Formation of Annular Protofibrillar Assembly by Cysteine Tripeptide: Unraveling the Interactions with NMR, FTIR, and Molecular Dynamics

Both hydrogen-bonding and hydrophobic interactions play a significant role in molecular assembly,including self-assembly of proteins and peptides. In this study, we report the formation of annular protofibrillar structure (diameter ∼500 nm) made of a newly synthesized s-benzylprotected cysteine tripeptide, which was primarily stabilized by hydrogen-bonding and hydrophobic interactions. Atomic force microscopy and field emission scanning electron microscopy analyses found small oligomers (diameter ∼60 nm) to bigger annular (outer diameter ∼300 nm; inner diameter, 100 nm) and protofibrillar structures after 1−2 days of incubation. Rotating-frame Overhauser spectroscopic (ROESY) analysis revealed the presence of several nonbonded proton−proton interactions among the residues, such as amide protons with methylene group, aromatic protons with tertiary butyl group, and methylene protons with tertiary butyl group. These added significant stability to bring the peptides closer to form a well-ordered assembled structure. Hydrogen−deuterium exchange NMR measurement further suggested that two individual amide protons among the three amide groups were strongly engaged with the adjacent tripeptide via H-bond interaction. However, the remaining amide proton was found to be exposed to solvent and remained noninteracting with other tripeptide molecules. In addition to chemical shift values, a significant change in amide bond vibrations of the tripeptide was found due to the formation of the self-assembled structure. The amide I mode of vibrations involving two amide linkages appeared at 1641 and 1695 cm−1 in the solid state. However, in the assembled state, the stretching band at 1695 cm−1 became broad and slightly shifted to ∼1689 cm−1. On the contrary, the band at 1641 cm−1 shifted to 1659 cm−1 and indicated that the −CO bond associated with this vibration became stronger in the assembled state. These changes in Fourier transform infrared spectroscopy frequency clearly indicated changes in the amide backbone conformation and the associated hydrogen-bonding pattern due to the formation of the assembled structure. In addition to hydrogen bonding, molecular dynamics simulation indicated that the number of π−π interactions also increased with increasing number of tripeptides participated in the self-assembly process. Combined results envisaged a cross β-sheet assembly unit consisting of four intermolecular hydrogen bonds. Such noncovalent peptide assemblies glued by hydrogen-bonding and other weak forces may be useful in developing nanocapsule and related materials

Molecular Details of Acetate Binding to a New Diamine Receptor by NMR and FT-IR Analyses

Acetate anion plays an important role in several biochemical functions such as enzyme reaction, antibody response, and action of receptor molecules. This investigation reports the synthesis and molecular details of a unique receptor, 2-amino-N-(2- amino-benzyl)-benzamide (R) that senses selectively acetate via simultaneous involvement of one aromatic amine group and an amide proton of the receptor molecule. Solution-state NMR, steady-state fluorescence, and FT-IR examinations established that the acetate anion binds to the receptor with 1:1 ratio with high specificity. The binding was stabilized by two H-bond formations between the oxygen atoms of acetate anion and two H atoms, one from amide group and the other from the amine group of the receptor. The binding interaction caused significant changes in the chemical shift of the receptor protons, and the evaluated affinity constant, from the NMR measurements, was found to be 1.87 × 104 M−1. Density functional theory (DFT) analysis further showed a significant rotation of one of the two aromatic rings leading to formation of a 10-member ring involving the acetate anion, amide proton, and the one amine group attached to aromatic ring. The H-bond patterns observed in the crystal structure were significantly changed due to complex formation. However, the changes in the geometrical arrangement in the complex caused a small but significant increase of the fluorescence emission. Acetate geometry and unique positioning of the amide and amine groups of the receptor render the recognition feasible, and DFT analysis estimated ∼30 kJ M−1 stabilization due to 1:1 complexation. Such positioning and geometrical arrangement may make the receptor very specific to bind acetate anion, and as such R became a very relevant molecule in detection and function of the acetate anion present in complex biochemical system

Conformation and Cytotoxicity of a Tetrapeptide Constellated with Alternative D- and L-proline

Proline containing peptides are highly important due to their natural abundance in various secondary structural elements like turns (b turn and c turn etc.) in proteins. Here the conformation, cytotoxicity and structure of a unique tetrapeptide composed of alternative D- and L-proline residues are discussed. The peptide showed a polyproline II like conformation in dilute aqueous solution. The aqueous solution of the peptide self-assembled to form spheroidal oligomers with a diameter of y90 nm. The morphological features were confirmed by bright field confocal images, TEM analysis and AFM. The alternative D- and Lproline residues in the peptide showed toxicity towards cancer cell lines and y50% cell death was recorded against three different types of cancer cells (Neura 2a, HEK 293 and Hep G2)

Deciphering the Structural Intricacy in Virulence Effectors for Proton-motive Force Mediated Unfolding in Type-III Protein Secretion

Given that the protein unfolding requisite for type-III secretion system (T3SS)-mediated secretion is an energetically unfavorable process, the question of how do pathogenic bacteria unfold and secrete hundreds of toxic proteins in seconds remain largely unknown. In this study, a systematic effort combining experimental and computational approaches has been employed to get some mechanistic insights on the unfolding of effectors in T3SS secretion. The in-depth analysis of pH-dependent folding and stability of a T3SS effector ExoY revealed that proton-concentration gradient (~pH 5.8–6.0) generated by proton-motive force (PMF) had significantly affected folding and structural stability of this protein without significant loss of the free energy of unfolding. Importantly, the lower energetic cost associated with the global unfolding of ExoY was mainly due to its inherent stereo-chemical frustrations embedded within its native-like structure as observed from its core structural analysis. These observations suggest that the cooperation between the evolved structural features of ExoY and pH-mediated unfolding is crucial for PMF-mediated T3SS secretion. From a comprehensive computational analysis of 371 T3SS effectors it was concluded that many of these effectors belong to the category of intrinsically disordered proteins (IDPs) and have similar conserved structural archetypes to facilitate early-stage unfolding process as observed in ExoY. We had also provided details of folding, stability, and molecular evolution in T3SS effectors and established the role of evolved structural archetypes in early-stage unfolding events of this effector for maintaining balance in secretion and function trade-off

C_α–H Carries Information of a Hydrogen Bond Involving the Geminal Hydroxyl Group: A Case Study with a Hydrogen-Bonded Complex of 1,1,1,3,3,3-Hexafluoro-2-propanol and Tertiary Amines

Experimental measurement of the contribution of H-bonding to intermolecular and intramolecular interactions that provide specificity to biological complex formation is an important aspect of macromolecular chemistry and structural biology. However, there are very few viable methods available to determine the energetic contribution of an individual hydrogen bond to binding and catalysis in biological systems. Therefore, the methods that use secondary deuterium isotope effects analyzed by NMR or equilibrium or kinetic isotope effect measurements are attractive ways to gain information on the H-bonding properties of an alcohol system, particularly in a biological environment. Here, we explore the anharmonic contribution to the C–H group when the O–H group of 1,1,1,3,3,3-hexafluoro-2-propanol (HFP) forms an intermolecular H-bond with the amines by quantum mechanical calculations and by experimentally measuring the H/D effect by NMR. Within the framework of density functional theory, ab initio calculations were carried out for HFP in its two different conformational states and their H-bonded complexes with tertiary amines to determine the ¹³C chemical shielding, change in their vibrational equilibrium distances, and the deuterium isotope effect on ¹³C2 (secondary carbon) of HFP upon formation of complexes with tertiary amines. When C2–OH was involved in hydrogen bond formation (O–H as hydrogen donor), it weakened the geminal C2–H bond; it was reflected in the NMR chemical shift, coupling constant, and the equilibrium distances of the C–H bond. The first derivative of nuclear shielding at C2 in HFP was −48.94 and −50.73 ppm Å^–1 for anti and gauche conformations, respectively. In the complex, the values were −50.28 and −50.76 ppm Å^–1, respectively. The C–H stretching frequency was lower than the free monomer, indicating enhanced anharmonicity in the C–H bond in the complex form. In chloroform, HFP formed a complex with the amine; δC2 was 69.107 ppm for HFP–triethylamine and 68.766 ppm for HFP-<i>d</i>₂–triethylamine and the difference in chemical shift, the ΔδC2 was 341 ppb. The enhanced anharmonicity in the hydrogen-bonded complex resulted in a larger vibrational equilibrium distance in C–H/D bonds. An analysis with the Morse potential function indicated that the enhanced anharmonicity encountered in the bond was the origin of a larger isotope effect and the equilibrium distances. Change in vibrational equilibrium distance and the deuterium isotope effect, as observed in the complex, could be used as parameters in monitoring the strength of the H-bond in small model systems with promising application in biomacromolecules

Synthesis, Characterization and Cytotoxicity study of Magnetic (Fe3O4)Nanoparticles and their Drug Conjugate

An easy synthesis of magnetic nanoparticles (Fe3O4) is described. Transmission electron microscopy (TEM), atomic force microscopy (AFM), dynamic light scattering (DLS) and X-ray diffraction (XRD) have been used to study the well dispersed and uniformly spherical nanoparticles. 5-Fluorouracil (5-FU) has been successfully loaded onto the nanoparticles and cytotoxicity studies were performed using a standard MTT assay. The results indicate that 5-fluorouracil-loaded iron nanoparticles are a more potent anticancer drug versus 5-fluorouracil alon

Envisaging the Structural Elevation in the Early Event of Oligomerization of Disordered Amyloid β Peptide

In Alzheimer’s disease (AD), amyloid β (Aβ) protein plays a detrimental role in neuronal injury and death. Recent in vitro and in vivo studies suggest that soluble oligomers of the Aβ peptide are neurotoxic. Structural properties of the oligomeric assembly, however, are largely unknown. Our present investigation established that the 40-residue-long Aβ peptide (Aβ40) became more helical, ordered, and compact in the oligomeric state, and both the helical and β-sheet components were found to increase significantly in the early event of oligomerization. The band-selective two-dimensional NMR analysis suggested that majority of the residues from sequence 12 to 22 gained a higher-ordered secondary structure in the oligomeric condition. The presence of a significant amount of helical conformation was confirmed by Raman bands at 1650 and 1336 cm^–1. Other residues remained mostly in the extended polyproline II (PPII) and less compact β-conformation space. In the event of maturation of the oligomers into an amyloid fiber, both the helical content and the PPII-like structural components declined and ∼72% residues attained a compact β-sheet structure. Interestingly, however, some residues remained in the collagen triple helix/extended 2.5₁-helix conformation as evidenced by the amide III Raman signature band at 1272 cm^–1. Molecular dynamics analysis using an optimized potential for liquid simulation force field with the peptide monomer indicated that some of the residues may have preferences for helical conformation and this possibly contributed in the event of oligomer formation, which eventually became a β-sheet-rich amyloid fiber

A novel spirooxindole derivative inhibits the growth of Leishmania donovani parasite both in vitro and in vivo by targeting type IB topoisomerase

Visceral Leishmaniasis is a fatal parasitic disease and there is an emergent need for development of effective drugs against this neglected tropical disease. We report here development of a novel spirooxindole derivative N-benzyl 2, 2’ α 3, 3’, 5’, 6’, 7’, 7α,α'-octahydro-2methoxycarbonyl-spiro [indole-3, 3’ -pyrrolizidine]-2 one (Compound 4c) which inhibits Leishmania donovani topoisomerase IB (LdTopIB) and kills the wild type as well as drug-resistant parasite strains. This compound inhibits catalytic activity of LdTopIB in competitive manner. Unlike Camptothecin, the compound does not stabilize the DNA-topoisomerase IB cleavage complex; rather, they hinder drug-DNA-enzyme covalent complex formation. Fluorescence studies show stoichiometry of this compound binding to LdTopIB is 2:1 (mole/mole) with a dissociation constant of 6.65 μM. Molecular docking with LdTopIB using the stereoisomers of Compound 4c produced two probable hits for binding site: one in small subunit and the other in the hinge region of the large subunit of LdTopIB. This spirooxindole is highly cytotoxic to promastogotes of L. donovani and also induces apoptosis-like cell death in parasite. Treatment with compound 4c causes depolarization of mitochondrial membrane potential, formation of reactive oxygen species inside parasites and ultimately fragmentation of nuclear DNA. Compound 4c also effectively clears amastigote forms of wild type and drug-resistant parasites from infected mouse peritoneal macrophages but has less effect on host macrophages. Moreover compound 4c showed strong antileishmanial efficacies in BALB/c mice model of leishmaniasis. Potentially this compound can be used as a lead for developing excellent anileishmanial agent against emerging drug resistant strains of the parasite

Metal-Free Activation of Molecular Oxygen by Quaternary Ammonium-Based Ionic Liquid: A Detail Mechanistic Study

Most oxidation processes in common organic synthesis and chemical biology require transition metal catalysts or metalloenzymes. Herein, we report a detailed mechanistic study of a metal-free oxygen (O2) activation protocol on benzylamine/alcohols using simple quaternary alkylammonium-based ionic liquids to produce products such as amide, aldehyde, imine, and in some cases, even aromatized products. NMR and various control experiments established the product formation and reaction mechanism, which involved the conversion of molecular oxygen into a hydroperoxyl radical via a proton-coupled electron transfer process. Detection of hydrogen peroxide in the reaction medium using colorimetric analysis supported the proposed mechanism of oxygen activation. Furthermore, first-principles calculations using density functional theory (DFT) revealed that reaction coordinates and transition state spin densities have a unique spin conversion of triplet oxygen leading to formation of singlet products via a minimum energy crossing point. In addition to DFT, domain-based local pair natural orbital coupled cluster, (DLPNO–CCSD(T)), and complete active space self-consistent field, CASSCF(20,14) methods complemented the above findings. Partial density of states analysis showed stabilization of π* orbital of oxygen in the presence of ionic liquid, making it susceptible to hydrogen abstraction in a mild, metal-free condition. Inductively coupled plasma atomic emission spectroscopic (ICP-AES) analysis of reactant and ionic liquids clearly showed the absence of any significant transition metal contamination. The current results described the origin of O2 activation within the context of molecular orbital (MO) theory and opened up a new avenue for the use of ionic liquids as inexpensive, multifunctional and high-performance alternative to metal-based catalysts for O2 activation