Gurunath Ramanathan

Excited State Relaxation Dynamics of Model Green Fluorescent Protein Chromophore Analogs: Evidence for Cis–Trans Isomerism

Two green fluorescent protein (GFP) chromophore analogs (4Z)-4-(N,N-dimethylaminobenzylidene)-1-methyl-2-phenyl-1,4-dihydro-5H-imidazolin-5-one (DMPI) and (4Z)-4-(N,N-diphenylaminobenzylidene)-1-methyl-2-phenyl-1,4-dihydro-5H-imidazolin-5-one (DPMPI) were investigated using femtosecond fluorescence up-conversion spectroscopy and quantum chemical calculations with the results being substantiated by HPLC and NMR measurements. The femtosecond fluorescence transients are found to be biexponential in nature and the time constants exhibit a significant dependence on solvent viscosity and polarity. A multicoordinate relaxation mechanism is proposed for the excited state relaxation behavior of the model GFP analogs. The first time component (τ(1)) was assigned to the formation of twisted intramolecular charge transfer (TICT) state along the rotational coordinate of N-substituted amine group. Time resolved intensity normalized and area normalized emission spectra (TRES and TRANES) were constructed to authenticate the occurrence of TICT state in subpicosecond time scale. Another picosecond time component (τ(2)) was attributed to internal conversion via large amplitude motion along the exomethylenic double bond which has been enunciated by quantum chemical calculations. Quantum chemical calculation also forbids the involvement of hula-twist because of high activation barrier of twisting. HPLC profiles and proton-NMR measurements of the irradiated analogs confirm the presence of Z and E isomers, whose possibility of formation can be accomplished only by the rotation along the exomethylenic double bond. The present observations can be extended to p-HBDI in order to understand the role of protein scaffold in reducing the nonradiative pathways, leading to highly luminescent nature of GFP.

Biomineralization of N,N-dimethylformamide by Paracoccus sp. strain DMF

N,N-dimethylformamide (DMF) is a man-made compound that is widely used as a solvent for the synthesis of various organic compounds. In this study, a bacterial strain Paracoccus sp. DMF capable of using DMF as the sole carbon, nitrogen and energy source, was isolated from an enrichment culture developed using activated sludge from domestic waste water treatment unit as the source inoculum. The strain DMF was characterized by biochemical tests and 16S rDNA sequence analysis, to be belonging to the genus Paracoccus. Growth on DMF was accompanied with ammonia release and the total organic carbon (TOC) analysis indicated its extensive mineralization. Batch culture studies were conducted in the substrate range of 100-5000 mg L(-1) to determine the biokinetic constants. Strain Paracoccus sp. DMF could tolerate very high concentrations of DMF as the growth was observed even at 15000 mg L(-1). High (micro(max)) and (K(i)) showed the suitability of the strain for the treatment of DMF containing waste water. Transient accumulation of dimethylamine (DMA) in the medium during the growth on DMF and utilization of DMA and monomethylamine (MMA) as growth substrates by Paracoccus sp. strain DMF showed that the pathway of DMF degradation involves DMA and MMA as intermediates, ultimately leading to the formation of carbon dioxide (CO(2)) and ammonia (NH(3)).

Biomineralization of 3-nitrotoluene by Diaphorobacter species

Three bacterial strains utilizing 3-nitrotoluene (3-NT) as a sole source of carbon, nitrogen and energy were isolated from an industrial wastewater treatment plant. Biochemical tests and 16S rDNA sequence analysis revealed that the isolated strains belonged to Diaphorobacter sp. Detailed studies were carried out with Diaphorobacter sp. strain DS2. Degradation of 3-NT by Diaphorobacter sp. strain DS2 was accompanied by the release of nitrite in the culture broth with increase in biomass. Total organic carbon analysis confirmed the extensive mineralization of 3-NT. The strain could degrade 3-methylcatechol, 4-methylcatechol and catechol easily suggesting that the degradation pathway could involve these as possible intermediates. Successful PCR amplification of the oxygenase large subunit and the presence of high activity for catechol 2,3-dioxygenase in the crude cell lysate further confirmed that the degradation of 3-NT occurred through (methyl)catechol intermediates in strain DS2. The strain DS2 was found to degrade other isomers of mononitrotoluene (2-NT and 4-NT) and nitrobenzene as well.

Short CO⋯C intermolecular contacts for molecular assembly

A short CO⋯C intermolecular contact and a weak C–H⋯π hydrogen bonding hold the molecules of (4Z)-4-(4-methoxybenzylidene)-1,2-diphenyl-1,4-dihydro-5H-imidazolin-5-one in a zigzag steps type of molecular assembly.

Integrated sorting, concentration and real time PCR based detection system for sensitive detection of microorganisms

The extremely low limit of detection (LOD) posed by global food and water safety standards necessitates the need to perform a rapid process of integrated detection with high specificity, sensitivity and repeatability. The work reported in this article shows a microchip platform which carries out an ensemble of protocols which are otherwise carried in a molecular biology laboratory to achieve the global safety standards. The various steps in the microchip include pre-concentration of specific microorganisms from samples and a highly specific real time molecular identification utilizing a q-PCR process. The microchip process utilizes a high sensitivity antibody based recognition and an electric field mediated capture enabling an overall low LOD. The whole process of counting, sorting and molecular identification is performed in less than 4 hours for highly dilute samples.

Expression, purification and substrate specificities of 3-nitrotoluene dioxygenase from Diaphorobacter sp. strain DS2

3-Nitotoluene dioxygenase (3-NTDO) is the first enzyme in the degradation pathway of 3-nitrotoluene (3-NT) by Diaphorobacter sp. strain DS2. The complete gene sequences of 3-NTDO were PCR amplified from genomic DNA of Diaphorobacter sp., cloned, sequenced and expressed. The 3-NTDO gene revealed a multi component structure having a reductase, a ferredoxin and two oxygenase subunits. Clones expressing the different subunits were constructed in pET21a expression vector system and overexpressed in E. coli BL21(DE3) host. Each subunit was individually purified separately to homogeneity. The active recombinant enzyme was reconstituted in vitro by mixing all three purified subunits. The reconstituted recombinant enzyme could catalyse biotransformations on a variety of organic aromatics.

Introduction of an electron push-pull system yields a planar Red Kaede fluorescence protein chromophore analogue stabilized by a C = O…π interaction

AbstractCrystal structures of four red kaede fluorescence protein chromophore analogues are reported here. Molecules I-III adopt a non-planar geometry stabilized by π…π stacking and hydrogen bonding. Introduction of an electron push-pull system induces molecule IV to be planar and a C = O…π supramolecular interaction is observed as well. Strong electron withdrawing and donating groups also ensure formation of a higher order two and three dimensional supramolecular architecture through hydrogen bonds in molecules I and IV. All the analogues exhibit good photoluminescence properties and emit in the red region with excellent quantum yields. Graphical AbstractCrystal structures of four Red Fluorescence Protein (rfp) chromophore analogues are reported. Molecules I-III are non planar despite having a linear π-conjugated system. All these three structures are mainly stabilized by π…π interactions, C-H…π and hydrogen bonding. In molecule IV, incorporation of an electron push-pull system introduced a weak C=O…π interaction that made the molecule planar.

Structures and conformation of a benzo-12-crown-4 containing dipeptide.

Crystal structures of the dipeptide Boc‐12‐Crown‐4‐l‐DOPA‐Gly‐OMe (chi) and Boc‐12‐Crown‐4‐d/l‐DOPA‐Gly‐OMe (rac) were solved by single crystal X‐ray diffraction. Analysis of the packing differences in the crystal reveals that the presence of a water molecule in chi enables intermolecular contacts with the solvent that result in a different conformation of the dipeptide backbone itself. An uncommon NH…N interaction stabilizes the peptide in its solid state.

Tuning of intermolecular interactions results in packing diversity in imidazolin-5-ones

AbstractCrystal structures of four green fluorescent protein (GFP) chromophore analogues with different packing interactions could be tuned by appropriate substitutions around the imidazolin-5-one ring are reported here. Compound 1 was crystallized from tetrahydrofuran at room temperature while compounds 2-4 have been crystallized from a mixture of methanol and dichloromethane in 3:1 ratio. Molecule 1, 2 and 3 crystallized in monoclinic lattice while molecule 4 preferred to crystallize in a triclinic crystal system. The crystal packing of these molecules was stabilized by C-H…π stacking and C-H… O type of supramolecular interactions. The results reveal that packing diversity can be easily accomplished in these molecules by tuning the substituents around the imidazolin-5-one ring. Photophysical studies also reveal that all have good quantum yield and fluoresce typically in red region due to presence of electron donating groups around the imidazolin-5-one ring. Graphical AbstractCrystal structures of four green fluorescence protein (GFP) chromophore analogues are reported. Stabilization through packing and packing diversity in crystals are achieved by modulating supramolecular interaction by varying the substituents.

Protonation of the imino nitrogen deactivates the excited state of imidazolin-5-one in the solid state

The imino nitrogen of p-methoxybenzylideneimidazolinone (pMBDI) was protonated using aqueous