Proline Substitution of Dimer Interface β-strand Residues as a Strategy for?the Design of Functional Monomeric Proteins

Proteins that exist in monomer-dimer equilibrium can be found in all organisms ranging from bacteria to humans; this facilitates fine-tuning of activities from signaling to catalysis. However, studying the structural basis of monomer function that naturally exists in monomer-dimer equilibrium is challenging, and most studies to date on designing monomers have focused on disrupting packing or electrostatic interactions that stabilize the dimer interface. In this study, we show that disrupting backbone H-bonding interactions by substituting dimer interface β-strand residues with proline (Pro) results in fully folded and functional monomers, by exploiting proline’s unique feature, the lack of a backbone amide proton. In interleukin-8, we substituted Pro for each of the three residues that form H-bonds across the dimer interface β-strands. We characterized the structures, dynamics, stability, dimerization state, and activity using NMR, molecular dynamics simulations, fluorescence, and functional assays. Our studies show that a single Pro substitution at the middle of the dimer interface β-strand is sufficient to generate a fully functional monomer. Interestingly, double Pro substitutions, compared to single Pro substitution, resulted in higher stability without compromising native monomer fold or function. We propose that Pro substitution of interface β-strand residues is a viable strategy for generating functional monomers of dimeric, and potentially tetrameric and higher-order oligomeric proteins.     

Differential Reprogramming of Defense Network in Capsicum annum L. Plants Against Colletotrichum truncatum Infection by Phyllospheric and Rhizospheric Trichoderma Strains

Journal of Plant Growth Regulation - Induction of defense response in host plants by the Trichoderma spp. has been attributed as one of the major mechanisms leading to inhibition of the pathogenic...     

Performance of vesicular-arbuscular mycorrhizae in saline-alkali soil in relation to various amendments

The present study shows that vesicular-arbuscular mycorrhizal colonization in plants grown in saline-alkali soil is host dependent and it is significantly affected by various amendments given to reclaim such soils. Maximum reduction in vesicular-arbuscular mycorrhizal colonization was observed in the plants raised in saline-alkali soil amended with gypsum and maximum colonization was observed after plant-growth promoting rhizobacterium or farm yard manure amendments. Plants possessing high level of vesicular-arbuscular mycorrhizal colonization (50–70%) included Acacia nilotica, Albizia lebbeck and Dalbergia sissoo. Casuarina equisetifolia possessed moderate level of vesicular-arbuscular mycorrhizae colonization (30–50%) and Azadirachta indica possessed low level of colonization (20–40%).     

A lipopolysaccharide mutant of Bradyrhizobium japonicum that uncouples plant from bacterial differentiation.

The Tn5-containing fragment from a non-nodulating mutant of Bradyrhizobium japonicum, strain ML142, was introduced into B. japonicum strain 61A101c by marker exchange to construct strain JS314. Strain JS314 failed to nodulate several soybean varieties tested. However, on a few varieties nodulelike structures were induced to a frequency of 54% of the plants inoculated. The ultrastructure of these nodules was studied in detail by light and electron microscopy. The nodules were devoid of internal bacteria, possessed central vascular tissue (unlike the lateral vascular tissue of a normal nodule), and exhibited localized cell death of epidermal cells. Study of the cell surface polysaccharides of strain JS314 revealed that the exopolysaccharide of this strain was identical to that of the wild type. However, the lipopolysaccharide (LPS) of strain JS314 showed gross differences from that isolated from the wild-type strain. Specifically, the LPS of strain JS314 appeared to lack the high molecular weight LPS I form, strongly suggesting that the LPS lacks the O-chain. Glycosyl-composition analysis showed that the LPS of mutant JS314 lacked 2,3-di-O-methylrhamnose, 3-O-methylrhamnose, fucose, and quinovosamine. These results indicate that LPS I in B. japonicum is essential for bacterial infection of soybean, but is not required to initiate plant cortical cell division, an early plant response to infection.     

Metabolism of cationized lipoproteins by human fibroblasts: biochemical and morphologic correlations

Human plasma low density lipoprotein (LDL) that had been rendered polycationic by coupling with N, N-dimethyl-1, 3-propanediamine (DMPA) was shown by electron microscopy to bind in clusters to the surface of human fibroblasts. The clusters resembled those formed by polycationic ferritin (DMPA-feritin), a visual probe that binds to anionic site on the plasma membrane. Biochemical studies with (125)I-labeled DMPA-LDL showed that the membrane-bound lipoprotein was internalized and hydrolyzed in lysosomes. The turnover time for cell bound (125)I-DMPA-LDL, i.e., the time in which the amount of (125)I-DMPA-LDL degraded was equal to the steady-state cellular content of the lipoprotein, was about 50 h. Because the DMPA-LDL gained access to fibroblasts by binding nonspecifically to anionic sites on the cell surface rather than by binding to the physiologic LDL receptor, its uptake failed to be regulated under conditions in which the uptake of native LDL was reduced by feedback suppression of the LDL receptor. As a result, unlike the case with native LDL, the DMPA-LDL accumulated progressively within the cell, and this led to a massive increase in the cellular content of both free and esterified cholesterol. Studies with (14)C-oleate showed that at least 20 percent of the accumulated cholesteryl esters represented cholesterol that had been esterified within the cell. After 4 days of incubation with 10 μg/ml of DMPA-LDL, fibroblasts had accumulated so much cholesteryl ester that neutral lipid droplets were visible at the light microscope level with Oil Red O staining. By electron microscopy, these intracellular lipid droplets were observed to lack a tripartite limiting membrane. The ability to cause the overaccumulation of cholesteryl esters within cells by using DMPA-LDL provides a model system for study of the pathologic consequences at the cellular level of massive deposition of cholesteryl ester.     

Immobilization of Rhizopus oryzae lipase on magnetic Fe3O4-chitosan beads and its potential in phenolic acids ester synthesis

A novel and simple method was developed for the preparation of magnetic Fe₃O₄ nanoparticles by chemical co-precipitation method and subsequent coating with 3-aminopropyltrimethoxysilane (APTMS) through silanization process. Magnetic Fe₃O₄-chitosan particles were prepared by the suspension cross-linking and covalent technique to be used in the application of magnetic carrier technology. The synthesized immobilization supports were characterized by scanning electron microscopy (SEM), thermogravimetric analysis (TGA) and X-ray diffraction (XRD). Using glutaraldehyde as the coupling agent, the lipase from R. oryzae was successfully immobilized onto the functionalized magnetic Fe₃O₄-chitosan beads. The results showed that 86.60% of R. oryzae lipase was bound on the synthesized immobilization support. This immobilized lipase was successfully used for the esterification of phenolic acid which resulted in esterification of phenolic acid in isooctane solvent reaction system for 8 consecutive cycles (totally 384 h), 72.6% of its initial activity was retained, indicating a high stability in pharmaceutical and industrial applications.     

Relationships between lake ecology and morphological characters in Icelandic Arctic charr,Salvelinus alpinus

The common occurrence of parallel phenotypic patterns suggests that a strong relationship exists between ecological dynamics and micro‐evolution. Comparative studies from a large number of populations under varying sets of ecological drivers could contribute to a better understanding of this relationship. We used data on morphology of arctic charr (Salvelinus alpinus) and ecological factors from 35 Icelandic lakes to test the hypothesis that morphological patterns among monomorphic charr populations from different lakes are related to interlake variation in ecological characteristics. There is extensive phenotypic diversity among populations of Icelandic charr, and populations are easily distinguished based on overall body morphology. The results obtained in the present study showed that the morphological diversity of charr was related to large‐scale diversity in lake ecology. Variation in charr morphology was related to water origin (e.g. spring fed versus run‐off), bedrock age, and fish community structure. The present study shows how various ecological factors can shape the biological diversity that we observe. © 2011 The Linnean Society of London, Biological Journal of the Linnean Society, 2011, 103 , 761–771.