In Vitro Characterization of Histone Chaperones using Analytical, Pull-Down and Chaperoning Assays.

Histone proteins associate with DNA to form the eukaryotic chromatin. The basic unit of chromatin is a nucleosome, made up of a histone octamer consisting of two copies of the core histones H2A, H2B, H3, and H4, wrapped around by the DNA. The octamer is composed of two copies of an H2A/H2B dimer and a single copy of an H3/H4 tetramer. The highly charged core histones are prone to non-specific interactions with several proteins in the cellular cytoplasm and the nucleus. Histone chaperones form a diverse class of proteins that shuttle histones from the cytoplasm into the nucleus and aid their deposition onto the DNA, thus assisting the nucleosome assembly process. Some histone chaperones are specific for either H2A/H2B or H3/H4, and some function as chaperones for both. This protocol describes how in vitro laboratory techniques such as pull-down assays, analytical size-exclusion chromatography, analytical ultra-centrifugation, and histone chaperoning assay could be used in tandem to confirm whether a given protein is functional as a histone chaperone.

NMR Assisted Antimicrobial Peptide Designing: Structure Based Modifications and Functional Correlation of a Designed Peptide VG16KRKP.

Antimicrobial Peptides (AMPs), within their realm incorporate a diverse group of structurally and functionally varied peptides, playing crucial roles in innate immunity. Over the last few decades, the field of AMP has seen a huge upsurge, mainly owing to the generation of the so-called drug resistant 'superbugs' as well as limitations associated with the existing antimicrobial agents. Due to their resilient biological properties, AMPs can very well form the sustainable alternative for nextgeneration therapeutic agents. Certain drawbacks associated with existing AMPs are, however, issues of major concern, circumventing which are imperative. These limitations mainly include proteolytic cleavage and hence poor stability inside the biological systems, reduced activity due to inadequate interaction with the microbial membrane, and ineffectiveness because of inappropriate delivery among others. In this context, the application of naturally occurring AMPs as an efficient prototype for generating various synthetic and designed counterparts has evolved as a new avenue in peptide-based therapy. Such designing approaches help to overcome the drawbacks of the parent AMPs while retaining the inherent activity. In this review, we summarize some of the basic NMR structure based approaches and techniques which aid in improving the activity of AMPs, using the example of a 16-residue dengue virus fusion protein derived peptide, VG16KRKP. Using first principle based designing technique and high resolution NMR-based structure characterization we validate different types of modifications of VG16KRKP, highlighting key motifs, which optimize its activity. The approaches and designing techniques presented can support our peers in their drug development work.

AtFKBP53: a chimeric histone chaperone with functional nucleoplasmin and PPIase domains.

FKBP53 is one of the seven multi-domain FK506-binding proteins present in Arabidopsis thaliana, and it is known to get targeted to the nucleus. It has a conserved PPIase domain at the C-terminus and a highly charged N-terminal stretch, which has been reported to bind to histone H3 and perform the function of a histone chaperone. To better understand the molecular details of this PPIase with histone chaperoning activity, we have solved the crystal structures of its terminal domains and functionally characterized them. The C-terminal domain showed strong PPIase activity, no role in histone chaperoning and revealed a monomeric five-beta palm-like fold that wrapped over a helix, typical of an FK506-binding domain. The N-terminal domain had a pentameric nucleoplasmin-fold; making this the first report of a plant nucleoplasmin structure. Further characterization revealed the N-terminal nucleoplasmin domain to interact with H2A/H2B and H3/H4 histone oligomers, individually, as well as simultaneously, suggesting two different binding sites for H2A/H2B and H3/H4. The pentameric domain assists nucleosome assembly and forms a discrete complex with pre-formed nucleosomes; wherein two pentamers bind to a nucleosome.

Lysozyme's lectin-like characteristics facilitates its immune defense function.

Interactions between human lysozyme (HL) and the lipopolysaccharide (LPS) of Klebsiella pneumoniae O1, a causative agent of lung infection, were identified by surface plasmon resonance. To characterize the molecular mechanism of this interaction, HL binding to synthetic disaccharides and tetrasaccharides representing one and two repeating units, respectively, of the O-chain of this LPS were studied. pH-dependent structural rearrangements of HL after interaction with the disaccharide were observed through nuclear magnetic resonance. The crystal structure of the HL-tetrasaccharide complex revealed carbohydrate chain packing into the A, B, C, and D binding sites of HL, which primarily occurred through residue-specific, direct or water-mediated hydrogen bonds and hydrophobic contacts. Overall, these results support a crucial role of the Glu35/Asp53/Trp63/Asp102 residues in HL binding to the tetrasaccharide. These observations suggest an unknown glycan-guided mechanism that underlies recognition of the bacterial cell wall by lysozyme and may complement the HL immune defense function.

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.

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 ß-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.

Inhibition and Degradation of Amyloid Beta (Aß40) 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 (Aß40) protein but also disaggregates the preformed oligomers and mature Aß 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 Aß40. 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.

Mode of Action of a Designed Antimicrobial Peptide: High Potency against Cryptococcus neoformans.

There is a significant need for developing compounds that kill Cryptococcus neoformans, the fungal pathogen that causes meningoencephalitis in immunocompromised individuals. Here, we report the mode of action of a designed antifungal peptide, VG16KRKP (VARGWKRKCPLFGKGG) against C. neoformans. It is shown that VG16KRKP kills fungal cells mainly through membrane compromise leading to efflux of ions and cell metabolites. Intracellular localization, inhibition of in vitro transcription, and DNA binding suggest a secondary mode of action for the peptide, hinting at possible intracellular targets. Atomistic structure of the peptide determined by NMR experiments on live C. neoformans cells reveals an amphipathic arrangement stabilized by hydrophobic interactions among A2, W5, and F12, a conventional folding pattern also known to play a major role in peptide-mediated Gram-negative bacterial killing, revealing the importance of this motif. These structural details in the context of live cell provide valuable insights into the design of potent peptides for effective treatment of human and plant fungal infections.

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 Phe(11), Phe(19), Phe(23), and Tyr(25) 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.

Designing potent antimicrobial peptides by disulphide linked dimerization and N-terminal lipidation to increase antimicrobial activity and membrane perturbation: Structural insights into lipopolysaccharide binding.

HYPOTHESIS: The remarkable rise in multi-drug resistant Gram-negative bacterial pathogens is a major concern to the well being of humans as well as susceptible plants. In recent years, diseases associated with inflammation and septicemia have already become a global health issue. Therefore, there is a rising demand for the development of novel "super" antibiotics. In this context, antimicrobial peptides offer an attractive, alternate therapeutic solution to conventional antibiotics. EXPERIMENTS: Microbroth dilution assay was performed to investigate the antimicrobial activities of the two designed peptides against Gram negative bacterial pathogens. Fluorescence studies including NPN dye uptake assay, Calcein entrapped vesicle leakage assay, quenching and anisotropy in presence of lipopolysaccharide (LPS) were performed to elucidate binding interactions and enhanced membrane permeabilisation. Hemolytic assay and endotoxin/LPS neutralisation assay were performed to study the hemolytic effects and LPS scavenging abilities of the peptides. High resolution NMR studies were performed to obtain insights into LPS-peptide interaction at the molecular level. FINDINGS: Here, we report more potent analogues of previously designed peptide VG16KRKP, designed through dimerization via Cys-Cys disulphide linkage and N-terminal lipidation. Similar to the parent peptide, VG16KRKP, the modified analogue peptides are non hemolytic in nature, but possessed, 2-10-fold increase in antibacterial activities against E. coli, human pathogen Pseudomonas aeruginosa and the devastating plant pathogen, Xanthomonas campestris pv. campestris as well as membrane permeabilization, and endotoxin neutralization. LPS bound solution structure of both analogues, as determined by NMR spectroscopy, reveal that the conserved hydrophobic triad motif, formed by Trp5, Leu11 and Phe12 is compactly organized and stabilized either by the acyl chain or disulphide bond. This structural constraint accounts for the separation of polar face from the hydrophobic face of the peptides. Our novel peptides designed through Cys-Cys dimerization and N-terminal lipidation, will serve as a template to develop more potent antimicrobials in future, to control plant and human diseases.

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 ß-defensin-3 analogs.

Human beta defensin-3 (HßD-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 HßD-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 HßD3 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 HßD3 analogs with phospholipid vesicles composed of zwitterionic PC or anionic PE:PG vesicles and LPS; (ii) conformation of HßD3-peptide analogs in the presence of PC or PE:PG vesicles; (iii) ability of HßD3 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.

Sequence context induced antimicrobial activity: insight into lipopolysaccharide permeabilization.

Lactoferrampin (WR17, Trp 268-Arg 284), an antimicrobial peptide, is known to have significant antibacterial and candidacidal activities. However, there are no previous studies explaining how WR17 permeabilizes the outer membrane of Gram negative bacteria and neutralizes endotoxins. In this study we used a series of assays like antimicrobial activity, calcein leakage, NPN dye uptake and endotoxin neutralization assay to show that the sequence context of WR17 modulates its multi-faceted activities. We determined the high resolution NMR structure of WR17 in LPS and found that the N-ter region forms a helix (Trp1-Phe11) and orients itself at an angle of 45° into the lipopolysaccharide (LPS) micelle, whereas the C-ter region (Lys13-Arg17) remains as a flexible extended random coil. We also verified this result through in silico molecular modeling simulation. Isothermal titration calorimetry showed that the interaction of WR17 and its analogues with LPS was primarily endothermic in nature. Using several fluorescence techniques such as anisotropy and red edge excitation shift assay we revealed motional restriction for Trp1 of WR17 in LPS. The distance between the indole ring of Trp1 of WR17 and the polar head group of LPS is around 7 Å, as obtained from the depth of insertion assay. Additionally, MD simulation demonstrated that the incorporation of the peptide in LPS is achieved with the help of the K(13)xK(15)xR(17) motif at the C-terminus. This novel anchoring "K(13)NKSR(17)" motif is currently being utilized in our ongoing research to design novel anti-endotoxic molecules.

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.