Competitive metal-binding stoichiometry between calcium and strontium by cell wall proteins of Neurospora crassa.

Cell wall proteins from Neurospora crassa were isolated and evaluated to demonstrate their metal ability to bind Ca2+ /Sr2+ by loading the solubilized protein fraction on to immobilized metal affinity chromatography (IMAC) column pre-equilibrated with Ca2+ /Sr2+ . The sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis IMAC eluent, revealed ∼18 proteins with a similarity in the proteome pattern of Ca2+ /Sr2+ fractions. Diethyl aminoethyl chromatography showed five proteins in common in binding to Ca2+ and Sr2+ , were subjected to N-terminal sequencing. The sequence analysis was studied for the determination of metal-binding site prediction by CHED software indicating that all five were found to have a high affinity toward Ca2+ . From these five, two were randomly selected and denoted as CWP-A (possess five Ca binding sites of six metal-binding sites) and CWP-B (possess six binding sites of eight metal-binding sites). They were selected for further characterization studies to determine their Ca2+ bound Sr2+ binding properties. Surprisingly, these proteins were able to bind Sr2+ ions (29 µmol) with equal affinity as to Ca2+ ions (42 µmol) by means of direct binding, and/or by displacing calcium as observed in metal-dependent proteolytic protection, fluorescence-based metal exchange assays, and molecular simulation studies. From the results, we demonstrate for the first time, that there is a stoichiometry between Ca2+ (an essential macro elemental metal ion) and Sr2+ ions (a nonessential element for which no reported metabolic activity is reported) for the metal-binding sites on cell wall proteins. This stoichiometry could be due to similar atomic dimensions and metal-protein structure stabilizing properties of Sr2+ compared to Ca2+ .

Evaluating the role of a trypsin inhibitor from soap nut (Sapindus trifoliatus L. Var. Emarginatus) seeds against larval gut proteases, its purification and characterization.

BACKGROUND: The defensive capacities of plant protease Inhibitors (PI) rely on inhibition of proteases in insect guts or those secreted by microorganisms; and also prevent uncontrolled proteolysis and offer protection against proteolytic enzymes of pathogens. METHODS: An array of chromatographic techniques were employed for purification, homogeneity was assessed by electrophoresis. Specificity, Ki value, nature of inhibition, complex formation was carried out by standard protocols. Action of SNTI on insect gut proteases was computationally evaluated by modeling the proteins by threading and docking studies by piper using Schrodinger tools. RESULTS: We have isolated and purified Soap Nut Trypsin Inhibitor (SNTI) by acetone fractionation, ammonium sulphate precipitation, ion exchange and gel permeation chromatography. The purified inhibitor was homogeneous by both gel filtration and polyacrylamide gel electrophoresis (PAGE). SNTI exhibited a molecular weight of 29 kDa on SDS-PAGE, gel filtration and was negative to Periodic Acid Schiff's stain. SNTI inhibited trypsin and pronase of serine class. SNTI demonstrated non-competitive inhibition with a Ki value of 0.75 ± 0.05×10-10 M. The monoheaded inhibitor formed a stable complex in 1:1 molar ratio. Action of SNTI was computationally evaluated on larval gut proteases from Helicoverpa armigera and Spodoptera frugiperda. SNTI and larval gut proteases were modeled and docked using Schrodinger software. Docking studies revealed strong hydrogen bond interactions between Lys10 and Pro71, Lys299 and Met80 and Van Der Waals interactions between Leu11 and Cys76amino acid residues of SNTI and protease from H. Armigera. Strong hydrogen bonds were observed between SNTI and protease of S. frugiperda at positions Thr79 and Arg80, Asp90 and Gly73, Asp2 and Gly160 respectively. CONCLUSION: We conclude that SNTI potentially inhibits larval gut proteases of insects and the kinetics exhibited by the protease inhibitor further substantiates its efficacy against serine proteases.

Expression and functional characterisation of TNC, a high-affinity nickel transporter from Neurospora crassa.

Our previous in silico studies identified a high-affinity nickel transporter, TNC, from the metal transportome of Neurospora crassa. A knockout mutant of the tnc gene in N. crassa failed to transport nickel, showed phenotypic growth defects and diminished urease activity under physiological levels of nickel. Transport assays conducted in wild type and knockout mutant strains showed that TNC transports nickel with high affinity but exhibits selectivity for other transition metal ions like cobalt. Heterologous complementation of Schizosaccharomyces pombe nickel uptake mutant by TNC further substantiates its nickel transport function. Transcriptional analysis of the nickel transporter encoding gene, tnc in N. crassa by qRT-PCR showed its constitutive expression in various phases of its life cycle. However, levels of the corresponding protein TNC were down-regulated only by increasing the nickel, but not cobalt concentration in the media. Immunolocalisation data suggested that TNC is distributed in the plasma membrane of N. crassa. Thus, the present study establishes TNC as a functional plasma membrane nickel transporter necessary for physiological acquisition of nickel in the multicellular fungi N. crassa.

Expression of a Neurospora crassa zinc transporter gene in transgenic Nicotiana tabacum enhances plant zinc accumulation without co-transport of cadmium.

Zinc (Zn) is an essential micronutrient required for growth and development of all organisms. Deficiency of Zn in humans is widespread, affecting 25% of world population and efforts are underway to develop crop plants with high levels of Zn in their edible parts. When strategies for enhancing Zn in crop plants are designed, it is essential to exclude cadmium (Cd), a toxic analogue of Zn. In the present work, a high affinity and high specificity zinc transporter gene (tzn1) from Neurospora crassa was cloned and introduced into Nicotiana tabacum with the objective of enhancing the potential of plants for zinc acquisition. When grown in hydroponic medium spiked with (65) Zn, transgenic plants showed enhanced accumulation of Zn (up to 11 times) compared to control plants, which was confirmed further by environmental scanning electron microscopy coupled with Energy Dispersive X-ray analysis. More importantly, no significant difference in uptake of Cd(2+), Fe(2+), Ni(2+), Cu(2+), Mn(2+) and Pb(2+) between the transgenic and control plants was observed. The present studies have shown that Neurospora crassa tzn1 is a potential candidate gene for developing transgenic plants for improving Zn uptake, without co-transport of Cd and may have implications in Zn phytofortification and phytoremediation.