Tryptophan end-tagging for promoted lipopolysaccharide interactions and anti-inflammatory effects

The objective of the present study is the investigation of possibilities for boosting peptide anti-inflammatory effects by tryptophan end-tagging, including identification of underlying mechanisms for this. In doing so, effects of tryptophan end-tagging of KYE21 (KYEITTIHNLFRKLTHRLFRR), a peptide derived from heparin co-factor II, on membrane and lipopolysaccharide (LPS) interactions were investigated by ellipsometry, NMR, fluorescence spectroscopy, and circular dichroism measurements. Through its N-terminal W stretch, WWWKYE21 displays higher membrane binding, liposome rupture, and bacterial killing than unmodified KYE21. Analogously, W-tagging promotes binding to E. coli LPS and to its endotoxic lipid A moiety. Furthermore, WWWKYE21 causes more stable peptide/LPS complexes than KYE21, as evidenced by detailed NMR studies, adopting a pronounced helical conformation, with a large hydrophobic surface at the N-terminus due to the presence of W-residues, and a flexible C-terminus due to presence of several positively charged arginine residues. Mirroring its increased affinity for LPS and lipid A, WWWKYE21 displays strongly increased anti-inflammatory effect due to a combination of direct lipid A binding, peptide-induced charge reversal of cell membranes for LPS scavenging, and peptide-induced fragmentation of LPS aggregates for improved phagocytosis. Importantly, potent anti-inflammatory effects were observed at low cell toxicity, demonstrated for both monocytes and erythrocytes

Conformational Aspects of High Content Packing of Antimicrobial Peptides in Polymer Microgels

Successful use of microgels as delivery systems of antimicrobial peptides (AMPs) requires control of factors determining peptide loading and release to/from the microgels as well as of membrane interactions of both microgel particles and released peptides. Addressing these, we here investigate effects of microgel charge density and conformationally induced peptide amphiphilicity on AMP loading and release using detailed nuclear magnetic resonance (NMR) structural studies combined with ellipsometry, isothermal titration calorimetry, circular dichroism, and light scattering. In parallel, consequences of peptide loading and release for membrane interactions and antimicrobial effects were investigated. In doing so, poly(ethyl acrylate-co-methacrylic acid) microgels were found to incorporate the cationic AMPs EFK17a (EFKRIVQRIKDFLRNLV) and its partially d-amino acid-substituted variant EFK17da (E(dF)KR(dI)VQR(dI)KD(dF)LRNLV). Peptide incorporation was found to increase with increasing with microgel charge density and peptide amphiphilicity. After microgel incorporation, which appeared to occur preferentially in the microgel core, NMR showed EFK17a to form a helix with pronounced amphiphilicity, while EFK17da displayed a folded conformation, stabilized by a hydrophobic hub consisting of aromatic/aromatic and aliphatic/aromatic interactions, resulting in much lower amphiphilicity. Under wide ranges of peptide loading, the microgels displayed net negative z-potential. Such negatively charged microgels do not bind to, nor lyse, bacteria-mimicking membranes. Instead, membrane disruption in these systems is mediated largely by peptide release, which in turn is promoted at higher ionic strength and lower peptide amphiphilicity. Analogously, antimicrobial effects against Escherichia coli were found to be dictated by peptide release. Taken together, the findings show that peptide loading, packing, and release strongly affect the performance of microgels as AMP delivery systems, effects that can be tuned by (conformationally induced) peptide amphiphilicity and by microgel charge density

Role of aromatic amino acids in lipopolysaccharide and membrane interactions of antimicrobial peptides for use in plant disease control

KYE28 (KYEITTIHNLFRKLTHRLFRRNFGYT-LR), the representative sequence of helix D of heparin co-factor II, was demonstrated to be potent against agronomically important Gram-negative plant pathogens Xanthomonas vesicatoria and Xanthomonas oryzae, capable of inhibiting disease symptoms in detached tomato leaves. NMR studies in the presence of lipopolysaccharide provided structural insights into the mechanisms underlying this, notably in relationship to outer membrane permeabilization. The three-dimensional solution structure of KYE28 in LPS is characterized by an N-terminal helical segment, an intermediate loop followed by another short helical stretch, and an extended C terminus. The two termini are in close proximity to each other via aromatic packing interactions, whereas the positively charged residues form an exterior polar shell. To further demonstrate the importance of the aromatic residues for this, a mutant peptide KYE28A, with Ala substitutions at Phe11 , Phe19 , Phe23 , and Tyr25 was designed, which showed attenuated antimicrobial activity at high salt concentrations, as well as lower membrane disruption and LPS binding abilities compared with KYE28. In contrast to KYE28, KYE28A adopted an extended helical structure in LPS with extended N and C termini. Aromatic packing interactions were completely lost, although hydrophobic interaction between the side chains of hydrophobic residues were still partly retained, imparting an amphipathic character and explaining its residual antimicrobial activity and LPS binding as observed from ellipsometry and isothermal titration calorimetry. We thus present key structural aspects of KYE28, constituting an aromatic zipper, of potential importance for the development of novel plant protection agents and therapeutic agents

Use of a small peptide fragment as an inhibitor of insulin fibrillation process: a study by high and low resolution spectroscopy.

A non-toxic, nine residue peptide, NIVNVSLVK is shown to interfere with insulin fibrillation by various biophysical methods. Insulin undergoes conformational changes under certain stress conditions leading to amyloid fibrils. Fibrillation of insulin poses a problem in its long-term storage, reducing its efficacy in treating type II diabetes. The dissociation of insulin oligomer to monomer is the key step for the onset of fibrillation. The time course of insulin fibrillation at 62°C using Thioflavin T fluorescence shows an increase in the lag time from 120 min without peptide to 236 min with peptide. Transmission electron micrographs show branched insulin fibrils in its absence and less inter-fibril association in its presence. Upon incubation at 62°C and pH 2.6, insulin lost some α-helical structure as seen by Fourier transformed infra-red spectroscopy (FT-IR), but if the peptide is added, secondary structure is almost fully maintained for 3 h, though lost partially at 4 h. FT-IR spectroscopy also shows that insulin forms the cross beta structure indicative of fibrils beyond 2 h, but in the presence of the peptide, α-helix retention is seen till 4 h. Both size exclusion chromatography and dynamic light scattering show that insulin primarily exists as trimer, whose conversion to a monomer is resisted by the peptide. Saturation transfer difference nuclear magnetic resonance confirms that the hydrophobic residues in the peptide are in close contact with an insulin hydrophobic groove. Molecular dynamics simulations in conjunction with principal component analyses reveal how the peptide interrupts insulin fibrillation. In vitro hemolytic activity of the peptide showed insignificant cytotoxicity against HT1080 cells. The insulin aggregation is probed due to the inter play of two key residues, Phe(B24) and Tyr(B26) monitored from molecular dynamics simulations studies. Further new peptide based leads may be developed from this nine residue peptide

Hemolytic and cytotoxity assay of NK9 peptide.

<p>(A) Percentage hemolysis of NK9 peptide on human red blood cells exhibiting less than 0.5% hemolysis for different concentrations (25, 50, 100, 150, 200 and 250 µM) of peptide been tested. Blank corresponds to untreated red blood cells (without peptide). The positive control for this experiment was 1% Triton X 100. (B) Percentage viability of HT1080 cells upon treatment with varying concentrations (5, 10, 25 and 50 µM) of NK9 peptide showing less than 5% cytotoxicity in each case, against untreated cells taken as blank. The positive control for this experiment was 0.5% Triton X 100.</p

Negative-staining transmission electron micrographs: (A) Bovine insulin and (B) Bovine insulin in presence of NK9.

<p>Molar ratio of NK9 to insulin is 1∶1. Insulin fibrils were grown at pH 2.6 and temperature 62°C for 8 hours. Scale bars ∼200 nm.</p

Time course of insulin fibrillation in the presence and absence of NK9, monitored using ThT fluorescence.

<p>(A) Temporal evolution of the ThT fluorescence intensity during incubation of insulin (350 μM) in fibrillation condition (pH 2.6, 62°C) in presence and absence of NK9 at different concentrations. ThT curves of insulin in respective NK9 concentrations are mentioned in the figure. (B) Effect of NK9 on fibrillation kinetics of insulin. NK9 (100 μM) was added to the buffer solutions (pH 2.6) of insulin (350 μM) at 62°C at 0, 15, 20 and 30 min of incubation. Molar ratio of NK9 to insulin is 0.28∶1. Inset showed a plot of lag time of insulin fibrillation in presence of 100 μM NK9 against incubation time.</p

Changes in the association state of insulin in presence and absence of NK9.

<p>Absorbance profiles at 220 nm from size exclusion chromatography experiments with insulin (350 μM): (A) Absence and (B) Presence of 350 µM NK9 at different time of incubation. Molar ratio of NK9 to insulin is 1∶1. Panel C shows SEC profile of insulin in presence of NK9 at 280 nm wavelength to distinguish insulin peak from the NK9.</p

Inhibition and Degradation of Amyloid Beta (Aβ40) Fibrillation by Designed Small Peptide: A Combined Spectroscopy, Microscopy, and Cell Toxicity Study

A designed nontoxic, nonhemolytic 11-residue peptide, NF11 (NAVR­WSLM­RPF), not only inhibits the aggregation of amyloid beta (Aβ40) protein but also disaggregates the preformed oligomers and mature Aβ fibrils, thereby reducing associated-toxicity. NMR experiments provide evidence of NF11’s ability to inhibit fibril formation, primarily through interaction with the N-terminus region as well as the central hydrophobic cluster of Aβ40. NF11 has micromolar binding affinity toward both monomeric and aggregated species for efficient clearance of toxic aggregates. From these in vitro results, the future development of a next generation peptidomimetic therapeutic agent for amyloid disease may be possible

Structural and Dynamic Insights into a Glycine-Mediated Short Analogue of a Designed Peptide in Lipopolysaccharide Micelles: Correlation Between Compact Structure and Anti-Endotoxin Activity

In this study, we report an interaction study of a 13-residue analogue peptide VG13P (VARGW­GRKC­PLFG), derived from a designed VG16KRKP peptide (VARGWK­RKCPLF­GKGG), with a Lys6Gly mutation and removal of the last three residues Lys¹⁴-Gly¹⁵-Gly¹⁶, in lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria and responsible for sepsis or septic shock. VG13P displays an enhanced anti-endotoxin property as evident from significant reduction in LPS-induced TNF-α gene expression levels in a monocytic cell line, while it retains almost unchanged antimicrobial activity as its parent VG16KRKP against Gram-negative bacterial as well as fungal pathogens. In addition, <i>in vitro</i> LPS binding properties of VG13P in comparison to its parent VG16KRKP also remained unhindered, suggesting that the flexible C-terminal end of VG16KRKP may not play a major role in its observed antibacterial and LPS binding properties. An NMR-resolved solution structure of VG13P in LPS reveals two consecutive β-turns: one at the N-terminus, followed by another at the central region, closely resembling a rocking chair. The crucial Lys6Gly mutation along with C-terminal truncation from VG16KRKP reorients the hydrophobic hub in VG13P in a unique way so as to fold the N-terminal end back on itself, forming a turn and allowing Val1 and Ala2 to interact with Leu11 and Phe12 to bring the hydrophobic residues closer together to form a more compact hub compared to its parent. The hub is further strengthened via CH-π interaction between Gly4 and Phe12. This accounts for its improved anti-endotoxin activity as well as to its uninterrupted antimicrobial activity