeneous type II beta-turn distribution is compatible with the formation of a beta-spiral structure, which provides the protein with elastic properties. The beta-turns and thus the beta-spiral are stabilized by hydrogen bonds within and between turns. Reformation of this hydrogen bonding network after, e.g., mechanical disruption may be important for the elastic properties of gluten proteins.     

Novel peptide surface for reversible immobilization of concanavalin A

Concanavalin A (Con A) was spontaneously adsorbed on polymyxin B surface. This peptide-lectin interaction was strong, K(D)=1.9 x 10(-10), based predominantly on creation of hydrophobic bonds, and was completely reversible. Concanavalin A on polymyxin B (PmB) retained higher binding capacity for yeast mannan, compared with covalently immobilized lectin. Kinetics of mannan-concanavalin A interaction were significantly different in dependence on type of concanavalin A immobilization.     

Self-assembly and secondary structure of beta-casein

The secondary structure alterations during isothermal and temperature guided beta-casein micellization were studied by dynamic light scattering, circular dichroism and Fourier transform infrared spectroscopy techniques. Micelle formation induced by the increase in the protein concentration at constant temperature is accompanied by the formation of a small number of additional peptide hydrogen bonds, preliminary assigned to the intraprotein beta-structure. The heating results in more pronounced but qualitatively different changes consisted in dehydration of the peptide groups and disruption of the polyproline II helix segments with the subsequent conversion to the random conformation and the beta-turns. Nevertheless, in both cases the total number of residues involved in the transition is very few and cannot be regarded as a decisive factor for casein micellization.     

Effect of polymyxin B on ion permeability of bacterial membranes

Effect of polymyxin B on the movement of K+ and H+ in polymyxin-sensitive cells of E. coli under different metabolic states has been studied. It was shown that polymyxin B induced the efflux of K+, decreased the efflux of H+ and inhibited the consumption of oxygen in bacterial cells. The effect of antibiotic on ion movement was independent of respiratory conditions. It was suggested that polymyxin B increased ion permeability and destroyed lipid-protein interactions of the respiratory chain simultaneously.     

Isolation and Partial Characterization of Antagonistic Peptides Produced by Paenibacillus sp. Strain B2 Isolated from the Sorghum Mycorrhizosphere

Paenibacillus sp. strain B2, isolated from the mycorrhizosphere of sorghum colonized by Glomus mosseae, produces an antagonistic factor. This factor has a broad spectrum of activity against gram-positive and gram-negative bacteria and also against fungi. The antagonistic factor was isolated from the bacterial culture medium and purified by cation-exchange, reverse-phase, and size exclusion chromatography. The purified factor could be separated into three active compounds following characterization by amino acid analysis and by combined reverse-phase chromatography and mass spectrometry (liquid chromatography-mass spectrometry and mass spectrometry-mass spectrometry). The first compound had the same retention time as polymyxin B₁, whereas the two other compounds were more hydrophobic. The molecular masses of the latter compounds are 1,184.7 and 1,202.7 Da, respectively, and their structure is similar to that of polymyxin B₁, with a cyclic heptapeptide moiety attached to a tripeptide side chain and a fatty acyl residue. They both contain threonine, phenylalanine, leucine, and 2,4-diaminobutyric acid residues. The peptide with a molecular mass of 1,184.7 contains a 2,3-didehydrobutyrine residue with a molecular mass of 101 Da replacing a threonine at the A2 position of the polymyxin side chain. This modification could explain the broader range of antagonistic activity of this peptide compared to that of polymyxin B.