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991.
Hyunbum Jang Fernando Teran Arce Srinivasan Ramachandran Ruth Nussinov 《Biophysical journal》2009,97(11):3029-3037
In Alzheimer's disease, calcium permeability through cellular membranes appears to underlie neuronal cell death. It is increasingly accepted that calcium permeability involves toxic ion channels. We modeled Alzheimer's disease ion channels of different sizes (12-mer to 36-mer) in the lipid bilayer using molecular dynamics simulations. Our Aβ channels consist of the solid-state NMR-based U-shaped β-strand-turn-β-strand motif. In the simulations we obtain ion-permeable channels whose subunit morphologies and shapes are consistent with electron microscopy/atomic force microscopy. In agreement with imaged channels, the simulations indicate that β-sheet channels break into loosely associated mobile β-sheet subunits. The preferred channel sizes (16- to 24-mer) are compatible with electron microscopy/atomic force microscopy-derived dimensions. Mobile subunits were also observed for β-sheet channels formed by cytolytic PG-1 β-hairpins. The emerging picture from our large-scale simulations is that toxic ion channels formed by β-sheets spontaneously break into loosely interacting dynamic units that associate and dissociate leading to toxic ionic flux. This sharply contrasts intact conventional gated ion channels that consist of tightly interacting α-helices that robustly prevent ion leakage, rather than hydrogen-bonded β-strands. The simulations suggest why conventional gated channels evolved to consist of interacting α-helices rather than hydrogen-bonded β-strands that tend to break in fluidic bilayers. Nature designs folded channels but not misfolded toxic channels. 相似文献
992.
Ishfaq Ahmed Sheikh Amit Kumar Singh Nagendra Singh Mau Sinha S. Baskar Singh Asha Bhushan Punit Kaur Alagiri Srinivasan Sujata Sharma Tej P. Singh 《The Journal of biological chemistry》2009,284(22):14849-14856
The crystal structure of the complex of lactoperoxidase (LPO) with its
physiological substrate thiocyanate (SCN–) has been
determined at 2.4Å resolution. It revealed that the
SCN– ion is bound to LPO in the distal heme cavity. The
observed orientation of the SCN– ion shows that the sulfur
atom is closer to the heme iron than the nitrogen atom. The nitrogen atom of
SCN– forms a hydrogen bond with a water (Wat) molecule at
position 6′. This water molecule is stabilized by two hydrogen bonds
with Gln423 Nε2 and Phe422 oxygen. In
contrast, the placement of the SCN– ion in the structure of
myeloperoxidase (MPO) occurs with an opposite orientation, in which the
nitrogen atom is closer to the heme iron than the sulfur atom. The site
corresponding to the positions of Gln423, Phe422 oxygen,
and Wat6′ in LPO is occupied primarily by the side chain of
Phe407 in MPO due to an entirely different conformation of the loop
corresponding to the segment Arg418–Phe431 of LPO.
This arrangement in MPO does not favor a similar orientation of the
SCN– ion. The orientation of the catalytic product
OSCN– as reported in the structure of
LPO·OSCN– is similar to the orientation of
SCN– in the structure of LPO·SCN–.
Similarly, in the structure of
LPO·SCN–·CN–, in which
CN– binds at Wat1, the position and orientation of
the SCN– ion are also identical to that observed in the
structure of LPO·SCN.Lactoperoxidase
(LPO4; EC 1.11.1.7) is
a Fe3+ heme enzyme that belongs to the mammalian peroxidase family
(1). The family of mammalian
peroxidases comprises lactoperoxidase
(2), eosinophil peroxidase
(3), thyroid peroxidase
(4), and myeloperoxidase (MPO)
(5). LPO, eosinophil
peroxidase, and MPO are responsible for antimicrobial function and innate
immune responses
(6–8),
whereas thyroid peroxidase plays a key role in thyroid hormone biosynthesis
(9). These peroxidases are
different from plant and fungal peroxidases because unlike plant and fungal
enzymes, the prosthetic heme group in mammalian peroxidases is covalently
linked to the protein (10).
There are also several striking structural and functional differences among
the mammalian peroxidases
(11). The heme group in MPO is
attached to the protein via three covalent linkages
(12), whereas LPO
(12,
13), eosinophil peroxidase
(12), and thyroid peroxidase
(12) contain only two ester
linkages. These covalent and various non-covalent linkages contribute
differentially to the high stability of the heme core as well as for the
peculiar values of their redox potentials
(2,
14). Furthermore, MPO consists
of two disulfide-linked protein chains, whereas LPO, eosinophil peroxidase,
and thyroid peroxidase are single chain proteins, although their chain lengths
differ greatly. In addition, their sequences contain several critical amino
acid differences that may also contribute to the variations in the
stereochemical environments of the substrate-binding sites. As a consequence
of these differences, the mammalian enzymes oxidize various inorganic ions
such as SCN–, Br–, Cl–, and
I– with differing specificities and potencies. Biochemical
studies have shown that LPO catalyzes preferentially the conversion of
SCN– to OSCN–
(15,
16), whereas MPO uses halides
(17,
18) with a preference for
chloride ion as the substrate. The preferences of eosinophil peroxidase and
thyroid peroxidase are bromide and iodide, respectively. However, the
stereochemical basis of the reported preferences for the substrates by
mammalian heme peroxidases is still unclear. So far, the structures of only
two mammalian enzymes, MPO and LPO, have been determined
(12,
13). It is of considerable
importance to identify the structural parameters that are responsible for the
subtle specificities. In the present work, we have attempted to address this
question through the new crystal structures of LPO complexes with
SCN– ions using goat, bovine, and buffalo lactoperoxidases.
Because the overall structures of complexes of SCN– with LPO
from all three species were found to be identical, the structure of the
complex of buffalo LPO with SCN– and the ternary complex with
SCN– and CN– will be discussed here, and
buffalo LPO will be termed hereafter as LPO. To highlight the factors
pertaining to binding specificity of SCN–, a comparison of
the structures of LPO·SCN– and
MPO·SCN– has also been made, revealing many valuable
differences pertaining to the observed orientations of the common substrate,
SCN– ion, when bound at the substrate-binding site in the
distal heme cavity of the two structures. The structures of
LPO·SCN– and MPO·SCN– clearly
show that the bound SCN– ions are present in the distal heme
cavity of two enzymes with opposite orientations. In the structure of
LPO·SCN–, the sulfur atom is closer to the heme iron
than the nitrogen atom, whereas in that of MPO·SCN–,
the nitrogen atom is closer to the heme iron than the sulfur atom. As a result
of this, the interactions of the SCN– ion in the distal site
of two proteins differ drastically. Gln423, a conserved water (Wat)
molecule at position 6′, and a well aligned carbonyl oxygen of
Phe422 in the proximity of the substrate-binding site in LPO
against a protruding Phe407 in MPO seem to play the key roles in
inducing the observed orientations of SCN– ions in LPO and
MPO. The structure of LPO·SCN– has also been compared
with the structure of its ternary complex with SCN– and
CN– ions. 相似文献
993.
Nazish Abdullah Bharani Srinivasan Nir Modiano Apurba Kumar Sau 《Journal of molecular biology》2009,386(3):690-298
Unlike other GTPases, interferon-gamma-induced human guanylate binding protein-1 has the ability to hydrolyze GTP to both GDP and GMP, with GMP being the major product of the reaction. This protein has two domains, an N-terminal globular domain and a C-terminal helical domain. These two domains are connected by a short intermediate region consisting of a two-stranded β-sheet and a helix. As human guanylate binding protein-1 has been shown to undergo stimulated GTPase activity without external GTPase-activating protein, we sought to understand the roles of each of the two individual domains, the intermediate region, a conserved motif (103DXEKGD108), and the mechanism of the stimulation of GTPase activity. The steady-state assays using radiolabeled [α-32P]GTP on the wild-type protein suggest that the stimulation of activity primarily occurs during the cleavage of the second phosphate of GTP rather than the first, through allosteric interaction. Using several truncated and mutant proteins, we demonstrate for the first time that both the α-helix of the intermediate region and the 103DXEKGD108 motif play critical roles for the hydrolysis to GMP, but they appear to act in different ways: α-helix acts through structural stabilization by allosteric interaction and, thus, acts as an internal GTPase-activating protein, whereas the motif might act by providing necessary catalytic residues. Our data also show that the N-terminal globular domain is able to perform only the first catalysis (GTP to GDP, an activity associated with basal level), but the helical domain in the full-length protein stimulates the hydrolysis of GTP to GMP with higher GMP formation by preventing the dissociation of GDP-bound enzyme dimer. 相似文献
994.
Patke S Maheshwari R Litt J Srinivasan S Aguilera JJ Colón W Kane RS 《Biochemistry》2012,51(14):3092-3099
The fibrillar deposition of serum amyloid A (SAA) has been linked to the disease amyloid A (AA) amyloidosis. We have used the SAA isoform, SAA2.2, from the CE/J mouse strain, as a model system to explore the inherent structural and biophysical properties of SAA. Despite its nonpathogenic nature in vivo, SAA2.2 spontaneously forms fibrils in vitro, suggesting that SAA proteins are inherently amyloidogenic. However, whereas the importance of the amino terminus of SAA for fibril formation has been well documented, the influence of the proline-rich and presumably disordered carboxy terminus remains poorly understood. To clarify the inherent role of the carboxy terminus in the oligomerization and fibrillation of SAA, we truncated the proline-rich final 13 residues of SAA2.2. We found that unlike full-length SAA2.2, the carboxy-terminal truncated SAA2.2 (SAA2.2ΔC) did not oligomerize to a hexamer or octamer, but formed a high molecular weight soluble aggregate. Moreover, SAA2.2ΔC also exhibited a pronounced decrease in the rate of fibril formation. Intriguingly, when equimolar amounts of denatured SAA2.2 and SAA2.2ΔC were mixed and allowed to refold together, the mixture formed an octamer and exhibited rapid fibrillation kinetics, similar to those for full-length SAA2.2. These results suggest that the carboxy terminus of SAA, which is highly conserved among SAA sequences in all vertebrates, might play important structural roles, including modulating the folding, oligomerization, misfolding, and fibrillation of SAA. 相似文献
995.
SS Oliver CA Musselman R Srinivasan JP Svaren TG Kutateladze JM Denu 《Biochemistry》2012,51(33):6534-6544
The chromodomain, helicase, DNA-binding protein 5 (CHD5) is a chromatin remodeling enzyme which is implicated in tumor suppression. In this study, we demonstrate the ability of the CHD5 PHD fingers to specifically recognize the unmodified N-terminus of histone H3. We use two distinct modified peptide-library platforms (beads and glass slides) to determine the detailed histone binding preferences of PHD(1) and PHD(2) alone and the tandem PHD(1-2) construct. Both domains displayed similar binding preferences for histone H3, where modification (e.g., methylation, acetylation, and phosphorylation) at H3R2, H3K4, H3T3, H3T6, and H3S10 disrupts high-affinity binding, and the three most N-terminal amino acids (ART) are crucial for binding. The tandem CHD5-PHD(1-2) displayed similar preferences to those displayed by each PHD finger alone. Using NMR, surface plasmon resonance, and two novel biochemical assays, we demonstrate that CHD5-PHD(1-2) simultaneously engages two H3 N-termini and results in a 4-11-fold increase in affinity compared with either PHD finger alone. These studies provide biochemical evidence for the utility of tandem PHD fingers to recruit protein complexes at targeted genomic loci and provide the framework for understanding how multiple chromatin-binding modules function to interpret the combinatorial PTM capacity written in chromatin. 相似文献
996.
Rengachari S Bezerra GA Riegler-Berket L Gruber CC Sturm C Taschler U Boeszoermenyi A Dreveny I Zimmermann R Gruber K Oberer M 《Biochimica et biophysica acta》2012,1817(7):1012-1021
In order to prevent photodestruction by high light, photosynthetic organisms have evolved a number of mechanisms, known as non-photochemical quenching (NPQ), that deactivate the excited states of light harvesting pigments. Here we investigate the NPQ mechanism in the cyanobacterium Synechocystis sp. PCC 6803 mutant deficient in both photosystems. Using non-linear laser fluorimetry, we have determined molecular photophysical characteristics of phycocyanin and spectrally distinct forms of allophycocyanin for the cells in non-quenched and quenched states. Our analysis of non-linear fluorescence characteristics revealed that NPQ activation leads to an ~2-fold decrease in the relaxation times of both allophycocyanin fluorescence components, F660 and F680, and a 5-fold decrease in the effective excitation cross-section of F680, suggesting an emergence of a pathway of energy dissipation for both types of allophycocyanin. In contrast, NPQ does not affect the rates of singlet-singlet exciton annihilation. This indicates that, upon NPQ activation, the excess excitation energy is transferred from allophycocyanins to quencher molecules (presumably 3'hydroxyechinenone in the orange carotenoid protein), rather than being dissipated due to conformational changes of chromophores within the phycobilisome core. Kinetic measurements of fluorescence quenching in the Synechocystis mutant revealed the presence of several stages in NPQ development, as previously observed in the wild type. However, the lack of photosystems in the mutant enhanced the magnitude of NPQ as compared to the wild type, and allowed us to better characterize this process. Our results suggest a more complex kinetics of the NPQ process, thus clarifying a multistep model for the formation of the quenching center. 相似文献
997.
Oxidative stress has been suggested as a contributory factor in development and complication of diabetes. The aim of the study was to evaluate the effect of diosmin (DS) in oxidative stress in streptozotocin-nicotinamide (STZ-NA)-induced diabetic rats by measuring the lipid peroxidation (LPO) as well as the ameliorative properties. Experimental diabetes was induced by a single intraperitoneal (i.p) injection of STZ (45 mg/kg body weight (b.w.)) dissolved in 0.1 mol/L citrate buffer (pH 4.5), 15 min after the i.p administration of NA (110 mg/kg b.w.). Diabetic rats exhibited increased plasma glucose with significant decrease in plasma insulin levels. The activities of antioxidant enzymes superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione-S-transferase (GST) and the levels of low-molecular weight antioxidants vitamin C, vitamin E and reduced glutathione (GSH) were decreased while increases in the levels of LPO markers were observed in liver and kidney tissues of diabetic control rats as compared to normal control rats. Oral treatment with DS (100mg/kg/day) for a period of 45 days showed significant ameliorative effects on all the biochemical parameters studied. Biochemical findings were supported by histological studies. These results indicated that DS has potential ameliorative effects in addition to its antidiabetic effect in type 2 diabetic rats. 相似文献
998.
Lateral gene transfer (LGT) is essential for generating between-strain genomic recombinants of Chlamydia trachomatis to facilitate the organism's evolution. Because there is no reliable laboratory-based gene transfer system for C. trachomatis, in vitro generation of recombinants from antibiotic-resistant strains is being used to study LGT. However, selection pressures imposed on in vitro recombinants likely affect statistical properties of recombination relative to naturally occurring clinical recombinants, including prevalence at particular loci. We examined multiple loci for 16 in vitro-derived recombinants of ofloxacin- and rifampin-resistant L(1) and D strains, respectively, grown with both antibiotics, and compared these with the same sequenced loci among 11 clinical recombinants. Breakpoints and recombination frequency were examined using phylogenetics, bioinformatics, and statistics. In vitro and clinical isolates clustered perfectly into two groups, without misclassification, using Ward's minimum variance based on breakpoint data. As expected, gyrA (confers ofloxacin resistance) and rpoB (confers rifampin resistance) had significantly more breakpoints among in vitro recombinants than among clinical recombinants (P < 0.0001 and P = 0.02, respectively, using the Wilcoxon rank sum test). Unexpectedly, trpA also had significantly more breakpoints for in vitro recombinants (P < 0.0001). There was also significant selection at other loci. The strongest bias was for ompA in strain D (P = 3.3 × 10(-8)). Our results indicate that the in vitro model differs statistically from natural recombination events. Additional genomic studies are needed to determine the factors responsible for the observed selection biases at unexpected loci and whether these are important for LGT to inform approaches for genetically manipulating C. trachomatis. 相似文献
999.
Doma M Abhayankar G Reddy VD Kavi Kishor PB 《Indian journal of experimental biology》2012,50(7):484-490
Leaves of Withania somnifera contained more withaferin A and withanolide A than roots indicating that these compounds mainly accumulate in leaves. With an increase in age of the plant, withaferin A was enhanced with a corresponding decrease in withanolide A. Hairy root cultures were induced from leaf explants using Agrobacterium rhizogenes and the transgenic nature of hairy roots was confirmed by partial isolation and sequencing of rolB gene, which could not be amplified in untransformed plant parts. In hairy roots, withaferin A accumulated at 2, 3 and 4% but not at 6% sucrose, the highest amount being 1733 microg/g dry weight at 4% level. High and equal amounts of withaferin A and withanolide A accumulated (890 and 886 microg/g dry tissue respectively) only at 3% sucrose. Increasing concentrations of glucose enhanced withaferin A and it peaked at 5% level (3866 microg/g dry tissue). This amount is 2842 and 34% higher compared to untransformed roots and leaves (collected from 210-day-old plants) respectively. Withanolide A was detected at 5% glucose but not at other concentrations. While chitosan and nitric oxide increased withaferin A, jasmonic acid decreased it. Acetyl salicylic acid stimulated accumulation of both withaferin A and withanolide A at higher concentrations. Triadimefon, a fungicide, enhanced withaferin A by 1626 and 3061% (not detected earlier) compared to hairy and intact roots respectively. 相似文献
1000.
Lee SK Potempa M Kolli M Özen A Schiffer CA Swanstrom R 《The Journal of biological chemistry》2012,287(16):13279-13290
Processing of the human immunodeficiency virus type 1 (HIV-1) Gag and Gag-Pro-Pol polyproteins by the HIV-1 protease (PR) is essential for the production of infectious particles. However, the determinants governing the rates of processing of these substrates are not clearly understood. We studied the effect of substrate context on processing by utilizing a novel protease assay in which a substrate containing HIV-1 matrix (MA) and the N-terminal domain of capsid (CA) is labeled with a FlAsH (fluorescein arsenical hairpin) reagent. When the seven cleavage sites within the Gag and Gag-Pro-Pol polyproteins were placed at the MA/CA site, the rates of cleavage changed dramatically compared with that of the cognate sites in the natural context reported previously. The rate of processing was affected the most for three sites: CA/spacer peptide 1 (SP1) (≈10-fold increase), SP1/nucleocapsid (NC) (≈10-30-fold decrease), and SP2/p6 (≈30-fold decrease). One of two multidrug-resistant (MDR) PR variants altered the pattern of processing rates significantly. Cleavage sites within the Pro-Pol region were cleaved in a context-independent manner, suggesting for these sites that the sequence itself was the determinant of rate. In addition, a chimera consisting of SP1/NC P4-P1 and MA/CA P1'-P4' residues (ATIM↓PIVQ) abolished processing by wild type and MDR proteases, and the reciprocal chimera consisting of MA/CA P4-P1 and SP1/NC P1'-4' (SQNY↓IQKG) was cleaved only by one of the MDR proteases. These results suggest that complex substrate interactions both beyond the active site of the enzyme and across the scissile bond contribute to defining the rate of processing by the HIV-1 PR. 相似文献