1Hß chemical shift-based phase modulated NMR methods for fast identification of amino acid types in proteins.

Here we report two phase modulated NMR experiments: PM-2D HN(CACBHB) and PM-2D HN(HB), that use 1Hß chemical shifts to rapidly identify amino acid type in proteins. The magnetization on the 1Hß spins during the experiments is allowed to evolve for a fixed evolution period that results in phase modulation (positive or negative) of the cross peaks corresponding to various amino acid residues on their 2D HN projections, resembling a typical 2D [1H-15N]-HSQC spectrum. All amino acids except glycine can be categorized into three discernible groups based on their 1Hß chemical shifts, resulting in unique phase patterns at different fixed evolution periods for 1Hß, thus facilitating their identification. Remarkably, the PM-2D HN(HB) stands out among all amino acid type identification NMR techniques for its applicability with cost-effective and most routinely employed 15N-labeled protein samples for NMR studies. Furthermore, when combined effectively with the 13Cß chemical shift-based phase modulated NMR method (PM-2D HN(CACB)), these methods resolved the identification of large groups of amino acids into relatively smaller groups. Moreover, these techniques can accelerate the sequence-specific sequential resonance assignment (SSRA) process and would help in fast tracking of assigned NMR signals exhibiting chemical shift perturbation on the 2D [1H-15N]-HSQC spectrum of proteins during various experiments (e.g., temperature change, pH change, and protein or ligand or cofactor binding) as well as in site-directed mutagenesis.

Evaluation of antibiofilm properties of dehydroacetic acid (DHA) grafted spiro-oxindolopyrrolidines synthesized via multicomponent 1,3-dipolar cycloaddition reaction.

The current work involves the use of dehydroacetic acid based chalcone derivatives for the synthesis of spirooxindole grafted pyrrolidine moieties. All the synthesized compounds have been characterized using spectroscopic techniques such as NMR (1H-NMR and 13C-NMR), IR, mass and elemental analysis. Molecular mechanics studies were performed to comprehend the regioselectivity in the product formation. Molecular docking of the synthesized compounds was performed with few bacterial proteins of Bacillus subtilis and Pseudomonas aeruginosa responsible for biofilm formation followed by molecular dynamics simulations with the potential lead compound. Further, to corroborate the results obtained via in silico study, anti-biofilm activity etc. of the synthesized compounds (4a-e) was checked for effectiveness against biofilm formation. Taken together, this study opens up to explore these compounds' multiple roles in diverse fields in the arena of medical sciences.

Screening of potential inhibitors against structural proteins from Monkeypox and related viruses of Poxviridae family via docking and molecular dynamics simulation.

Monkeypox virus (MPXV) is an orthopoxvirus which causes zoonotic infection in humans. Even though sporadic cases of this infection are limited to the African continent, but if the infection continues to increase unabated, it can be a cause of serious concern for the human populace. Smallpox vaccination has been in use against monkeypox infection but it only provides mild protection. In the current study, we have screened novel small molecules (estrone fused heterocycles (EH1-EH7)) exhibiting good binding with monkeypox virus protein and related proteins from Poxviridae family of viruses via computational approaches. EH1-7 series of small molecules selected for the work have been synthesized via cycloaddition methodology. Docking and Molecular Dynamics (MD) results highlight EH4 compound to have strong binding affinity towards monkeypox and other related viral proteins selected for the study. Thus, computational outcomes suggest EH4 as a good candidate against monkeypox. Currently, no antiviral medication has been approved against monkeypox and the treatment is only via therapeutics available for smallpox and related conditions that may be helpful against monkeypox. Our study is thus an attempt to screen novel compounds against monkeypox infection, which would, in turn, facilitate development of novel therapeutics against Poxviridae family. HIGHLIGHTSMonkeypox infection is a public health emergency and necessitates immediate drug discovery.Molecular docking study to screen estrone-fused heterocycles compounds against Monkeypox and other orthopoxviruses.Molecular dynamics simulations revealed interaction/high binding affinities among EH4 heterocyclic compound and profilin-like protein from the monkeypox virus.Estrone-fused heterocycles compounds are promising anti-viral agents as per our in silico analysis.Our study provides evidence for investigating estrone-fused heterocycles compounds for further pharmacological interventions.Communicated by Ramaswamy H. Sarma.

Monkeypox: This orthopoxvirus leads to mpox (monkeypox) disease which shows symptoms similar to that smallpox, however to less severe extent.Poxviridae family: This is commonly a family of double-stranded DNA viruses. The natural hosts for these viruses are arthropods and Vertebrates.Molecular Dynamic simulation: MD simulation is crucial for determining the ligand's stability and revealing the duration of its interaction with the respective macromolecular structure.Molecular Docking: Molecular docking aids in determining specific sites where the ligand binds with the macromolecule as well as its binding affinity. Bioinformatics tools such as docking have been widely employed for aiding drug discovery efforts.Protein binding energy: On docking protein with the ligand, the binding energy shows the free energy change during binding process between protein-ligand.

Comparative structure, dynamics and evolution of acyl-carrier proteins from Borrelia burgdorferi, Brucella melitensis and Rickettsia prowazekii.

Acyl carrier proteins (ACPs) are small helical proteins found in all kingdoms of life, primarily involved in fatty acid and polyketide biosynthesis. In eukaryotes, ACPs are part of the fatty acid synthase (FAS) complex, where they act as flexible tethers for the growing lipid chain, enabling access to the distinct active sites in FAS. In the type II synthesis systems found in bacteria and plastids, these proteins exist as monomers and perform various processes, from being a donor for synthesis of various products such as endotoxins, to supplying acyl chains for lipid A and lipoic acid FAS (quorum sensing), but also as signaling molecules, in bioluminescence and activation of toxins. The essential and diverse nature of their functions makes ACP an attractive target for antimicrobial drug discovery. Here, we report the structure, dynamics and evolution of ACPs from three human pathogens: Borrelia burgdorferi, Brucella melitensis and Rickettsia prowazekii, which could facilitate the discovery of new inhibitors of ACP function in pathogenic bacteria.

NMR structure and dynamics of inhibitory repeat domain variant 12, a plant protease inhibitor from Capsicum annuum, and its structural relationship to other plant protease inhibitors.

Although several plant protease inhibitors have been structurally characterized using X-ray crystallography, very few have been studied using NMR techniques. Here, we report an NMR study of the solution structure and dynamics of an inhibitory repeat domain (IRD) variant 12 from the wound-inducible Pin-II type proteinase inhibitor from Capsicum annuum. IRD variant 12 (IRD12) showed strong anti-metabolic activity against the Lepidopteran insect pest, Helicoverpa armigera. The NMR-derived three-dimensional structure of IRD12 reveals a three-stranded anti-parallel ß-sheet rigidly held together by four disulfide bridges and shows structural homology with known IRDs. It is interesting to note that the IRD12 structure containing ∼75% unstructured part still shows substantial amount of rigidity of N-H bond vectors with respect to its molecular motion.Communicated by Ramaswamy H. Sarma.

Statistical analysis of intermolecular interactions in trypsin-inhibitor complexes.

Communicated by Ramaswamy H. Sarma.

1H, 13C and 15N NMR assignments of two plant protease inhibitors (IRD7 and IRD12) from the plant Capsicum annuum.

Helicoverpa species are polyphagous pests, with the larval stages causing major damage to economically valuable crops such as cotton, tomato, corn, sorghum, peas, sunflower, wheat and other pulses. Over the years, Helicoverpa armigera has developed resistance to most classes of chemical insecticides, and consequently it is now largely controlled on cotton plants via the use of Bt transgenic crops that express insecticidal Cry toxins which in-turn expedited resistance development in a number of pest species including H. armigera. In a hope to provide other eco-friendly alternatives solutions to counter the effect of the pest, people have identified a number of protease inhibitors (PIs) from the domesticated capsicum species Capsicum annuum, several of which potently inhibited H. armigera gut proteases and impeded growth of H. armigera larva. With a view to explore and enhance the specific nature or properties of these PIs on the mechanism of inhibition, structural and functional characterization of these PIs are inevitable. Towards this goal, we have carried out complete 1H, 13C and 15N resonance assignments of two of these PIs, identified as IRD7 and IRD12, using a suite of 2D and 3D multi-dimensional and multi-nuclear NMR experiments.