Enhanced Amphiphilic Profile of a Short β-Stranded Peptide Improves Its Antimicrobial Activity

SB056 is a novel semi-synthetic antimicrobial peptide with a dimeric dendrimer scaffold. Active against both Gram-negative and -positive bacteria, its mechanism has been attributed to a disruption of bacterial membranes. The branched peptide was shown to assume a β-stranded conformation in a lipidic environment. Here, we report on a rational modification of the original, empirically derived linear peptide sequence [WKKIRVRLSA-NH₂, SB056-lin]. We interchanged the first two residues [KWKIRVRLSA-NH₂, β-SB056-lin] to enhance the amphipathic profile, in the hope that a more regular β-strand would lead to a better antimicrobial performance. MIC values confirmed that an enhanced amphiphilic profile indeed significantly increases activity against both Gram-positive and -negative strains. The membrane binding affinity of both peptides, measured by tryptophan fluorescence, increased with an increasing ratio of negatively charged/zwitterionic lipids. Remarkably, β-SB056-lin showed considerable binding even to purely zwitterionic membranes, unlike the original sequence, indicating that besides electrostatic attraction also the amphipathicity of the peptide structure plays a fundamental role in binding, by stabilizing the bound state. Synchrotron radiation circular dichroism and solid-state ¹⁹F-NMR were used to characterize and compare the conformation and mobility of the membrane bound peptides. Both SB056-lin and β-SB056-lin adopt a β-stranded conformation upon binding POPC vesicles, but the former maintains an intrinsic structural disorder that also affects its aggregation tendency. Upon introducing some anionic POPG into the POPC matrix, the sequence-optimized β-SB056-lin forms well-ordered β-strands once electro-neutrality is approached, and it aggregates into more extended β-sheets as the concentration of anionic lipids in the bilayer is raised. The enhanced antimicrobial activity of the analogue correlates with the formation of these extended β-sheets, which also leads to a dramatic alteration of membrane integrity as shown by ³¹P-NMR. These findings are generally relevant for the design and optimization of other membrane-active antimicrobial peptides that can fold into amphipathic β-strands.     

A critical evaluation of the conformational requirements of fusogenic peptides in membranes

It is generally assumed that fusogenic peptides would require a certain conformation, which triggers or participates in the rate-determining step of membrane fusion. Previous structure analyses of the viral fusion peptide from gp41 of HIV-1 have yielded contradictory results, showing either an α-helical or a β-stranded conformation under different conditions. To find out whether either of these conformations is relevant in the actual fusion process, we have placed sterically demanding substitutions into the fusion peptide FP23 to prevent or partially inhibit folding and self-assembly. A single substitution of either D- or L-CF₃-phenylglycine was introduced in different positions of the sequence, and the capability of these peptide analogues to fuse large unilamellar vesicles was monitored by lipid mixing and dynamic light scattering. If fusion proceeds via a β-stranded oligomer, then the D- and L-epimers are expected to differ systematically in their activity, since the D-epimers should be unable to form β-structures due to sterical hindrance. If an α-helical conformation is relevant for fusion, then the D-epimers would be slightly disfavoured compared to the L-forms, hence a small systematic difference in fusion activity should be observed. Interestingly, we find that (1) all D- and L-epimers are fusogenically active, though to different extents compared to the wild type, and – most importantly – (ii) there is no systematic preference for either the D- or L-forms. We therefore suggest that a well-structured α-helical peptide conformation or a β-stranded oligomeric assembly can be excluded as the rate-determining state. Instead, fusion appears to involve conformationally disordered peptides with a pronounced structural plasticity. Dedicated to Prof. K. Arnold on the occasion of this 65th birthday.     

Conformational space search of Neuromedin C using replica exchange molecular dynamics and molecular dynamics

The present study involves the utilization of replica exchange molecular dynamics (REMD) methodology to explore the conformational space of Neuromedin C (NMC) using implicit (REMD^implicit) and explicit (REMD^explicit) water models. Comparison of the structures obtained from these simulations indicate that REMD^explicit trajectory display a greater tendency to induce β‐turns and bent structures as compared to those obtained from the REMD^implicit simulation. Moreover, two additional MD trajectories performed using Langevin (MD^Lang) and Berendsen (MD^Berend) algorithms under generalized born (GB) solvent conditions were also suitably competent to sample similar kinds of conformations, although the extent of beta turns was low compared to those observed in REMD^explicit simulation. Finally, the comparison of results obtained from all the trajectories and those derived from the NMR studies of Ni(II) complex of NMC indicates that the REMD under explicit conditions is more efficient in sampling the conformations, and show good agreement with the experimental results. Copyright © 2010 European Peptide Society and John Wiley & Sons, Ltd.     

Concentration-dependent realignment of the antimicrobial peptide PGLa in lipid membranes observed by solid-state 19F-NMR

The membrane-disruptive antimicrobial peptide PGLa is found to change its orientation in a dimyristoyl-phosphatidylcholine bilayer when its concentration is increased to biologically active levels. The alignment of the alpha-helix was determined by highly sensitive solid-state NMR measurements of (19)F dipolar couplings on CF(3)-labeled side chains, and supported by a nonperturbing (15)N label. At a low peptide/lipid ratio of 1:200 the amphiphilic peptide resides on the membrane surface in the so-called S-state, as expected. However, at high peptide concentration (>/=1:50 molar ratio) the helix axis changes its tilt angle from approximately 90 degrees to approximately 120 degrees , with the C-terminus pointing toward the bilayer interior. This tilted "T-state" represents a novel feature of antimicrobial peptides, which is distinct from a membrane-inserted I-state. At intermediate concentration, PGLa is in exchange between the S- and T-state in the timescale of the NMR experiment. In both states the peptide molecules undergo fast rotation around the membrane normal in liquid crystalline bilayers; hence, large peptide aggregates do not form. Very likely the obliquely tilted T-state represents an antiparallel dimer of PGLa that is formed in the membrane at increasing concentration.     

Peptide-Lipid Interactions of the Stress-Response Peptide TisB That Induces Bacterial Persistence

The bacterial stress-response peptide TisB in Escherichia coli has been suggested to dissipate the transmembrane potential, such that the depletion of ATP levels induces the formation of dormant persister cells which can eventually form biofilms. We studied the structure and membrane interactions of TisB to find out whether it forms pores or other proton-selective channels. Circular dichroism revealed an amphiphilic α-helical structure when reconstituted in lipid vesicles, and oriented circular dichroism showed that the helix assumes a transmembrane alignment. The addition of TisB to dye-loaded vesicles caused leakage only at very high peptide concentration, notably with a Hill coefficient of 2, which suggests that dimers must be involved. Coarse-grained molecular dynamics simulations showed that membrane binding of monomeric TisB is rapid and spontaneous, and transmembrane insertion is energetically feasible. When TisB oligomers are assembled as transmembrane pores, these channels collapse during the simulations, but transmembrane dimers are found to be stable. Given the pattern of charges on the amphiphilic TisB helix, we postulate that antiparallel dimers could be assembled via a ladder of salt bridges. This electrostatic charge-zipper could enable protons to pass along a wire of trapped water molecules across the hydrophobic membrane.     

A novel dendrimeric peptide with antimicrobial properties: structure-function analysis of SB056

The novel antimicrobial peptide with a dimeric dendrimer scaffold, SB056, was empirically optimized by high-throughput screening. This procedure produced an intriguing primary sequence whose structure-function analysis is described here. The alternating pattern of hydrophilic and hydrophobic amino acids suggests the possibility that SB056 is a membrane-active peptide that forms amphiphilic β-strands in a lipid environment. Circular dichroism confirmed that the cationic SB056 folds as β-sheets in the presence of anionic vesicles. Lipid monolayer surface pressure experiments revealed unusual kinetics of monolayer penetration, which suggest lipid-induced aggregation as a membranolytic mechanism. NMR analyses of the linear monomer and the dendrimeric SB056 in water and in 30% trifluoroethanol, on the other hand, yielded essentially unstructured conformations, supporting the excellent solubility and storage properties of this compound. However, simulated annealing showed that many residues lie in the β-region of the Ramachandran plot, and molecular-dynamics simulations confirmed the propensity of this peptide to fold as a β-type conformation. The excellent solubility in water and the lipid-induced oligomerization characteristics of this peptide thus shed light on its mechanism of antimicrobial action, which may also be relevant for systems that can form toxic β-amyloid fibrils when in contact with cellular membranes. Functionally, SB056 showed high activity against Gram-negative bacteria and some limited activity against Gram-positive bacteria. Its potency against Gram-negative strains was comparable (on a molar basis) to that of colistin and polymyxin B, with an even broader spectrum of activity than numerous other reference compounds.     

Effect of maternal micronutrients (folic acid, vitamin B12) and omega 3 fatty acids on liver fatty acid desaturases and transport proteins in Wistar rats

A disturbed fatty acid metabolism increases the risk of adult non-communicable diseases. This study examines the effect of maternal micronutrients on the fatty acid composition, desaturase activity, mRNA levels of fatty acid desaturases and transport proteins in the liver. Pregnant female rats were divided into 6 groups at 2 levels of folic acid both in the presence and absence of vitamin B(12). The vitamin B(12) deficient groups were supplemented with omega 3 fatty acid. An imbalance of maternal micronutrients reduces liver docosahexaenoic acid, increases Δ5 desaturase activity but decreases mRNA levels, decreases Δ6 desaturase activity but not mRNA levels as compared to control. mRNA level of Δ5 desaturase reverts back to the levels of the control group as a result of omega 3 fatty acid supplementation. Our data for the first time indicates that maternal micronutrients differentially alter the activity and expression of fatty acid desaturases in the liver.     

Some cases of onychomycosis from North India in different working environments

Three fungi, Alternaria humicola, A. pluriseptata and Aspergillus niger are being reported as new probable etiologic filamentous fungi, causing onychomycosis. The morphology and physiology of these fungi is discussed.     

Synergistic transmembrane alignment of the antimicrobial heterodimer PGLa/magainin

The antimicrobial activity of amphipathic alpha-helical peptides is usually attributed to the formation of pores in bacterial membranes, but direct structural information about such a membrane-bound state is sparse. Solid state (2)H-NMR has previously shown that the antimicrobial peptide PGLa undergoes a concentration-dependent realignment from a surface-bound S-state to a tilted T-state. The corresponding change in helix tilt angle from 98 to 125 degrees was interpreted as the formation of PGLa/magainin heterodimers residing on the bilayer surface. Under no conditions so far, has an upright membrane-inserted I-state been observed in which a transmembrane helix alignment would be expected. Here, we have demonstrated that PGLa is able to assume such an I-state in a 1:1 mixture with magainin 2 at a peptide-to-lipid ratio as low as 1:100 in dimyristoylphosphatidylcholine/dimyristoylphosphatidylglycerol model membranes. This (2)H-NMR analysis is based on seven orientational constraints from Ala-3,3,3-d(3) substituted in a non-perturbing manner for four native Ala residues as well as two Ile and one Gly. The observed helix tilt of 158 degrees is rationalized by the formation of heterodimers. This structurally synergistic effect between the two related peptides from the skin of Xenopus laevis correlates very well with their known functional synergistic mode of action. To our knowledge, this example of PGLa is the first case where an alpha-helical antimicrobial peptide is directly shown to assume a transmembrane state that is compatible with the postulated toroidal wormhole pore structure.