Heme coordination states of unfolded ferrous cytochrome C

The structural changes of ferrous Cyt-c that are induced by binding to SDS micelles, phospholipid vesicles, DeTAB, and GuHCl as well as by high temperatures and changes in the pH have been studied by RR and UV-Vis absorption spectroscopies. Four species have been identified in which the native methionine-80 ligand is removed from the heme iron. This coordination site is either occupied by a histidine (His-33 or His-26) to form a 6cLS configuration, which is the prevailing species in GuHCl at pH 7.0 and ambient temperature, or remains vacant to yield a 5cHS configuration. The three identified 5cHS species differ with respect to the hydrogen-bond interactions of the proximal histidine ligand (His-18) and include a nonhydrogen-bonded, a hydrogen-bonded, and a deprotonated imidazole ring. These structural motifs have been found irrespective of the unfolding conditions used. An unambiguous spectroscopic distinction of these 5cHS species is possible on the basis of the Fe-N(imidazole) stretching vibrations, the RR bands in the region between 1300 and 1650 cm(-1), and the electronic transitions in the Soret- and Q-band regions. In acid and neutral solutions, the species with a hydrogen-bonded and a nonhydrogen-bonded His-18 prevail, whereas in alkaline solutions a configuration with a deprotonated His-18 ligand is also observed. Upon lowering the pH or increasing the temperature in GuHCl solutions, the structure on the proximal side of the heme is perturbed, resulting in a loss of the hydrogen-bond interactions of the His-18 ligand. Conversely, the hydrogen-bonded His-18 of ferrous Cyt-c is stabilized by electrostatic interactions which increase in strength from phospholipid vesicles to SDS micelles. The results here suggest that unfolding of Cyt-c is initiated by the rupture of the Fe-Met-80 bond and structural reorganizations on the distal side of the heme pocket, whereas the proximal part is only affected in a later stage of the denaturation process.     

Nitric oxide, cytochrome c oxidase and myoglobin: competition and reaction pathways

It is relevant to cell physiology that nitric oxide (NO) reacts with both cytochrome oxidase (CcOX) and oxygenated myoglobin (MbO(2)). In this respect, it has been proposed [Pearce, L.L., et al. (2002) J. Biol. Chem. 277, 13556-13562] that (i) CcOX in turnover out-competes MbO(2) for NO, and (ii) NO bound to reduced CcOX is "metabolized" in the active site to nitrite by reacting with O(2). In contrast, rapid kinetics experiments reported in this study show that (i) upon mixing NO with MbO(2) and CcOX in turnover, MbO(2) out-competes the oxidase for NO and (ii) after mixing nitrosylated CcOX with O(2) in the presence of MbO(2), NO (and not nitrite) dissociates from the enzyme causing myoglobin oxidation.     

Biological and polymeric self-assembled hybrid systems: Structure and properties of thylakoid/polyelectrolyte complexes

A novel hybrid system composed of biological components and synthetic polymer, thylakoid/polycation complex, has been formed and studied. Effects of complex formation on the structure, electrostatics and functioning of thylakoid membranes have been examined. Thylakoids from bean leaves were used to form complexes with polycation polyallylamine hydrochloride (PAAH) in two systems: (i) thylakoid/polycation complexes formed in an aqueous bulk phase, and (ii) immobilized thylakoid/polycation planar complexes. Immobilized on a solid substrate surface, thylakoid/polycation complexes were prepared using layer-by-layer stepwise alternate adsorption technique, i.e., via the sequential alternate adsorption of thylakoids and polycation molecules. The morphology of built up structures was investigated by scanning electron microscopy. Light-induced electron transport in chloroplasts was studied by the electron paramagnetic resonance (EPR) method. Spin probe technique was employed to study the structural and electrostatic characteristics of thylakoid membranes. We have found that efficiency of light-induced electron transport in thylakoid membranes and membrane structure were not changed noticeably by PAAH binding to thylakoids in a wide range of PAAH concentrations. The data obtained indicate the physiologically-soft character of polycation interactions with thylakoid membranes and demonstrate effectiveness of interfacial self-assembly approach to fabrication of complex planar functional nanostructures from biological components and synthetic polymers.     

Relating surfactant properties to activity and solubilization of the human adenosine a3 receptor

The effects of various surfactants on the activity and stability of the human adenosine A3 receptor (A3) were investigated. The receptor was expressed using stably transfected HEK293 cells at a concentration of 44 pmol functional receptor per milligram membrane protein and purified using over 50 different nonionic surfactants. A strong correlation was observed between a surfactant's ability to remove A3 from the membrane and the ability of the surfactant to remove A3 selectively relative to other membrane proteins. The activity of A3 once purified also correlates well with the selectivity of the surfactant used. The effects of varying the surfactant were much stronger than those achieved by including A3 ligands in the purification scheme. Notably, all surfactants that gave high efficiency, selectivity and activity fall within a narrow range of hydrophile-lipophile balance values. This effect may reflect the ability of the surfactant to pack effectively at the hydrophobic transmembrane interface. These findings emphasize the importance of identifying appropriate surfactants for a particular membrane protein, and offer promise for the development of rapid, efficient, and systematic methods to facilitate membrane protein purification.     

Solution properties of tetramethylrhodamine-modified G-actin

In the recently solved structure of TMR-modified ADP-G-actin, the nucleotide cleft is in a closed state conformation, and the D-loop contains an alpha-helix (L. R. Otterbein, P. Graceffa, and R. Dominguez, 2001, Science, 293:708-711). Subsequently, questions were raised regarding the possible role of the TMR label on Cys(374) in determining these aspects of G-actin structure. We show here that the susceptibility of D-loop on G-actin to subtilisin cleavage, and ATP/ADP-dependent changes in this cleavage, are not affected by TMR-labeling of actin. The TMR modification inhibits nucleotide exchange, but has no effect on DNase I binding and the fast phase of tryptic digestion of actin. These results show an absence of allosteric effects of TMR on subdomain 2, while confirming ATP/ADP-dependent changes in D-loop structure. In conjunction with similar results obtained on actin-gelsolin segment 1 complex, this works reveals the limitations of solution methods in probing the putative open and closed nucleotide cleft states of G-actin.     

Structural and kinetic properties of Rhodobacter sphaeroides photosynthetic reaction centers containing exclusively Zn-coordinated bacteriochlorophyll as bacteriochlorin cofactors

The Zn-BChl-containing reaction center (RC) produced in a bchD (magnesium chelatase) mutant of Rhodobacter sphaeroides assembles with six Zn-bacteriochlorophylls (Zn-BChls) in place of four Mg-containing bacteriochlorophylls (BChls) and two bacteriopheophytins (BPhes). This protein presents unique opportunities for studying biological electron transfer, as Zn-containing chlorins can exist in 4-, 5-, and (theoretically) 6-coordinate states within the RC. In this paper, the electron transfer perturbations attributed exclusively to coordination state effects are separated from those attributed to the presence, absence, or type of metal in the bacteriochlorin at the H_A pocket of the RC. The presence of a 4-coordinate Zn^2 + ion in the H_A bacteriochlorin instead of BPhe results in a small decrease in the rates of the P* → P⁺H_A⁻ → P⁺Q_A⁻ electron transfer, and the charge separation yield is not greatly perturbed; however coordination of the Zn^2 + by a fifth ligand provided by a histidine residue results in a larger rate decrease and yield loss. We also report the first crystal structure of a Zn-BChl-containing RC, confirming that the H_A Zn-BChl was either 4- or 5-coordinate in the two types of Zn-BChl-containing RCs studied here. Interestingly, a large degree of disorder, in combination with a relatively weak anomalous difference electron density was found in the H_B pocket. These data, in combination with spectroscopic results, indicate partial occupancy of this binding pocket. These findings provide insights into the use of BPhe as the bacteriochlorin pigment of choice at H_A in both BChl- and Zn-BChl-containing RCs found in nature.     

Investigation of the interaction of gold(III)-alkyldiamine complexes with l-histidine and imidazole ligands by H and C NMR, and UV spectrophotometry

Reactions of Au(III)-alkyldiamine complex with l-histidine and imidazole were carried out and monitored time-dependant by ¹H and ¹³C NMR. Kinetics for the [Au(en)Cl₂]⁺ reaction with l-histidine was determined by initial rate method at constant pH and 25 °C using UV-Vis absorption technique, and found to be first order with respect to each component, with a pseudo second order rate constant of 39 ± 3 M⁻¹ s⁻¹. Reaction rates of l-histidine and imidazole reactions with the [Au(en)Cl₂]⁺ complex was found to be strongly dependant on pD. The pD also has profound effect on the stability of the complex. It was observed that concurrent redox reactions also take place in solution in which Au(III) is reduced to metallic Au(0), while l-histidine and imidazole are oxidized to oxy and hydroxyl products. The optimization of the structure of [(His)Au(en)]³⁺ complex was carried out by gaussian03 at the RB3LYP level that showed a distorted square pyramid with the histidine carboxyl group at the pyramid top.     

Association of TNF-alpha,IL-6 and IL-1beta gene polymorphisms with polycystic ovary syndrome: a meta-analysis

Background

Several studies on the association of TNF-alpha (−308 G/A), IL-6 (−174 G/C) and IL-1beta (−511 C/T) polymorphisms with polycystic ovary syndrome (PCOS) risk have reported conflicting results. The aim of the present study was to assess these associations by meta-analysis.

Results

A total of 14 eligible articles (1665 cases/1687 controls) were included in this meta-analysis. The results suggested that there was no obvious association between the TNF-alpha (−308 G/A) polymorphism and PCOS in the overall population or subgroup analysis by ethnicity, Hardy–Weinberg equilibrium (HWE) in controls, genotyping method, PCOS diagnosis criteria, and study sample size. Also, no obvious association was found between the TNF-alpha (−308 G/A) polymorphism and obesity in patients with PCOS (body mass index [BMI] ≥ 25 kg/m² vs. BMI < 25 kg/m²). Regarding the IL-6 (−174 G/C) polymorphism, also no association was found in the overall population in heterozygote comparison, dominant model, and recessive model. Even though an allelic model (odds ratio [OR] = 0.63, 95% confidence interval [CI] = 0.41–0.96) and a homozygote comparison (OR = 0.52, 95% CI = 0.30–0.93) showed that the IL-6 (−174 G/C) polymorphism was marginally associated with PCOS. Further subgroup analysis suggested that the effect size was not significant among HWE in controls (sample size ≤ 200) and genotyping method of pyrosequencing under all genetic models. Similarly, there was no association between the IL-1beta (−511 C/T) polymorphism and PCOS in the overall population or subgroup analysis under all genetic models. Furthermore, no significant association was found between the IL-1beta (−511 C/T) polymorphism and several clinical and biochemical parameters in patients with PCOS.

Conclusions

The results of this meta-analysis suggest that the TNF-alpha (−308 G/A), IL-6 (−174 G/C), and IL-1beta (−511 C/T) polymorphisms may not be associated with PCOS risk. However, further case–control studies with larger sample sizes are needed to confirm our results.

Electronic supplementary material

The online version of this article (doi:10.1186/s12863-015-0165-4) contains supplementary material, which is available to authorized users.     

基因拷贝数和甲醇浓度对重组毕赤酵母产华根霉脂肪酶的影响

将华根霉脂肪酶基因克隆到甲基营养型毕赤酵母中表达,以甲醇利用快型菌株为宿主,在7 L发酵罐水平对脂肪酶基因拷贝数分别为3、5、6的3株基因重组菌——XY RCL-3、XY RCL-5、XY RCL-6进行高密度发酵调控,同时研究了甲醇浓度对表达华根霉脂肪酶的影响。结果表明,XY RCL-5在相同条件下发酵产酶能力高于XY RCL-6和XY RCL-3,最适甲醇诱导浓度控制在0.1%±0.02%时,酶活可达到12 500 U/mL,菌体干重达到204 g/L,蛋白浓度也能达到8.02 g/L。     

Functional properties of type I and type II cytochromes c3 from Desulfovibrio africanus

Type I cytochrome c₃ is a key protein in the bioenergetic metabolism of Desulfovibrio spp., mediating electron transfer between periplasmic hydrogenase and multihaem cytochromes associated with membrane bound complexes, such as type II cytochrome c₃. This work presents the NMR assignment of the haem substituents in type I cytochrome c₃ isolated from Desulfovibrio africanus and the thermodynamic and kinetic characterisation of type I and type II cytochromes c₃ belonging to the same organism. It is shown that the redox properties of the two proteins allow electrons to be transferred between them in the physiologically relevant direction with the release of energised protons close to the membrane where they can be used by the ATP synthase.     

Low-dimensional compounds containing cyano groups. XIX. Crystal structure, spectroscopic, thermal and magnetic properties of {[Cu(tn)2]3[Pt(CN)4]2}[Pt(CN)4] (tn = 1,3-diaminopropane) complex

Violet prismatic crystals of {[Cu(tn)₂]₃[Pt(CN)₄]₂}[Pt(CN)₄] (tn = 1,3-diaminopropane) were crystallized from the water-methanol solution containing CuCl₂·2H₂O, tn and K₂[Pt(CN)₄]·3H₂O. Prepared complex was characterized using elemental analysis, infrared and UV-Vis spectroscopy, magnetic measurement and thermal analysis. X-ray analysis revealed an ionic character of the complex containing mononuclear square planar [Pt(CN)₄]²⁻ complex anions and penta-nuclear [Cu(tn)₂-Pt(CN)₄-Cu(tn)₂-Pt(CN)₄-Cu(tn)₂]²⁺ complex cations. The inner Cu(II) atom of the complex cation is hexa-coordinated, whereas two crystallographically equivalent peripheral Cu(II) atoms are penta-coordinated in the shape of a deformed square pyramid. Four v(CN) absorption bands observed in the IR spectrum are in agreement with the higher number of crystallographically different cyano groups and a broad highly asymmetric band observed in the reflectance UV-Vis spectrum is consistent with the presence of both hexa- and penta-coordinated Cu(II) atoms in the structure. The temperature dependence of the inverse susceptibility suggests the presence of a weak antiferromagnetic exchange coupling between Cu(II) ions. The complex is stable up to 210 °C when its two-stage thermal decomposition starts.     

Mutation and expression analysis of the IDH1, IDH2, DNMT3A, and MYD88 genes in colorectal cancer

Colorectal cancer (CRC) is one of the leading causes of death around the world. Its genetic mechanism was intensively investigated in the past decades with findings of a number of canonical oncogenes and tumor-suppressor genes such as APC, KRAS, and TP53. Recent genome-wide association and sequencing studies have identified a series of promising oncogenes including IDH1, IDH2, DNMT3A, and MYD88 in hematologic malignancies. However, whether these genes are involved in CRC remains unknown. In this study, we screened the hotspot mutations of these four genes in 305 CRC samples from Han Chinese by direct sequencing. mRNA expression levels of these genes were quantified by quantitative real-time PCR (RT-qPCR) in paired cancerous and paracancerous tissues. Association analyses between mRNA expression levels and different cancerous stages were performed. Except for one patient harboring IDH1 mutation p.I99M, we identified no previously reported hotspot mutations in colorectal cancer tissues. mRNA expression levels of IDH1, DNMT3A, and MYD88, but not IDH2, were significantly decreased in the cancerous tissues comparing with the paired paracancerous normal tissues. Taken together, the hotspot mutations of IDH1, IDH2, DNMT3A, and MYD88 gene were absent in CRC. Aberrant mRNA expression of IDH1, DNMT3A, and MYD88 gene might be actively involved in the development of CRC.     

Characterization of 10 MADS-box genes from Pyrus pyrifolia and their differential expression during fruit development and ripening

We cloned 10 Japanese pear (Pyrus pyrifolia) MIKC-type II MADS-box genes, and analyzed their expression during fruit development and ripening. PpMADS2-1 was APETALA (AP)1-like; PpMADS3-1 was FRUITFULL (FUL)/SQUAMOSA (SQUA)-like; PpMADS4-1 was AGAMOUS-like (AGL)6; PpMADS5-1 and PpMADS8-1 were SUPPRESSOR OF OVEREXPRESSION OF CONSTANS (SOC)-like; PpMADS9-1, PpMADS12-1, PpMADS14-1 and PpMADS16-1 were SEPALLATA (SEP)-like; while PpMADS15-1 was AGL/SHATTERPROOF (SHP)-like. Phylogenetic analysis showed their grouping into five major clades (and 10 sub-clades) that was consistent with their diverse functional types. Expression analysis in flower tissue revealed their distinct putative homeotic functional classes: A-class (PpMADS2-1, PpMADS3-1, PpMADS4-1, and PpMADS14-1), C-class (PpMADS15-1), E-class (PpMADS9-1, PpMADS12-1, and PpMADS16-1) and E (F)-class (PpMADS5-1 and PpMADS8-1). Differential gene expression was observed in different fruit tissues (skin, cortex and core) as well as in the cortex during the course of fruit development and ripening. Collectively, our results suggest their involvement in the diverse aspects of plant development including flower development and the course of fruit development and ripening.     

Characterization of the complete mitochondrial genome of Chilo auricilius and comparison with three other rice stem borers

The mitogenome of Chilo auricilius (Lepidoptera: Pyraloidea: Crambidae) was a circular molecule made up of 15,367 bp. Sesamia inferens, Chilo suppressalis, Tryporyza incertulas, and C. auricilius, are closely related, well known rice stem borers that are widely distributed in the main rice-growing regions of China. The gene order and orientation of all four stem borers were similar to that of other insect mitogenomes. Among the four stem borers, all AT contents were below 83%, while all AT contents of tRNA genes were above 80%. The genomes were compact, with only 121–257 bp of non-coding intergenic spacer. There are 56 or 62-bp overlapping nucleotides in Crambidae moths, but were only 25-bp overlapping nucleotides in the noctuid moth S. inferens. There was a conserved motif ‘ATACTAAA’ between trnS2 (UCN) and nad1 in Crambidae moths, but this same region was ‘ATCATA’ in the noctuid S. inferens. And there was a 6-bp motif ‘ATGATAA’ of overlapping nucleotides, which was conserved in Lepidoptera, and a 14-bp motif ‘TAAGCTATTTAAAT’ conserved in the three Crambidae moths (C. suppressalis, C. auricilius and T. incertulas), but not in the noctuid. Finally, there were no stem-and-loop structures in the two Chilo moths.     

Complete hunting cycle of Dionaea muscipula: Consecutive steps and their electrical properties

The total hunting cycle of the Venus flytrap consists of five stages: 1. Open state → 2. Closed state → 3. Locked state → 4. Constriction and digestion → 5. Semi-open state → 1. Open state. The opening of the trap after digestion consists of two steps: opening of the lobes, and changing of their curvature from concave to convex shape. Uncouplers carbonylcyanide-4-trifluoromethoxyphenyl hydrazone (FCCP) and carbonylcyanide-3-chlorophenylhydrazone (CCCP) inhibit the trap from opening for two weeks and antracene-9-carboxylic acid inhibits the trap from constricting. Different stages of the hunting cycle have different electrical characteristics. The biologically closed electrochemical circuits in the Venus flytrap are analyzed using the charged capacitor method. If the initial voltage applied to the Venus flytrap is 0.5 V or greater, changing the polarity of the electrodes between the midrib and one of the lobes results in a rectification effect and in different kinetics of discharge capacitance. These effects can be caused by the fast transport of ions through ion channels. The electrical properties of the Venus flytrap were investigated and equivalent electrical circuits within the upper leaf were proposed to explain the experimental data.     

Genome-wide screening of alpha-tocopherol sensitive genes in heart tissue from alpha-tocopherol transfer protein null mice (ATTP(-/-))

Alpha-tocopherol transfer protein (ATTP) null mice (ATTP(-/-)) have a systemic deficiency of alpha-tocopherol (AT). The heart AT levels of ATTP(-/-) are <10% of those in ATTP(+/+) mice. The genomic responses of heart to AT deficiency were determined in 3 months old male ATTP(-/-) mice and compared with their ATTP(+/+) littermate controls using Affymetrix 430A 2.0 high density oligonucleotide arrays. Differential analysis of approximately 13000 genes identified repression of genes related to immune system and activation of genes related to lipid metabolism and inflammation with no significant change in the expression of classical antioxidant genes (catalase, superoxide dismutase, glutathione peroxidase) in ATTP(-/-) as compared to ATTP(+/+) mice. The present data identifies novel classes of AT sensitive genes in heart tissue.     

Slow formation of stable complexes during coincubation of minimal rRNA and ribosomal protein S4

Ribosomal protein S4 binds and stabilizes a five-helix junction or five-way junction (5WJ) in the 5′ domain of 16S ribosomal RNA (rRNA) and is one of two proteins responsible for nucleating 30S ribosome assembly. Upon binding, both protein S4 and 5WJ reorganize their structures. We show that labile S4 complexes rearrange into stable complexes within a few minutes at 42 °C, with longer coincubation leading to an increased population of stable complexes. In contrast, prefolding the rRNA has a smaller effect on stable S4 binding. Experiments with minimal rRNA fragments show that this structural change depends only on 16S residues within the S4 binding site. SHAPE (selective 2′-hydroxyl acylation analyzed by primer extension) chemical probing experiments showed that S4 strongly stabilizes 5WJ and the helix (H) 18 pseudoknot, which become tightly folded within the first minute of S4 binding. However, a kink in H16 that makes specific contacts with the S4 N-terminal extension, as well as a right-angle motif between H3, H4, and H18, requires a minute or more to become fully structured. Surprisingly, S4 structurally reorganizes the 530-loop and increases the flexibility of H3, which is proposed to undergo a conformational switch during 30S assembly. These elements of the S4 binding site may require other 30S proteins to reach a stable conformation.     

Characterization of the complete mitochondrial genome of Tryporyza incertulas, in comparison with seven other Pyraloidea moths

The complete 15,223-bp mitochondrial genome (mitogenome) of Tryporyza incertulas (Walker) (Lepidoptera: Pyraloidea: Crambidae) was determined, characterized and compared with seven other species of superfamily Pyraloidea. The order of 37 genes was typical of insect mitochondrial DNA sequences described to date. Compared with other moths of Pyraloidea, the A + T biased (77.0%) of T. incertulas was the lowest. Eleven protein-coding genes (PCGs) utilized the standard ATN, but cox1 used CGA and nad4 used AAT as the initiation codons. Ten protein-coding genes had the common stop codon TAA, except nad3 having TAG as the stop codon, and cox2, nad4 using T, TA as the incomplete stop codons, respectively. All of the tRNA genes had typical cloverleaf secondary structures except trnS1(AGN), in which the dihydrouridine (DHU) arm did not form a stable stem-loop structure. There was a spacer between trnQ and nad2, which was common in Lepidoptera moths. A 6-bp motif ‘ATACTA’ between trnS2(UCN) and nad1, a 7-bp motif “AGC(T)CTTA” between trnW and trnC and a 6-bp motif “ATGATA” of overlapping region between atp8 and atp6 were found in Pyraloidea moths. The A + T-rich region contained an ‘ATAGT(A)’-like motif followed by a poly-T stretch. In addition, two potential stem-loop structures, a duplicated 19-bp repeat element, and two microsatellites ‘(TA)₁₂’ and ‘(TA)₉’ were observed in the A + T-rich region of T. incertulas mitogenome. Finally, the phylogenetic relationships of Pyraloidea species were constructed based on amino acid sequences of 13 PCGs of mitogenomes using Bayesian inference (BI) and maximum likelihood (ML) methods. These molecular-based phylogenies supported the morphological classification on relationships within Pyraloidea species.