Biodegradation of octogen and hexogen by Pelomonas aquatica strain WS2-R2A-65 under aerobic condition.

Biodegradation ability of a native bacterial species Pelomonas aquatica strain WS2-R2A-65, isolated from nitramine explosive-contaminated effluent, for octogen (HMX) and hexogen (RDX) under aerobic condition has been explored in this study. Scanning electron microscopy indicated that the isolate WS2-R2A-65 retained its morphology both in the presence and absence of HMX or RDX. During an incubation period of 20 days, the isolate cometabolically degraded 78 and 86% of HMX and RDX with initial concentrations 6 and 60 mg L-1, respectively. The degradation mechanism followed the first-order kinetics for both the nitramines with a 50% degradation time of 9.9 and 7.7 days for HMX and RDX, respectively. Positive electrospray ionisation mass spectroscopy indicates that biodegradation of nitamines follows multiple degradation pathways with one involving ring cleavage via single-electron transfer to nitramines leading to the elimination of single nitrite ion as evident from the formation of methylenedinitramine (MEDINA) and its methyl derivatives. The other pathways involve the reduction of both the nitramines to their nitroso, hydroxylamino and amino derivatives. These metabolites get further ring cleaved to give secondary metabolites viz. N-hydroxymethylmethylenedintramine, N-nitrosoamino and hydrazinyl derivatives leading to simpler less hazardous end products. Thus, the isolate WS2-R2A-65 proves to be an efficient microbial species for bioremediation of nitramines-contaminated effluent.

Aerobic biodegradation of HMX by Planomicrobium flavidum.

In this report, aerobic biodegradation of octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine or high melting explosive (HMX), a highly explosive chemical by Planomicrobium flavidum strain S5-TSA-19, an isolate from an explosive-contaminated soil, was investigated. The isolate S5-TSA-19 degraded 70% of HMX in 20 days during which time nitrite ion was produced with the subsequent formation of metabolites, viz. methylenedintramine and N-methyl-N,N'-dinitromethanediamine with molecular weights 136 Da and 149 Da, respectively. The degradation mechanism was found to follow first-order kinetics with a half-life of 11.55 days and formation of above intermediates indicate single nitrite elimination pathway. The proliferation of isolate S5-TSA-19 in the absence of nitramines indicates the cometabolic degradation of HMX. Isolate S5-TSA-19 can thus be used as futuristic microbe for degradation of HMX at explosive-contaminated site.

Resonance energy transfer and ligand binding studies on pH-induced folded states of human serum albumin.

Human serum albumin (HSA) is a very important transporter protein in the circulatory system. It is a multi-domain binding protein, which binds a wide variety of ligands in its multiple binding sites and aids in transport, distribution and metabolism of many endogenous and exogenous ligands. With change in pH, HSA is known to undergo conformational transformation, which is very essential for picking up and releasing them at sites of differing pH inside physiological system. Hence, the characterization of ligand binding to these pH-induced conformers is extremely important. We have explored binding interaction of a ligand protoporphyrin IX (PPIX), which is demonstrated (X-ray crystallography) to reside in domain-IB at the various pH-induced folded states of HSA. The ligand PPIX is found to remain attached to all the HSA conformers which offers an opportunity to use Förster's resonance energy transfer (FRET) between an intrinsic donor fluorophore (Trp214) located in domain-IIA to the acceptor ligand PPIX to characterize the inter-domain separation between IB and IIA. Additionally FRET between an extrinsic fluorophore 2-p-toluidinylnaphthalene-6-sulfonate (TNS) located in domain-IIIA and PPIX is also undertaken to quantify the inter-domain separation between IB and IIIA. Circular dichroism (CD) and dynamic light scattering (DLS) studies have been done in conjunction with picosecond time resolved fluorescence and polarization-gated spectroscopy to determine, respectively, the secondary and tertiary structures of various pH-induced folded states of the protein. Severe structural perturbation including swelling of the protein is observed in the low pH-induced conformer of HSA as evidenced from all the techniques used.

Spectroscopic studies on the effect of temperature on pH-induced folded states of human serum albumin.

Human serum albumin (HSA) is a very important multi-domain transporter protein in the circulatory system responsible for carriage of various kinds of ligands within the physiological system. HSA is also known to undergo conformational transformation at different pH(s) and temperatures. In this report we have studied the binding interactions of a photosensitizing drug, protoporphyrin IX (PPIX) with various conformers of HSA at different temperatures using picosecond time-resolved fluorescence spectroscopy. Also, using dynamic light scattering (DLS) and circular dichroism (CD) spectroscopy we have followed the structural transition of various conformers of HSA at different temperatures. Ensuring the intact binding of PPIX to various conformers of HSA at different temperatures as revealed through time-resolved fluorescence anisotropy decay and significant spectral overlap of emission of Trp214 residue (donor) in domain-IIA and absorption of PPIX (acceptor) bound to domain-IB of HSA, we have applied Förster's resonance energy transfer (FRET) technique to determine the interdomain separation under various environmental conditions. The alkali-induced conformer of HSA shows almost no change in donor-acceptor distance in contrast to the native and acid-induced conformers of HSA, which show a decrease in distance with increase in temperature. Through this study the non-covalently bound PPIX is shown to be an efficient FRET probe in reporting the different temperature-induced folded states of HSA in buffer solutions of widely differing pH values.

Fluorescence relaxation dynamics of acridine orange in nanosized micellar systems and DNA.

In this paper, we report a detailed study of the fluorescence relaxation dynamics of a well-known fluorescent DNA intercalator, acridine orange (AO), in reverse micelles (RM), micelles, and DNA using picosecond resolved fluorescence spectroscopy. Solvation studies of AO in AOT reverse micelles (RM) containing water indicate the locations of AO close to the interface and those in RM containing NaOH; there are two types of AO--one in the nonpolar oil phase and the other at the interface. The bound water at the reverse micellar interface is found to be much more rigid than that at the micellar interface of sodium dodecyl sulfate (SDS) micelles. Dynamic light scattering (DLS) studies allow for the determination of the hydrodynamic radius and the overall tumbling motion of the macromolecules. Wobbling-in-cone data analysis of the temporal fluorescence anisotropy decay allows for determination of restriction on the motion of fluorophores attached to the macromolecules. This model further applied to AO-intercalated genomic DNA and synthetic oligonucleotides within their structural integrity (as confirmed through circular dichroism (CD) studies) shows that AO experiences less restriction in genomic salmon sperm DNA compared with that in synthetic oligonucleotides, and among the oligonucleotides, the ones with AT base pairs are much more rigid. This study would invoke further research on the dynamical nature of AO in restricted environments.

Activity of Subtilisin Carlsberg in macromolecular crowding.

Enzymatic activity of a proteolytic enzyme Subtilisin Carlsberg (SC) in anionic sodium dodecyl sulfate (SDS) micellar medium has been explored and found to be retarded compared to that in bulk buffer. Circular dichroism (CD) study reveals that SDS, which is a potential protein denaturant, has an insignificant denaturation effect on SC. The structural integrity of the protein offers an opportunity to study the functionality of the enzyme SC in a macromolecular crowding of micelles. Dynamic light scattering (DLS) data indicates no sandwich-like micelle-SC complex formation ruling out the possibility of interaction of the enzyme with the hydrophobic core of the micelle. However, steady state and time resolved emission studies on specific and nonspecific fluorescent probes indicate the proximity effect at the surface of the enzyme due to macromolecular crowding of the micelles. The agreement of retarded enzymatic activity in the micellar crowd with a theoretical model ascribed to the facts that substrates are compartmentalized in the micelles and enzyme interacts with the micelle through stern layer.

Direct observation of protein folding in nanoenvironments using a molecular ruler.

We observe folding of horse heart cytochrome c in various environments including nano-compartments (micelles and reverse micelles). Using picosecond-resolved Förster resonance energy transfer (FRET) dynamics of an extrinsic covalently attached probe dansyl (donor) at the surface of the protein to a heme group (acceptor) embedded inside the protein, we measured angstrom-resolved donor-acceptor distances in the environments. The overall structural perturbations of the protein revealed from the FRET experiments are in close agreement with our circular dichroism (CD) and dynamic light scattering (DLS) studies on the protein in a variety of solution conditions. The change of segmental motion of the protein due to imposed restriction in the nano-compartments compared to that in bulk buffer is also revealed by temporal fluorescence anisotropy of the dansyl probe.

Ultrafast photoinduced deligation and ligation dynamics: DCM in micelle and micelle-enzyme complex.

We report studies on diffusion controlled deligation and ligation dynamics of a probe ligand 4-(dicyanomethylene)-2-methyl-6-(p-dimethylamino-styryl) 4H-pyran (DCM) with cationic cetyltrimethylammonium bromide (CTAB) micelles. In order to investigate the effect of spatial heterogeneity on the dynamics we study the DCM labeled micelle upon complexation with an enzyme alpha-chymotrypsin (CHT). The variation of fluorescence line-width (Gamma(t)) of DCM in the complex and also in the micelle indicates the diffusion dynamics of DCM through various environments of different polarities. The temporal behavior of Gamma(t) reveals that at 50 mM CTAB concentration the excited DCM traverses 6.5 Angstrom distance from the surface of a host micelle (deligation) before entering to a stern layer of another adjacent micelle (ligation). From neutron scattering experiment the distance 6.5 Angstrom is found to be the thickness of a stern layer of CTAB micelle. No indication of ligation has been found at 2 mM CTAB concentration as the intermicellar distance is estimated to be very large (416 Angstrom) compared to the previous case. The dynamical behavior of Gamma(t) is also indicative of significantly slower diffusion of the ligand molecules (DCM) at the surface of the micelle in presence and absence of the enzyme compared to that in the bulk buffer. We have also studied the dynamics of solvation and local geometrical restriction on the probe DCM at the micellar surface with and without CHT. With picosecond time resolution, we found time constants of the solvation relaxation processes of the DCM labeled enzyme-micelle complex to be 230 ps (45%) and 870 ps (55%), which were comparable to those of the micelle without the enzyme. The time dependent anisotropy revealing local orientational motions of the probe in the complex was also found to be similar to that of DCM at the micellar surface in absence of CHT. These studies attempt to link the dynamical features for insight into the ligand mediated intercellular communication and the biological function of the enzyme alpha-chymotrypsin upon complexation with the CTAB micelle.

Ultrafast dynamics in a nanocage of enzymes: solvation and fluorescence resonance energy transfer in reverse micelles.

In this contribution we report studies of the nature of solvation and resonance energy transfer processes in a reverse micelle (RM) upon encapsulation of a digestive enzyme, alpha-chymotrypsin (CHT). We have used one donor, Coumarin 500 (C500), and three acceptors Rhodamine 123 (R123, cationic), ethidium bromide (EtBr, cationic), and Merocyanine 540 (MC540, anionic). By selectively exciting the donor at the surface of the RM with a proper excitation wavelength we have examined solvation dynamics in the microenvironment. The solvation correlation function in the RM without CHT exhibits single-exponential decay with time constant approximately 660 ps, which is similar to that of the CHT-included RM. However, in the case of CHT-included RM (w(0)=10), the time-resolved anisotropy and spectral linewidth analysis of the surface-bound donor reveal the existence of an annular aqueous channel of thickness approximately 2.5 A between the enzyme surface and the inner surface of the RM. The aqueous channel is a potential host for the water-soluble substrate and also is involved in maintaining the proper functionality of RM encapsulated CHT. The studies use both steady-state and time-resolved fluorescence resonance energy transfer (FRET) techniques to measure donor-acceptor distances in the RM and also emphasize the danger of using steady-state fluorescence quenching as a method in careful estimation of the distances. The local geometrical restriction on the donor and acceptor molecules was estimated from time-resolved polarization (anisotropy) measurements. The time-resolved anisotropy of the donor and acceptor molecules also revealed significant randomization of the relative orientation of transition dipoles of the donor and acceptor, justifying the use of 2/3 as the value of the orientation factor kappa2. These studies attempt to elucidate the excellence of the RM as a nanohost of biological macromolecules.

Ultrafast surface solvation dynamics and functionality of an enzyme alpha-chymotrypsin upon interfacial binding to a cationic micelle.

In this contribution we report studies on enzymatic activity of alpha-chymotrypsin (CHT) upon complexation with cationic cetyltrimethylammonium bromide (CTAB) micelle. With picosecond time resolution, we examined solvation dynamics at the interface of CHT-micelle complex, and rigidity of the binding. We have used 5-(dimethyl amino) naphthalene-1-sulfonyl chloride (dansyl chloride; DC) that is covalently attached to the enzyme at the surface sites. The solvation processes at the surface of CHT in buffer solution are found to be mostly in the sub-50 ps time scale. However, at the interface the solvation correlation function decays with time constant 150 ps (65%) and 500 ps (35%), which is significantly different from those found at the enzyme and micellar surfaces. The binding structure of the enzyme-micelle complex was examined by local orientational motion of the probe DC and compared with the case without micelle. The orientational dynamics of the probe DC in the complex reveals a structural perturbation at the surface sites of CHT upon complexation, consistent with other reported structural studies. We also found possible entanglement of charge transfer dynamics of the probe DC on the measured solvation processes by using time-resolved area normalized emission spectroscopic technique. The interfacial solvation process and complex rigidity elucidate the strong recognition mechanism between CHT and the micelle, which is important to understand the biological function of CHT upon complexation with the micelle.