Bactericidal Potency and Extended Serum Life of Stereo-Chemically Engineered Peptides Against Mycobacterium

International Journal of Peptide Research and Therapeutics - Tuberculosis is one of the leading causes of death, with an annual mortality rate of 2 million. The present treatment regimen...     

Structure‐based barcoding of proteins

A reduced representation in the format of a barcode has been developed to provide an overview of the topological nature of a given protein structure from 3D coordinate file. The molecular structure of a protein coordinate file from Protein Data Bank is first expressed in terms of an alpha‐numero code and further converted to a barcode image. The barcode representation can be used to compare and contrast different proteins based on their structure. The utility of this method has been exemplified by comparing structural barcodes of proteins that belong to same fold family, and across different folds. In addition to this, we have attempted to provide an illustration to (i) the structural changes often seen in a given protein molecule upon interaction with ligands and (ii) Modifications in overall topology of a given protein during evolution. The program is fully downloadable from the website http://www.iitg.ac.in/probar/ .     

Biokinetics and <Emphasis Type="Italic">In Vivo</Emphasis> Distribution Behaviours of Silica-Coated Cadmium Selenide Quantum Dots

Recently, quantum dots derived from trace elements like cadmium and selenium have attracted widespread interest in biology and medicine. They are rapidly being used as novel tools for both diagnostic and therapeutic purposes. In this report, we evaluated the distribution of silica-coated cadmium selenide (CdSe) quantum dots (QDs) following intravenous injection into male Swiss albino mice as a model system for determining tissue localization using in vivo fluorescence and ex vivo elemental analysis by inductively coupled plasma optical emission spectroscopy (ICP-OES). Trioctylphosphine oxide-capped CdSe quantum dots were synthesized and rendered water soluble by overcoating with silica, using aminopropyl silane (APS) as silica precursor. ICP-OES was used to measure the cadmium content to indicate the concentration of QDs in blood, organs and excretion samples collected at predetermined time intervals. Meanwhile, the distribution and aggregation state of QDs in tissues were also investigated in cryosections of the organs by fluorescence microscopy. We have demonstrated that the liver and kidney were the main target organs for QDs. Our systematic investigation clearly shows that most of the QDs were metabolized in the liver and excreted via faeces and urine in vivo. A fraction of free QDs, maintaining their original form, could be filtered by glomerular capillaries and excreted via urine as small molecules within 5 days.     

bPE toolkit: toolkit for computational protein engineering

We present a computational toolkit consisting of five utility tools, for performing basic operations on a protein structure file in PDB format. The toolkit consists of five different programs which can be integrated as part of a pipeline for computational protein structure characterization or as a standalone analysis package. The programs include tools for chirality check for amino acids (ProChiral), contact map generation (CoMa), data redundancy (DaRe), hydrogen bond potential energy (HyPE) and electrostatic interaction energy (EsInE). All programs in the toolkit can be accessed and downloaded through the following link: http://www.iitg.ac.in/bpetoolkit/.     

The interplay of sequence and stereochemistry in defining conformation in proteins and polypeptides

Sequential specification of conformation in proteins and polypeptides is a triangular interplay involving the system of linked peptides, the sequences in side chains, and water as solvent. Stereochemistry in side chain linkages is obviously important in the interaction between all of the players, but no specification of its explicit role, if any, in linking sequence with conformation has been made. Flory and coworkers made a puzzling observation in 1967 that, when mutated from poly-L to alternating-L,D stereochemical structure, polypeptides will suffer a reduction in overall dimension or characteristic ratio by an astonishing factor of 10 and to a value even lower than that predicted for free rotation (Miller, W. G.; Brant, D. A.; Flory, P. J. J Mol Biol 1967, 23, 67-80). Enquiring into this longstanding puzzle, Durani and coworkers found that the stereochemical modification will also abolish conformational sensitivity in polypeptide structure to solvent, because electrostatic interactions in the system of linked peptides are transformed from a condition of mutual conflict to one of harmony (Ramakrishnan, V.; Ranbhor, R.; Kumar, A.; Durani, S. J Phys Chem B 2006, 110, 9314-9323). Thus, poly-L stereochemistry could be the fulcrum linking sequences with phi,psis in protein and polypeptide structures, via dielectric arbitrations in a conflicting type of interpeptide electrostatics, in agreement with the electrostatic screening model of Avbelj and Moult (Avbelj, F.; Moult, J. Biochemistry 1995, 34, 755-764).     

Selective photothermal efficiency of citrate capped gold nanoparticles for destruction of cancer cells

Gold nanoparticles are recently having much attention because of their increased applications in biomedical fields. In this paper, we demonstrated the photothermal efficacy of citrate capped gold nanoparticles (AuNPs) for the destruction of A431 cancer cells. Citrate capped AuNPs were synthesized successfully and characterized by UV–visible–NIR spectrophotometry and High Resolution Transmission Electron Microscopy (HR-TEM). Further, AuNPs were conjugated with epidermal growth factor receptor antibody (anti-EGFR) and applied for the selective photothermal therapy (PTT) of human epithelial cancer cells, A431. PTT experiments were conducted in four groups, Group I—control cells, Group II—cells treated with laser light alone, Group III—cells treated with unconjugated AuNP and further laser irradiation and Group IV—anti-EGFR conjugated AuNP treated cells irradiated by laser light. After laser irradiation, cell morphology changes that were examined using phase contrast microscopy along with the relevant biochemical parameters like lactate dehydrogenase activity, reactive oxygen species generation and caspase-3 activity were studied for all the groups to determine whether cell death occurs due to necrosis or apoptosis. From these results we concluded that, these immunotargeted nanoparticles could selectively induce cell death via ROS mediated apoptosis when cells were exposed to a low power laser light.     

Geofold: topology-based protein unfolding pathways capture the effects of engineered disulfides on kinetic stability

Protein unfolding is modeled as an ensemble of pathways, where each step in each pathway is the addition of one topologically possible conformational degree of freedom. Starting with a known protein structure, GeoFold hierarchically partitions (cuts) the native structure into substructures using revolute joints and translations. The energy of each cut and its activation barrier are calculated using buried solvent accessible surface area, side chain entropy, hydrogen bonding, buried cavities, and backbone degrees of freedom. A directed acyclic graph is constructed from the cuts, representing a network of simultaneous equilibria. Finite difference simulations on this graph simulate native unfolding pathways. Experimentally observed changes in the unfolding rates for disulfide mutants of barnase, T4 lysozyme, dihydrofolate reductase, and factor for inversion stimulation were qualitatively reproduced in these simulations. Detailed unfolding pathways for each case explain the effects of changes in the chain topology on the folding energy landscape. GeoFold is a useful tool for the inference of the effects of disulfide engineering on the energy landscape of protein unfolding.     

Peptido-mimetic Approach in the Design of Syndiotactic Antimicrobial Peptides

Biocompatibility, low toxicity and high selectivity towards bacterial cells has been the hallmark of peptide-based antibiotics. The innate immune system has been employing such molecular systems against invading pathogens as a successful defense strategy. In this study, we attempt to develop topologically constrained antimicrobial peptides with syndiotactic stereochemical arrangement, by incorporating L and D amino acids successively in its amino acid sequence. Acetylated versions of the designed peptides were also examined for its influence on bactericidal potency, against Gram-positive and Gram-negative bacteria. Syndiotactic stereochemical arrangement of the polypeptide main chain mimics stereochemistry of Gramicidin, a naturally occurring antimicrobial peptides. Gramicidin is a class of penta-deca-peptides isolated from soil bacteria Bacillus brevis, but their utility as antibiotic was limited to topical use due to high levels of hemotoxicity. Activity profiles of the four de novo designed peptide variants show higher specificity towards Gram-positive bacteria than Gram-negative variants, matching earlier reports on the therapeutic potential of gramicidin as a broad spectrum antibiotic. Significantly, our hemolytic assay confirms very low (<1%) levels of toxicity for the designed peptides unlike gramicidin. Earlier reports confirm that incorporation of D amino acids effectively negates the possibility of proteolytic degradation, thus pointing to the potential utility of de novo designed peptides with diversified stereochemistry as a promising new approach in the generation of novel antibiotic peptides.     

Simulated folding in polypeptides of diversified molecular tacticity: implications for protein folding and de novo design

Stereochemistry could be a powerful variable for conformational tune up of polypeptides for de novo design. It may be also useful probe of possible role of interamide energetics in selection and stabilization of conformation. The homopolypeptides Ac-Xxx30-NHMe, with Xxx = Ala, Val, and Leu, of diversified stereochemical structure are generated by simulated racemization with a modified GROMOS-96 force field. The polypeptides, and other systematic stereochemical variants, are folded by simulated annealing with another modified GROMOS-96 force field under the dielectric constant values 1, 4, and 10. The resultant 15,000 molecular folds of isotactic (poly-L-chiral), syndiotactic (alternating L,D-chiral), and heterotactic (random-L,D-chiral) stereochemical structure, belonging to three polypeptide series, achieved under three different folding conditions, are assessed statistically for structure-to-energy-to-conformation relationship. The results suggest that interamide electrostatics could be a major factor in secondary-structure selection in polypeptides while main-chain stereochemistry could dictate molecular packing and therefore the relative magnitude of hydrogen-bond and Lennard-Jones (LJ) contributions in conformational energy. A method for computational design of heterotactic molecular folds in polypeptide structure has been developed, and the first road map for a chiral tune up of polypeptide structure based on stereochemical engineering has been laid down. Broad implications for protein structure, folding, and de novo design are briefly discussed.