Molecular replacement studies on crystal structure of allophycocyanin from red algae Porphyra yezoensis

Using the crystal structure of allophycocyanin from cyanobacterium Spirulina platensis (APC-SP) as a search model,the crystal structure of allophycocyanin from red algae Porphyra yezoensis (APC-PY) has been studied by molecular replacement methods.The APC-PY crystals (Form 3) belong to the space group of R32,cell dimensions a=b= 10.53 nm,c=18.94 nm,α= β= 90°,γ=120°;there is one αβ monomer in each crystallographic asymmetric unit in the cell.The translation function search gave a unique peak with a correlation coefficient (Cc) of 67.0% and an R-factor of 36.1% for reflection data from 1.0 to 0.4 nm.Using the results by molecular replacement,the initial model of APC-PY was built,and the coincidence of the chromophore in APC-PY initial model with its 2Fo-Fc OMIT map further confirms the results by molecular replacement.     

Synthesis and characterization of aspartic acid complexes of antimony and bismuth triiodide

Summary. New bioinorganic complexes of the aspartic acid with the antimony or bismuth triiodide were synthesized by a direct solid–solid reaction at room temperature. The formula of the complex is MI₃[OOCCH₂CH(NH₂)CO]_2.5 · 2.5H₂O (M = Sb, Bi). The complex may be a dimer with bridge structure. The crystal structure of the complexes belongs to a triclinic system. The lattice parameters are a = 0.9883 nm, b = 1.4284 nm, c = 2.0114 nm, α = 94.46°, β = 99.76° and γ = 100.1° for the complex of antimony and a = 0.9756 nm, b = 1.4560 nm, c = 1.9875 nm, α = 94.18°, β = 97.25° and γ = 101.16° for the complex of bismuth. The infrared spectra and thermal analyses can demonstrate the complex formation between the aspartic acid and the antimony or bismuth ion.     

Crystal structure determination of a neutral neurotoxin BmK M4 fromButhus martensii Karsch at 0.20 nm

BmK M4 is a neutral neurotoxin in the BmK toxin series. It is medially toxic and belongs to group III cc-toxins. The purified sample was crystallized in rhombic space group P6 Using an X-ray diffraction technique, the crystal structure of BmK M4 was revealed by molecular replacement at 0.20 nm resolution. The model was refined. The final crystallographic R factor was 0.142 and the free R factor was 0.173. The root mean square deviation is 0.001 5 nm for the bond length and 1.753° for the bond angles. 64 water molecules were added to the asymmetric unit. The refined structure showed an unusual non-prolyl cis peptide bond at residue 10. The structure was compared with group II a-toxin BmK M8 (an acidic, weak toxin). The potential structural implications of the cis peptide bond were discussed.     

Purification, crystallization and preliminary crystallographic investigations of selenium-containing phycocyanin from selenium-rich algae ( Spirulina platensis )

The selenium-containing phycocyanin from the selenium-rich algae (Spirulina platensis) has been crystallized in two crystal forms by the hanging-drop vapor diffusion techniques. A chromatographic procedure of gel filtration and anion exchange was used for purification. Form I crystal with space group P2₁ and cell parameters a =108.0 ?, b= 117.0 ?, c = 184.0?, β= 90.2° and 12(αβ) units in the asymmetric unit was obtained by using (NH₄)₂SO₄ as precipitant. These crystals diffract up to 2.8 ?. Form II crystal obtained by using PEG4000 as precipitant belongs to space group P6₃ with unit cell constants a = 155.0 ?, c = 40.3 ?, γ =120.0° and one(αβ) unit in the asymmetric unit. The crystals diffract beyond 2.9 ?. The possible stacking forms of phycocyanin molecules in the first crystal form were discussed.     

Divergence of the F1-ATP Synthase Complex in the Ice Worm, Mesenchytraeus solifugus (Annelida, Clitellata, Enchytraeidae)

The F₁-ATP synthase complex constitutes the catalytic component of F₁F₀-ATP synthase, the primary ATP synthetic enzyme in the cell. Previous studies indicate that the glacier ice worm, Mesenchytraeus solifugus, maintains unusually high ATP levels that continue to rise as temperatures decline, suggesting that molecular changes within ice worm F₁-ATP synthase subunits may contribute to this energetic anomaly. In this report, we compared ice worm F₁-ATP synthase subunits (α, β, γ) with homologues across metazoan phyla (arthropod, chordate, nematode) and among a group of clitellate annelids (Enchytraeus albidus, Enchytraeus buchholzi, Lumbriculus variegatus, Theromyzon tessulatum). Amino acid alignments indicated that ice worm F₁-ATP α and F₁-ATP β subunits share strong sequence homology with their mesophilic counterparts, respectively, but that ATP γ has diverged more rapidly. Moreover, F₁-ATP α and F₁-ATP β displayed amino acid compositional changes consistent with trends observed in other cold adapted proteins, while F₁-ATP γ diverged in unexpected directions (e.g., gains in size, charged residues). Several ice worm-specific amino acid substitutions map to positions near the F₁-ATP β catalytic site while others occur near subunit contact sites.     

Some characteristics of cytochromec-555 isolated from sulfide oxidizingXanthomonas sp. DY44

From a heterotrophic bacterium,Xanthomonas sp. DY44 which was previously reported to oxidize hydrogen sulfide (H₂S) to polysulfide, cytochromec-555 (cyt.c-555) responsible for oxidation of sulfide was purified by DEAE-Toyopearl and Sepadex G-75 column chromatography. Cyt.c-555 with a molecular weight of 12,500 showed maximum absorption at 555 nm (α-peak), 522 nm (β-peak) and 417 nm (γ-peak) for the reduced form which was prepared by addition of Na₂S₂O₄. Cyt.c-555 was also reduced by addition of sulfide (Na₂S and H₂S), and the oxidized products of sulfide by cyt.c-555 was identified as polysulfide. The reduced form of cyt.c-555 was suggested to be oxidized coupled with cyt.c oxidase which is tolerant to sulfide.     

Heterogeneity of α-cardiac myosin heavy chains in a small marsupial, Antechinus flavipes, and the effect of hypothyroidism on its ventricular myosins

The effect of drug-induced hypothyroidism on ventricular myosin gene expression was explored in a small marsupial, Antechinus flavipes. Pyrophosphate gel electrophoresis, SDS-PAGE and western blotting were used to analyse changes in native myosin isoforms and myosin heavy chains (MyHCs) in response to hypothyroidism. In some animals, five instead of the normal three native myosin components were found: V_1a, V_1b,V_1c, V₂ and V₃, in order of decreasing mobility. In western blots, V_1a, V_1b, and V_1c reacted with anti-α-MyHC antibody, but not with anti-β-MyHC, whereas V₂ and V₃ reacted with anti-β-MyHC antibody. SDS-PAGE of the unusual ventricular myosins revealed three MyHC isoforms, two of which bound anti-α-MyHC antibody while the third bound anti-β-MyHC antibody. We conclude that V_1a, V_1b, V_1c are triplets arising from the dimerization of two distinct α-MyHC isoforms. Hypothyroidism, verified by metabolic studies, decreased α-MyHC content significantly (t-test, P < 0.001) from 91.6 ± 5.9% (SEM, n = 4) in control animals to 67.2 ± 5.7% (SEM, n = 4) in hypothyroid animals, with a concomitant increase in β-MyHC content. We conclude that in adult marsupials, ventricular myosins are also responsive to changes in the thyroid state as found in eutherians, and suggest that evolution of the molecular mechanisms underlying this thyroid responsiveness predate the divergence of marsupials and eutherians.     

Biliprotein assembly in the disc-shaped phycobilisomes of Rhodella violacea electron microscopical and biochemical analyses of C-phycocyanin and allophycocyanin aggregates

C-phycocyanin and allophycocyanin from the red alga Rhodella violacea were investigated by electron microscopy and biochemical methods using samples taken from the same fractions.The molecular weights of the native biliprotein aggregates C-phycocyanin and allophycocyanin are about 139,000 (140,000) and 130,000 (145,000) as revealed by calibrated gel chromatography, gradient gel electrophoresis and morphological measurements on the basis of an average protein packing density. These molecular weights are direct evidence for a trimeric aggregation form ()₃ of these biliproteins. Independently, their monomers were determined to be about 34,400 (C-phycocyanin) and 33,900 (allophycocyanin).C-phycocyanin and allophycocyanin are ringshaped, six-membered, biliprotein aggregates with dimensions of about 10.2×3.0 nm and 10.0×3.0 nm, respectively. The aggregates are made up of six subunits, 3 and 3, which are assumed to be associated in alternating positions. They are arranged in regular hexagons in C₆ symmetry. Hexameric aggregates ()₆, so far only isolated for C-phycocyanin, originate by face to face association of two trimeric aggregates.     

Crystal structure of pokeweed antiviral protein from seeds of Phytolacca americana at 0.25 nm

Crystals of pokeweed antiviral protein (PAP) from seeds of Phytolacca americana with high diffraction ability were grown from high protein concentration (100 mg/mL) solution at high temperature (33℃). The crystal structure was solved by use of molecular replacement method and refined by use of molecular dynamic method at 0 25 nm to an R factor of 18.15% with standard deviations from standard geometry of 0.001 6 nm and 2.04° for bond lengths and bond angles, respectively. Comparison with two other PAPs revealed, near the active center, a sequence and structure variable region, consisting of the loop connecting the fifth β strand with the second α helix and including a proposed active residue, suggesting this loop probably to be related to difference in activity.$$$$     

Computational studies of the binding site of α<Subscript>1A</Subscript>-adrenoceptor antagonists

Aimed at achieving a good understanding of the 3-dimensional structures of human α_1A-adrenoceptor (α_1A-AR), we have successfully developed its homology model based on the crystal structure of β₂-AR. Subsequent structural refinements were performed to mimic the receptor’s natural membrane environment by using molecular mechanics (MM) and molecular dynamics (MD) simulations in the GBSW implicit membrane model. Through molecular docking and further simulations, possible binding modes of subtype-selective α_1A-AR antagonists, Silodosin, RWJ-69736 and (+)SNAP-7915, were examined. Results of the modeling and docking studies are qualitatively consistent with available experimental data from mutagenesis studies. The homology model built should be very useful for designing more potent subtype-selective α_1A-AR antagonists and for guiding further mutagenesis studies. Figure The superposition of β₂-AR crystal structure (gold ribbons) and α_1A-AR homology model (blue ribbons)     

Characteristics of protection by MgADP and MgATP of α3β3γ subcomplex of thermophilic Bacillus PS3 βY341W-mutant F1-ATPase from inhibition by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole support a bi-site mechanism of catalysis

MgADP and MgATP binding to catalytic sites of βY341W-α₃β₃Γ subcomplex of F₁-ATPase from thermophilic Bacillus PS3 has been assessed using their effect on the enzyme inhibition by 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl). It was assumed that NBD-Cl can inhibit only when catalytic sites are empty, and inhibition is prevented if a catalytic site is occupied with a nucleotide. In the absence of an activator, MgADP and MgATP protect βY341W-α₃β₃Γ sub-complex from inhibition by NBD-Cl by binding to two catalytic sites with an affinity of 37 μM and 12 mM, and 46 μM and 15 mM, respectively. In the presence of an activator lauryldimethylamine-N-oxide (LDAO), MgADP protects βY341W-α₃β₃Γ subcomplex from inhibition by NBD-Cl by binding to a catalytic site with a K _d of 12 mM. Nucleotide binding to a catalytic site with affinity in the millimolar range has not been previously revealed in the fluorescence quenching experiments with βY341W-α₃β₃Γ subcomplex. In the presence of activators LDAO or selenite, MgATP protects βY341W-α₃β₃Γ subcomplex from inhibition by NBD-Cl only partially, and the enzyme remains sensitive to inhibition by NBD-Cl even at MgATP concentrations that are saturating for ATPase activity. The results support a bi-site mechanism of catalysis by F₁-ATPases.     

Homology modeling and dynamics of the extracellular domain of rat and human neuronal nicotinic acetylcholine receptor subtypes α4β2 and α7

In recent years, it has become clear that the neuronal nicotinic acetylcholine receptor (nAChR) is a valid target in the treatment of a variety of diseases, including Alzheimer’s disease, anxiety, and nicotine addiction. As with most membrane proteins, information on the three-dimensional (3D) structure of nAChR is limited to data from electron microscopy, at a resolution that makes the application of structure-based design approaches to develop specific ligands difficult. Based on a high-resolution crystal structure of AChBP, homology models of the extracellular domain of the neuronal rat and human nAChR subtypes α4β2 and α7 (the subtypes most abundant in brain) were built, and their stability assessed with molecular dynamics (MD). All models built showed conformational stability over time, confirming the quality of the starting 3D model. Lipophilicity and electrostatic potential studies performed on the rat and human α4β2 and α7 nicotinic models were compared to AChBP, revealing the importance of the hydrophobic aromatic pocket and the critical role of the α-subunit Trp—the homolog of AChBP-Trp 143—for ligand binding. The models presented provide a valuable framework for the structure-based design of specific α4β2 nAChR subtype ligands aimed at improving therapeutic and diagnostic applications. Figure Electrostatic surface potential of the binding site cavity of the neuronal nicotinic acetylcholine receptor (nAChR). Nicotinic models performed with the MOLCAD program: a rat α7, b rat α4β2, c human α7, d human α4β2. All residues labeled are part of the α7 (a,c) or α4 (b,d) subunit with the exception of Phe 117, which belongs to subunit β2 (d). Violet Very negative, blue negative, yellow neutral, red very positive     

Isolation and characterization of a new phycoerythrin from the cyanobacterium <Emphasis Type="Italic">Synechococcus</Emphasis> sp. ECS-18

A new phycoerythrin, SCH-phycoerythrin, was purified from Synechococcus sp. ECS-18 by DEAE-Sephacel anion exchange chromatography and Sephacryl S-300 gel filtration. The protein pigment had an absorbance maximum at 542 nm and a fluorescence maximum at 565 nm. The native molecular mass was approximately 219 kDa as determined by gel filtration, and sodium dodecyl sulfate polyacrylamide gel electrophoresis demonstrated the presence of two subunits, with molecular mass of 19 and 17.9 kDa. These observations are consistent with the (αβ)₆ subunit composition that is characteristic of phycoerythrins. The α- and β-subunits showed immunological identity by Ouchterlony double immunodiffusion with an anti-phycoerythrin antiserum. The DNA sequence of the SCH-phycoerythrin gene was determined by PCR amplification using primers based on the conserved N-terminal amino acid sequence of the α- and β-subunits of phycoerythrins.     

Effect of mutation on helper T-cells and viral population: A computer simulation model for HIV

Summary A Monte Carlo simulation is proposed to study the dynamics of helper T-cells (N _H) and viral (N _V) populations in an immune response model relevant to HIV. Cellular states are binary variables and the interactions are described by logical expressions. Viral population shows a nonmonotonic growth before reaching a constant value while helper T-cells grow to a constant after a relaxation/reaction time. Initially, the population of helper cells grows with time with a power-law, N _H ∼t ^β, before reaching the steady-state; the growth exponent β increases systematically (β ≈ 1 – 2) with the mutation rate (P _mut≈0.1–0.4). The critical recovery time (t _c) increases exponentially with the viral mutation, t _c≈Ae ^αP _mut , with α=4.52±0.29 in low mutation regime and α=15.21±1.41 in high mutation regime. The equilibrium population of helper T-cell declines slowly with P _mut and collapses at ∼ 0.40; the viral population exhibits a reverse trend, i.e., a slow increase before the burst around the same mutation regime.     

Intensity of nitrification in soil as a function of time and soil moisture

Proceeding from three previously derived expressions for the intensity of nitrification in soil as a function of time (logΣN=K.logt+q), as a function of incubation moisture (logΣN=A.pF _i+B), as a function of initial moisture (logΣN=C.pF _v+D), it was shown that the nitrification intensity as a function of time and of moisture can be expressed by the bilinear function log ΣN=a.pF _i.logT+b.pF _i+c.logt+d; as a function of time and of initial moisture by the bilinear function logΣ=N=a.pF _v.logt+b.pF _v+c.logt+d; as a function of initial and incubation moisture by the bilinear function log ΣN=a.pF _ipF _v+b.pF _i+c.pF _v+d. The intensity of nitrification as a function of time, incubation moisture and initial moisture may be expressed by the multilinear function log ΣN=a.pF _i.pF _v.logt+b.pF _i.pF _v+c.pF _i.logt+d.pF _v.logt+e .pF _i+f.pF _v=g.logt+h. This function is valid for all the incubation moistures lying between pF _i 3.0 and 4.0 and for all initial moistures between 3.5 and 5.9 provided that the incubation temperature remains constant.     

Dehydration-mediated activation of the xanthophyll cycle in darkness: is it related to desiccation tolerance?

The development of desiccation tolerance by vegetative tissues was an important step in the plants’ conquest of land. To counteract the oxidative stress generated under these conditions the xanthophyll cycle plays a key role. Recent reports have shown that desiccation itself induces de-epoxidation of xanthophyll cycle pigments, even in darkness. The aim of the present work was to study whether this trait is a common response of all desiccation-tolerant plants. The xanthophyll cycle activity and the maximal photochemical efficiency of PS II (F _v/F _m) as well as β-carotene and α-tocopherol contents were compared during slow and rapid desiccation and subsequent rehydration in six species pairs (with one desiccation-sensitive and one desiccation-tolerant species each) belonging to different taxa. Xanthophyll cycle pigments were de-epoxidised in darkness concomitantly with a decrease in F _v/F _m during slow dehydration in all the desiccation-tolerant species and in most of the desiccation-sensitive ones. De-epoxidation was reverted in darkness by re-watering in parallel with the recovery of the initial F _v/F _m. The stability of the β-carotene pool confirmed that its hydroxylation did not contribute to zeaxanthin formation. The α-tocopherol content of most of the species did not change during dehydration. Because it is a common mechanism present in all the desiccation-tolerant taxa and in some desiccation-sensitive species, and considering its role in antioxidant processes and in excess energy dissipation, the induction of the de-epoxidation of xanthophyll cycle pigments upon dehydration in the dark could be understood as a desiccation tolerance-related response maintained from the ancestral clades in the initial steps of land occupation by plants.     

Heterogeneity and subunit composition of the haemoglobins of five tilapiine species (Teleostei, Cichlidae) of the genera Oreochromis and Sarotherodon

Haemoglobins of five tilapiine species of the genera Oreochromis and Sarotherodon were investigated. By gel filtration chromatography a molecular weight of 67–69 kDa was determined for the tetrameric molecules which remained stable between pH 5.0 and pH 9.1. When subjected to sodium dodecyl sulphate-Urea-polyacrylamide gel electrophoresis (PAGE), haemoglobins of all species each were split into monomers of three different molecular weights ranging between 16.3 kDA and 17.6 kDa. Subsequently, isoelectric focusing separated haemolysates into about 23 differently charged tetrameric haemoglobins that were arranged in species-specific patterns. This diversity was shown to result from the occurrence of different types of globin chains. By acidic urea PAGE a total of seven major α-globins and five major β-globins were detected and species-characteristic chain variants were identified. To determine the globin chain composition of particular haemoglobin tetramers, 26 bands were isolated by isoelectric focusing and analysed by acidic urea PAGE. Tetramers consisted of doublets of identical α- and identical β-chains (α₂β₂, symmetric tetramers), or combinations of three (α₂ββ*; αα*β₂) or four (αα*ββ*) distinct chains (asymmetric tetramers). Finally, globin chains of Oreochromis niloticus were subjected to partial N-terminal amino acid sequencing. Differences in the composition of the three major β-chains could be shown, whereas the α-chains were N-terminally blocked. Accepted: 12 September 1997     

Purification,characterization, and potential applications of formate oxidase from <Emphasis Type="Italic">Debaryomyces vanrijiae</Emphasis> MH201

Formate oxidase was found in cell-free extracts of Debaryomyces vanrijiae MH201, a soil isolate. After purification by column chromatography, the preparation showed a protein band corresponding to a molecular mass (MM) of 64 kDa on sodium dodecyl sulfate–polyacrylamide gel electrophoresis. The MM, estimated by a gel filtration, was 99 kDa. The preparation showed two and three bands on isoelectric focusing under denaturing and native conditions, respectively. These results suggest that the preparation contained three isoforms, each of which might be composed of αα, αβ, and ββ subunits with apparently similar MM. The preparation acted on formate with K _m and V _max values of 11.7 mM and 262 μmol min⁻¹ mg⁻¹, respectively, at pH 4.5 and 25°C, but showed no evidence of activity on the other compounds tested. The optimum pH and temperature were pH 4.0 and 35°C, respectively. The preparation showed activities of 85% of the initial activity after storage at pH 6.0 and 4°C for 8 weeks. When 10 mM formaldehyde was reacted with 2.0 U ml⁻¹ of the enzyme preparation at pH 5.5 and room temperature in the presence of 2.0 U ml⁻¹ of a microbial aldehyde oxidase and 100 U ml⁻¹ of catalase for 180 min, neither of formate nor formaldehyde was detected, suggesting that the reaction involved the quantitative conversion of formaldehyde to carbon dioxide.     

Purification and initial characterization of phycobiliproteins from the endophytic cyanobacterium of Azolla

The endophytic cyanobacterium, Anabaena azollae, isolated from laboratory cultures of Azolla caroliniana Willd., contains three spectroscopically distinct biliproteins. About 70% of the biliprotein is c-phycocyanin (_max 610 nm) and 13% is allophycocyanin (_max 647 nm, shoulder 620 nm). A third pigment corresponds to phycoerythrocyanin (_max 570 nm, shoulder 590 nm). In very dilute solutions of allophycocyanin, at constant pH and buffer strength, the 647 nm maximum disappears and a single _max occurs at 615–620 nm. The 647 nm absorption maximum reappears upon concentrating the dilute solution. Very dilute solutions of phycoerythrocyanin exhibit a broad peak between 570 and 590 nm. Absorption spectra of c-phycocyanin are not significantly altered upon dilution. Fluorescence emission maxima of phycoerythrocyanin, c-phycocyanin, and allophycocyanin occur at 630 nm, 643 nm and 660 nm respectively, using 540 nm excitation. Two subunits, of molecular weight 16,500 () and 20,600 (), are seen in c-phycocyanin upon dissociation with SDS. Dissociation of allophycocyanin and phycoerythrocyanin with SDS yields one sizeclass of subunits, with a molecular weight of about 17,500 for allophycocyanin and 18,000 for phycoerythrocyanin.Contribution No. 684 Offprint requests to: G. A. Peters