Mapping Key Residues of ISD11 Critical for NFS1-ISD11 Subcomplex Stability IMPLICATIONS IN THE DEVELOPMENT OF MITOCHONDRIAL DISORDER, COXPD19

Biogenesis of the iron-sulfur (Fe-S) cluster is an indispensable process in living cells. In mammalian mitochondria, the initial step of the Fe-S cluster assembly process is assisted by the NFS1-ISD11 complex, which delivers sulfur to scaffold protein ISCU during Fe-S cluster synthesis. Although ISD11 is an essential protein, its cellular role in Fe-S cluster biogenesis is still not defined. Our study maps the important ISD11 amino acid residues belonging to putative helix 1 (Phe-40), helix 3 (Leu-63, Arg-68, Gln-69, Ile-72, Tyr-76), and C-terminal segment (Leu-81, Glu-84) are critical for in vivo Fe-S cluster biogenesis. Importantly, mutation of these conserved ISD11 residues into alanine leads to its compromised interaction with NFS1, resulting in reduced stability and enhanced aggregation of NFS1 in the mitochondria. Due to altered interaction with ISD11 mutants, the levels of NFS1 and Isu1 were significantly depleted, which affects Fe-S cluster biosynthesis, leading to reduced electron transport chain complex (ETC) activity and mitochondrial respiration. In humans, a clinically relevant ISD11 mutation (R68L) has been associated in the development of a mitochondrial genetic disorder, COXPD19. Our findings highlight that the ISD11 R68A/R68L mutation display reduced affinity to form a stable subcomplex with NFS1, and thereby fails to prevent NFS1 aggregation resulting in impairment of the Fe-S cluster biogenesis. The prime affected machinery is the ETC complex, which showed compromised redox properties, causing diminished mitochondrial respiration. Furthermore, the R68L ISD11 mutant displayed accumulation of mitochondrial iron and reactive oxygen species, leading to mitochondrial dysfunction, which correlates with the phenotype observed in COXPD19 patients.

On the Structural Stability of Melt Spun Ribbons of Fe95-x Zr (x) B4Cu1 (x=7 and 9) Alloys and Correlation with Their Magnetic Properties

Melt spun ribbons of Fe95-x Zr (x) B4Cu1 with x = 7 (Z7B4) and 9 (Z9B4) alloys have been prepared, and their structure and magnetic properties have been evaluated using XRD, DSC, TEM, VSM, and Mossbauer spectroscopy. The glass forming ability (GFA) of both alloys has been calculated theoretically using thermodynamical parameters, and Z9B4 alloy is found to possess higher GFA than that of Z7B4 alloy which is validated by XRD results. On annealing, the amorphous Z7B4 ribbon crystallizes into nanocrystalline alpha-Fe, whereas amorphous Z9B4 ribbon shows two-stage crystallization process, first partially to bcc solid solution which is then transformed to nanocrystalline alpha-Fe and Fe2Zr phases exhibiting bimodal distribution. A detailed phase analysis using Mossbauer spectroscopy through hyperfine field distribution of phases has been carried out to understand the crystallization behavior of Z7B4 and Z9B4 alloy ribbons. In order to understand the phase transformation behavior of Z7B4 and Z9B4 ribbons, molar Gibbs free energies of amorphous, alpha-Fe, and Fe2Zr phases have been evaluated. It is found that in case of Z7B4, alpha-Fe is always a stable phase, whereas Fe2Zr is stable at higher temperature for Z9B4. (C) The Minerals, Metals & Materials Society and ASM International 2015

Analysis on conservation of disulphide bonds and their structural features in homologous protein domain families

Background: Disulphide bridges are well known to play key roles in stability, folding and functions of proteins. Introduction or deletion of disulphides by site-directed mutagenesis have produced varying effects on stability and folding depending upon the protein and location of disulphide in the 3-D structure. Given the lack of complete understanding it is worthwhile to learn from an analysis of extent of conservation of disulphides in homologous proteins. We have also addressed the question of what structural interactions replaces a disulphide in a homologue in another homologue. Results: Using a dataset involving 34,752 pairwise comparisons of homologous protein domains corresponding to 300 protein domain families of known 3-D structures, we provide a comprehensive analysis of extent of conservation of disulphide bridges and their structural features. We report that only 54% of all the disulphide bonds compared between the homologous pairs are conserved, even if, a small fraction of the non-conserved disulphides do include cytoplasmic proteins. Also, only about one fourth of the distinct disulphides are conserved in all the members in protein families. We note that while conservation of disulphide is common in many families, disulphide bond mutations are quite prevalent. Interestingly, we note that there is no clear relationship between sequence identity between two homologous proteins and disulphide bond conservation. Our analysis on structural features at the sites where cysteines forming disulphide in one homologue are replaced by non-Cys residues show that the elimination of a disulphide in a homologue need not always result in stabilizing interactions between equivalent residues. Conclusion: We observe that in the homologous proteins, disulphide bonds are conserved only to a modest extent. Very interestingly, we note that extent of conservation of disulphide in homologous proteins is unrelated to the overall sequence identity between homologues. The non-conserved disulphides are often associated with variable structural features that were recruited to be associated with differentiation or specialisation of protein function.

The effect of pH on the unfolding pathway and stability of ribosome-inactivating protein abrin-II

The effect of pH on the unfolding pathway acid the stability of the toxic protein abrin-II have been studied by increasing denaturant concentrations of guanidine hydrochloride and by monitoring the change in 8,1-anilino naphthalene sulfonic acid (ANS) fluorescence upon binding to the hydrophobic sites of the protein. Intrinsic protein fluorescence, far and near UV-circular dichroism (CD) spectroscopy and ANS binding studies reveal that the unfolding of abrin-II occurs through two intermediates at pH 7.2 and one intermediate at pH 4.5. At pH 7.2, the two subunits A and B of abrin-II unfold sequentially. The native protein is more stable at pH 4.5 than at pH 7.2. However, the stability of the abrin-II A-subunit is not affected by a change in pH. These observations may assist in an understanding of the physiologically relevant transmembrane translocation of the toxin.

Thermodynamic characterization of the conformational stability of the homodimeric protein, pea lectin

The conformational stability of the homodimeric pea lectin was determined by both isothermal urea-induced and thermal denaturation in the absence and presence of urea. The denaturation profiles were analyzed to obtain the thermodynamic parameters associated with the unfolding of the protein. The data not only conform to the simple A(2) double left right arrow 2U model of unfolding but also are well described by the linear extrapolation model for the nature of denaturant-protein interactions. In addition, both the conformational stability (Delta G(s)) and the Delta C-p for the protein unfolding is quite high, at about 18.79 kcal/ mol and 5.32 kcal/(mol K), respectively, which may be a reflection of the relatively larger size of the dimeric molecule (M-r 49 000) and, perhaps, a consequent larger buried hydrophobic core in the folded protein. The simple two-state (A(2) double left right arrow 2U) nature of the unfolding process, with the absence of any monomeric intermediate, suggests that the quaternary interactions alone may contribute significantly to the conformational stability of the oligomer-a point that may be general to many oligomeric proteins.

Tyrosine kinases and calcium dependent activation of endothelial cell phospholipase D by diperoxovanadate

Reactive oxygen species (ROS) mediated modulation of signal transduction pathways represent an important mechanism of cell injury and barrier dysfunction leading to the development of vascular disorders. Towards understanding the role of ROS in vascular dysfunction, we investigated the effect of diperoxovanadate (DPV), derived from mixing hydrogen peroxide and vanadate, on the activation of phospholipase D (PLD) in bovine pulmonary artery endothelial cells (BPAECs). Addition of DPV to BPAECs in the presence of .05% butanol resulted in an accumulation of [P-32] phosphatidylbutanol (PBt) in a dose- and time-dependent manner. DPV also caused an increase in tyrosine phosphorylation of several protein bands (Mr 20-200 kD), as determined by Western blot analysis with antiphosphotyrosine antibodies. The DPV-induced [P-32] PBt-accumulation was inhibited by putative tyrosine kinase inhibitors such as genistein, herbimycin, tyrphostin and by chelation of Ca2+ with either EGTA or BAPTA, however, pretreatment of BPAECs with the inhibitor PKC bisindolylmaleimide showed minimal inhibition. Also down-regulation of PKC alpha and epsilon, the major isotypes of PKC in BPAECs, by TPA (100 nM, 18 h) did not attenuate the DPV-induced PLD activation. The effects of putative tyrosine kinase and PKC inhibitors were specific as determined by comparing [P-32] PBt formation between DPV and TPA. In addition to tyrosine kinase inhibitors, antioxidants such as N-acetylcysteine and pyrrolidine dithiocarbamate also attenuated DPV-induced protein tyrosine phosphorylation and PLD stimulation. These results suggest that oxidation, prevented by reduction with thiol compounds, is involved in DPV-dependent protein tyrosine phosphorylation and PLD activation.

Effect of spatial dispersion of the dielectric on the binding energy of D- ion in Si and Ge

Using a multivalley effective mass theory, we obtain the binding energy of a D- ion in Si and Ge taking into account the spatial variation of the host dielectric function. We find that on comparison with experimental results the effect of spatial dispersion is important in the estimation of binding energy for the D- formed by As in Si and Ge. The effect is less significant for the case of D- formed by P and Sb donors.

Immunochemical approach to the mapping of an assembled epitope of human chorionic gonadotropin: Proximity of CTP-alpha to the receptor binding region of the beta-subunit

A single-step solid-phase RIA (SS-SPRIA) developed in our laboratory using hybridoma culture supernatants has been utilised for the quantitation of epitope-paratope interactions. Using SS-SPRIA as a quantitative tool for the assessment of epitope stability, it was found that several assembled epitopes of human chorionic gonadotropin (hCG) are differentially stable to proteolysis and chemical modification. Based on these observations an approach has been developed for identifying the amino acid residues constituting an epitopic region. This approach has now been used to map an assembled epitope at/near the receptor binding region of the hormone. The mapped site forms a part of the seat belt region and the cystine knot region (C34-C38-C88-C90-H106). The carboxy terminal region of the alpha-subunit forms a part of the epitope indicating its proximity to the receptor binding region. These results are in agreement with the reported receptor binding region identified through other approaches and the X-ray crystal structure of hCG.

Studies on the target conditioning for deposition of $LiCoO_{2}$ films

Deposition of good quality thin films of Lithium Cobalt Oxide (LiCoO2), by sputtering is preceded by target conditioning, which dictates the surface composition, morphology and electrochemical performance of the deposited film. Sputtering from a Virgin target surface, results in films with excess of the more reactive elements. The concentration of these reactive elements in the films decreases until the system reaches a steady state after sufficient sputtering from the target. This paper discusses the deposition kinetics in terms of target conditioning of LiCoO2. The composition, morphology and texturing of deposited film during various hours of sputtering were analyzed using X-ray photoelectron Spectroscopy (XPS) and Field Emission Scanning electron microscopy (FESEM). The compositional stability is not observed in the films formed during the initial hours or Sputtering from the fresh target, which becomes stable after several hours of sputtering. The Li and Co concentration in the Films deposited subsequently is found to be varying and possible causes are discussed. After the compositional stability is reached, electrochemical analysis of LiCoO2 thin films was performed, which shows a discharge capacity of 129 mu Ah/cm(2).

Hingeless-rotor aeromechanical stability in axial and forward flight with wake dynamics

A finite-state wake model is used to investigate aeromechanical stability of hingeless-rotor helicopters in the ground-contact, hover and trimmed-night conditions. The investigation covers three items: (1) the convergence of the damping with increasing number of wake harmonics for the lag regressing, and body pitch and roll modes; (2) a parametric study of the damping over a range of thrust level, advance ratio and number of blades; and (3) correlations, primarily with the damping and frequency measurements of these lag and body modes. The convergence and parametric studies are conducted in the hover and trimmed-flight conditions; they include predictions from the widely used dynamic inflow model. The correlations are conducted in the ground-contact conditions and include predictions from the dynamic inflow and vortex models; recently, this vortex model is proposed for the axial-flight conditions and is used to investigate the coupled free vibrations of rotor flapping and body modes. The convergence and parametric studies show that a finite-state wake model that goes well beyond the dynamic inflow model is required for fairly converged damping, Moreover, the correlations from the finite-state wake, dynamic inflow and vortex models are generally satisfactory.

Analytical approximation solutions for 3-D optical waveguides: Review

The rectangular dielectric waveguide is the most commonly used structure in integrated optics, especially in semi-conductor diode lasers. Demands for new applications such as high-speed data backplanes in integrated electronics, waveguide filters, optical multiplexers and optical switches are driving technology toward better materials and processing techniques for planar waveguide structures. The infinite slab and circular waveguides that we know are not practical for use on a substrate because the slab waveguide has no lateral confinement and the circular fiber is not compatible with the planar processing technology being used to make planar structures. The rectangular waveguide is the natural structure. In this review, we have discussed several analytical methods for analyzing the mode structure of rectangular structures, beginning with a wave analysis based on the pioneering work of Marcatili. We study three basic techniques with examples to compare their performance levels. These are the analytical approach developed by Marcatili, the perturbation techniques, which improve on the analytical solutions and the effective index method with examples.

Micro-scale anaerobic digestion of point source components of organic fraction of municipal solid waste

The fermentation characteristics of six specific types of the organic fraction of municipal solid waste (OFMSW) were examined, with an emphasis on properties that are needed when designing plug-flow type anaerobic bioreactors. More specifically, the decomposition patterns of a vegetable (cabbage), fruits (banana and citrus peels), fresh leaf litter of bamboo and teak leaves, and paper (newsprint) waste streams as feedstocks were studied. Individual OFMSW components were placed into nylon mesh bags and subjected to various fermentation periods (solids retention time, SRT) within the inlet of a functioning plug-flow biogas fermentor. These were removed at periodic intervals, and their composition was analyzed to monitor decomposition rates and changes in chemical composition. Components like cabbage waste, banana peels, and orange peels fermented rapidly both in a plug-flow biogas reactor (PFBR) as well as under a biological methane potential (BMP) assay, while other OFMSW components (leaf litter from bamboo and teak leaves and newsprint) fermented slowly with poor process stability and moderate biodegradation. For fruit and vegetable wastes (FVW), a rapid and efficient removal of pectins is the main cause of rapid disintegration of these feedstocks, which left behind very little compost forming residues (2–5%). Teak and bamboo leaves and newsprint decomposed only to 25–50% in 30 d. These results confirm the potential for volatile fatty acids accumulation in a PFBR’s inlet and suggest a modification of the inlet zone or operation of a PFBR with the above feedstocks.

Stability of Dimeric Interface in Banana Lectin: Insight from Molecular Dynamics Simulations

Banana lectin (Banlec) is a homodimeric non-glycosylated protein. It exhibits the b-prism I structure. High-temperature molecular dynamics simulations have been utilized to monitor and understand early stages of thermally induced unfolding of Banlec. The present study elucidates the behavior of the dimeric protein at four different temperatures and compares the structural and conformational changes to that of the minimized crystal structure. The process of unfolding was monitored by following the radius of gyration, the rms deviation of each residue, change in relative solvent accessibility and the pattern of inter- and intra-subunit interactions. The overall study demonstrates that the Banlec dimer is a highly stable structure, and the stability is mostly contributed by interfacial interactions. It maintains its overall conformation during high-temperature (400–500 K) simulations, with only the unstructured loop regions acquiring greater momentum under such condition. Nevertheless, at still higher temperatures (600 K) the tertiary structure is gradually lost which later extends to loss of secondary structural elements. The pattern of hydrogen bonding within the subunit and at the interface across different stages has been analyzed and has provided rationale for its intrinsic high stability.

Localisation of Plasmodium falciparum uroporphyrinogen III decarboxylase of the heme-biosynthetic pathway in the apicoplast and characterisation of its catalytic properties

Uroporphyrinogen decarboxylase (UROD) is a key enzyme in the heme-biosynthetic pathway and in Plasmodium falciparum it occupies a strategic position in the proposed hybrid pathway for heme biosynthesis involving shuttling of intermediates between different subcellular compartments in the parasite. In the present study, we demonstrate that an N-terminally truncated recombinant P. falciparum UROD (r(Δ)PfUROD) over-expressed and purified from Escherichia coli cells, as well as the native enzyme from the parasite were catalytically less efficient compared with the host enzyme, although they were similar in other enzyme parameters. Molecular modeling of PfUROD based on the known crystal structure of the human enzyme indicated that the protein manifests a distorted triose phosphate isomerase (TIM) barrel fold which is conserved in all the known structures of UROD. The parasite enzyme shares all the conserved or invariant amino acid residues at the active and substrate binding sites, but is rich in lysine residues compared with the host enzyme. Mutation of specific lysine residues corresponding to residues at the dimer interface in human UROD enhanced the catalytic efficiency of the enzyme and dimer stability indicating that the lysine rich nature and weak dimer interface of the wild-type PfUROD could be responsible for its low catalytic efficiency. PfUROD was localised to the apicoplast, indicating the requirement of additional mechanisms for transport of the product coproporphyrinogen to other subcellular sites for its further conversion and ultimate heme formation.