Aritreyee Datta

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, PheB24 and TyrB26 monitored from molecular dynamics simulations studies. Further new peptide based leads may be developed from this nine residue peptide.

Antimicrobial Peptides: Insights into Membrane Permeabilization, Lipopolysaccharide Fragmentation and Application in Plant Disease Control

The recent increase in multidrug resistance against bacterial infections has become a major concern to human health and global food security. Synthetic antimicrobial peptides (AMPs) have recently received substantial attention as potential alternatives to conventional antibiotics because of their potent broad-spectrum antimicrobial activity. These peptides have also been implicated in plant disease control for replacing conventional treatment methods that are polluting and hazardous to the environment and to human health. Here, we report de novo design and antimicrobial studies of VG16, a 16-residue active fragment of Dengue virus fusion peptide. Our results reveal that VG16KRKP, a non-toxic and non-hemolytic analogue of VG16, shows significant antimicrobial activity against Gram-negative E. coli and plant pathogens X. oryzae and X. campestris, as well as against human fungal pathogens C. albicans and C. grubii. VG16KRKP is also capable of inhibiting bacterial disease progression in plants. The solution-NMR structure of VG16KRKP in lipopolysaccharide features a folded conformation with a centrally located turn-type structure stabilized by aromatic-aromatic packing interactions with extended N- and C-termini. The de novo design of VG16KRKP provides valuable insights into the development of more potent antibacterial and antiendotoxic peptides for the treatment of human and plant infections.

Membrane Disruptive Antimicrobial Activities of Human β-Defensin-3 Analogs

Human beta defensin-3 (HβD-3) is a host-defense protein exhibiting antibacterial activity towards both Gram-negative and Gram-positive bacteria. There is considerable interest in the function of this protein due to its increased salt tolerance and activity against Gram-positive Staphylococcus aureus. In this study, analogs of HβD-3 devoid of N and C terminal regions are investigated to determine the influence of specific structural motif on antimicrobial activity and selectivity between Gram-positive and Gram-negative bacteria. Circular dichroism, fluorescence and solid-state NMR experiments have been used to investigate the conformation and mode of action of HβD3 analogs with various model membranes to mimic bacterial inner and outer membranes and also mammalian membranes. Our studies specifically focused on determining four major characteristics: (i) interaction of HβD3 analogs with phospholipid vesicles composed of zwitterionic PC or anionic PE:PG vesicles and LPS; (ii) conformation of HβD3-peptide analogs in the presence of PC or PE:PG vesicles; (iii) ability of HβD3 analogs to permeate phospholipid vesicles composed of PC or PE:PG; and (iv) activities on bacteria cells and erythrocytes. Our results infer that the linear peptide L25P and its cyclic form C25P are more active than L21P and C21P analogs. However, they are less active than the parent peptide, thus pointing towards the importance of the N terminal domain in its biological activity. The variation in the activities of L21P/C21P and L25P/C25P also suggest the importance of the positively charged residues at the C terminus in providing selectivity particularly to Gram-negative bacteria.

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.

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 NF11s 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.

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.

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, in vitro 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.

An Approach Towards Structure Based Antimicrobial Peptide Design for Use in Development of Transgenic Plants: A Strategy for Plant Disease Management

Antimicrobial peptides (AMPs), also known as host defense peptides (HDPs), are ubiquitous and vital components of innate defense response that present themselves as potential candidates for drug design, and aim to control plant and animal diseases. Though their application for plant disease management has long been studied with natural AMPs, cytotoxicity and stability related shortcomings for the development of transgenic plants limit their usage. Newer technologies like molecular modelling, NMR spectroscopy and combinatorial chemistry allow screening for potent candidates and provide new avenues for the generation of rationally designed synthetic AMPs with multiple biological functions. Such AMPs can be used for the control of plant diseases that lead to huge yield losses of agriculturally important crop plants, via generation of transgenic plants. Such approaches have gained significant attention in the past decade as a consequence of increasing antibiotic resistance amongst plant pathogens, and the shortcomings of existing strategies that include environmental contamination and human/animal health hazards amongst others. This review summarizes the recent trends and approaches used for employing AMPs, emphasizing on designed/modified ones, and their applications toward agriculture and food technology.

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.