Design of inhibitor peptide sequences based on the interfacial knowledge of the protein G-IgG crystallographic complex and their binding studies with IgG.

Protein-protein interactions (PPI) have emerged as valuable targets in medicinal chemistry due to their key roles in important biological processes. The modulation of PPI by small peptides offers an excellent opportunity to develop drugs against human diseases. Here, we exploited the knowledge of the binding interface of the IgG-protein G complex (PDB:1FCC) for designing peptides that can inhibit these complexes. Herein, we have designed several closely related peptides, and the comparison of results from experiments and computational studies indicated that all the peptides bind close to the expected binding site on IgG and the complexes are stable. A minimal sequence consisting of 11 amino acids (P5) with binding constants in the range of 100 nM was identified. We propose that the main affinity differences across the series of peptides arose from the presence of polar amino acid residues. Further, the molecular dynamic studies helped to understand the dynamic properties of complexes in terms of flexibility of residues and structural stability at the interface. The ability of P5 to compete with the protein G in recognizing IgG can help in the detection and purification of antibodies. Further, it can serve as a versatile tool for a better understanding of protein-protein interactions.

New Insights into the Role of Ligand-Binding Modes in GC-DNA Condensation through Thermodynamic and Spectroscopic Studies.

In biological systems, the unprompted assembly of DNA molecules by cationic ligands into condensed structures is ubiquitous. The ability of ligands to provoke DNA packaging is crucial to the molecular organization and functional control of DNA, yet their underlined physical roles have remained elusive. Here, we have examined the DNA condensation mechanism of four cationic ligands, including their primary DNA-binding modes through extensive biophysical studies. We observed contrasting changes for these ligands binding to poly[dGdC]:poly[dGdC] (GC-DNA) and poly[dAdT]:poly[dAdT] (AT-DNA). Based on a CD spectroscopic study, it was confirmed that only GC-DNA undergoes B- to Ψ-type DNA transformation in the presence of ligands. In the fluorescence displacement assay (FDA), the ability of ligands to displace GC-DNA-bound EtBr follows the order: protamine21+ > cohex3+ > Ni2+ > spermine4+, which indicates that there is no direct correlation between the ligand charge and its ability to displace the drug from the DNA, indicating that GC-DNA condensation is not just influenced by electrostatic interaction but ligand-specific interactions may also have played a crucial role. Furthermore, the detailed ITC-binding studies suggested that DNA-ligand interactions are generally driven by unfavorable enthalpy and favorable entropy. The correlations from various studies insinuate that cationic ligands show major groove binding as one of the preferred binding modes during GC-DNA condensation.

Formation of Protamine and Zn-Insulin Assembly: Exploring Biophysical Consequences.

The insulin-protamine interaction is at the core of the mode of action in many insulin formulations (Zn + insulin + protamine) and to treat diabetes, in which protamine is added to the stable form of hexameric insulin (Zn-insulin). However, due to the unavailability of quantitative data and a high-resolution structure, the binding mechanism of the insulin-protamine complex remains unknown. In this study, it was observed that Zn-insulin experiences destabilization as observed by the loss of secondary structure in circular dichroism (CD), and reduction in thermal stability in melting study, upon protamine binding. In isothermal titration calorimetry (ITC), it was found that the interactions were mostly enthalpically driven. This is in line with the positive ΔC m value (+880 cal mol-1), indicating the role of hydrophilic interactions in the complex formation, with the exposure of hydrophobic residues to the solvent, which was firmly supported by the 8-anilino-1-naphthalene sulfonate (ANS) binding study. The stoichiometry (N) value in ITC suggests the multiple insulin molecules binding to the protamine chain, which is consistent with the picture of the condensation of insulin in the presence of protamine. Atomic force microscopy (AFM) suggested the formation of a heterogeneous Zn-insulin-protamine complex. In fluorescence, Zn-insulin experiences strong Tyr quenching, suggesting that the location of the protamine-binding site is near Tyr, which is also supported by the molecular docking study. Since Tyr is critical in the stabilization of insulin self-assembly, its interaction with protamine may impair insulin's self-association ability and thermodynamic stability while at the same time promoting its flexible conformation desired for better biological activity.

Biochemical and Biophysical Characterisation of the Hepatitis E Virus Guanine-7-Methyltransferase.

Hepatitis E virus (HEV) is an understudied pathogen that causes infection through fecal contaminated drinking water and is prominently found in South Asian countries. The virus affects ~20 million people annually, leading to ~60,000 infections per year. The positive-stranded RNA genome of the HEV genotype 1 has four conserved open reading frames (ORFs), of which ORF1 encodes a polyprotein of 180 kDa in size, which is processed into four non-structural enzymes: methyltransferase (MTase), papain-like cysteine protease, RNA-dependent RNA polymerase, and RNA helicase. MTase is known to methylate guanosine triphosphate at the 5'-end of viral RNA, thereby preventing its degradation by host nucleases. In the present study, we cloned, expressed, and purified MTase spanning 33-353 amino acids of HEV genotype 1. The activity of the purified enzyme and the conformational changes were established through biochemical and biophysical studies. The binding affinity of MTase with magnesium ions (Mg2+) was studied by isothermal calorimetry (ITC), microscale thermophoresis (MST), far-UV CD analysis and, fluorescence quenching. In summary, a short stretch of nucleotides has been cloned, coding for the HEV MTase of 37 kDa, which binds Mg2+ and modulate its activity. The chelation of magnesium reversed the changes, confirming its role in enzyme activity.

Synthetic Tunability and Biophysical Basis for Fabricating Highly Fluorescent and Stable DNA Copper Nanoclusters.

The real motivation in the present work is to tune the synthesis variables that can result in a highly fluorescent and stable DNA copper nanocluster (CuNC) and also to understand the intricate mechanism behind this process. Here, carefully optimized concentrations of various reactants enabled the creation of a DNA-encapsulated CuNC for AT-DNA, displaying a size of <1.0 nm as confirmed by transmission electron microscopy and dynamic light scattering. The extremely small size of the AT-DNACuNC supports the discrete electronic transitions, also characterized by an exceptionally strong negative circular dichroism (CD) band around 350 nm, whose intensity is well correlated with the observed strong fluorescence emission intensity. This remarkably strong CD can open new applications in the detection and quantification of a specific DNACuNC. Further, time-dependent fluorescence analysis suggested stronger photostabilization of these DNACuNCs. The simulation study, based on Cu ion distribution, explained how AT-DNA is a better candidate for NC formation than GC-DNA. In conclusion, the better-tuned synthesis procedure has resulted in a highly compact, well-defined three-dimensional conformation that promotes a more favorable microenvironment to sequester a DNA-based CuNC with high brightness and outstanding photostability.

Crystalline and Amorphous Preparation of Aluminum Hydroxide Nanoparticles Enhances Protective Antigen Domain 4 Specific Immunogenicity and Provides Protection Against Anthrax.

INTRODUCTION: Aluminum salts, although they have been used as adjuvants in many vaccine formulations since 1926, exclusively induce a Th2-biased immune response, thereby limiting their use against intracellular pathogens like Mycobacterium tuberculosis. METHODS AND RESULTS: Herein, we synthesized amorphous and crystalline forms of aluminum hydroxide nanoparticles (AH nps) of 150-200 nm size range. Using Bacillus anthracis protective antigen domain 4 (D4) as a model antigen, we demonstrated that both amorphous and crystalline forms of AH nps displayed enhanced antigen D4 uptake by THP1 cells as compared to commercial adjuvant aluminum hydroxide gel (AH gel). In a mouse model, both amorphous and crystalline AH nps triggered an enhanced D4-specific Th2- and Th1-type immune response and conferred superior protection against anthrax spore challenge as compared to AH gel. Physicochemical characterization of crystalline and amorphous AH nps revealed stronger antigen D4 binding and release than AH gel. CONCLUSION: These results demonstrate that size and crystallinity of AH nps play important roles in mediating enhanced antigen presenting cells (APCs) activation and potentiating a strong antigen-specific immune response, and are critical parameters for the rational design of alum-based Th1-type adjuvant to induce a more balanced antigen-specific immune response.

In-vitro Detection of Phytopathogenic Fungal Cell Wall by Polyclonal Sera Raised Against Trimethyl Chitosan Nanoparticles.

PURPOSE: The objective of this research was to generate a tool for the first-line detection of fungal infection in plants. Chitin is one of the unique fungal cell wall polysaccharide which is naturally deacetylated to chitosan upon infection. It is said to be involved in the fungal cell wall modulation and plant-pathogen communication. Therefore, detection of chitosan could be potentially helpful in the detection of fungal contamination. METHODS: Five different phytopathogenic fungi strains were used for the study. Polyclonal sera were raised in the mice against Trimethylchitosan nanoparticles to generate an enhanced humoral immune response and generate a rich and heterogeneous repertoire of antibodies. The binding affinity of the sera with fungal cell wall was analyzed by ELISA, Langmuir isotherm, confocal microscopy and ITC (Isothermal Calorimetry). RESULTS: The raised polyclonal sera could detect chitosan in the fungal cell wall, as analyzed with the different techniques. However, the detection specificity varied among the strains in proportion to the chitin content of their cell wall. Fusarium oxysporum was detected with the highest affinity while Trichoderma reesei was detected with the least affinity by ELISA. Adsorption isotherm, as well as ITC, revealed the specific and high binding capacity. Confocal microscopy also confirmed the detection of all strains used in the study. CONCLUSION: This novel technique employing TMC nanoparticulate system could be potentially used as a source to raise sera against chitosan in an inexpensive and less laborious manner. Rapid detection of fungal contamination by the polyclonal antibodies could help in devising a quick solution. The polyclonal sera are expected to detect a span of epitopes and provide precise detection. The detection system could be advanced for future applications such as food quality control, crop protection, and human fungal infection detection and treatment.