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Capillary crystallization and molecular-replacement solution of haemoglobin II from the clam Lucina pectinata.

ABSTRACT: Haemoglobin II is one of three haemoglobins present in the cytoplasm of the Lucina pectinata mollusc that inhabits the Caribbean coast. Using HBII purified from its natural source, crystallization screening was performed using the counter-diffusion method with capillaries of 0.2 mm inner diameter. Crystals of HbII suitable for data collection and structure determination were grown in the presence of agarose at 0.1%(w/v) in order to improve their quality. The crystals belong to the tetragonal space group P4(2)2(1)2, with unit-cell parameters a = b = 73.92, c = 152.35 A, and diffracted X-rays to a resolution of better than 2.0 A. The asymmetric unit is a homodimer with a corresponding Matthews coefficient (VM) of 3.15 A3 Da(-1) and a solvent content of 61% by volume.

SUBMITTER: Gavira JA

PROVIDER: S-EPMC2197196 | biostudies-literature | 2006 Mar

REPOSITORIES: biostudies-literature

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Capillary crystallization and molecular-replacement solution of haemoglobin II from the clam Lucina pectinata.

Gavira José A JA de Jesus Walleska W Camara-Artigas Ana A López-Garriga Juan J García-Ruiz Juan M JM

Acta crystallographica. Section F, Structural biology and crystallization communications 20060210 Pt 3

Haemoglobin II is one of three haemoglobins present in the cytoplasm of the Lucina pectinata mollusc that inhabits the Caribbean coast. Using HBII purified from its natural source, crystallization screening was performed using the counter-diffusion method with capillaries of 0.2 mm inner diameter. Crystals of HbII suitable for data collection and structure determination were grown in the presence of agarose at 0.1%(w/v) in order to improve their quality. The crystals belong to the tetragonal spa ...[more]

PMID: 16511300

Similar Datasets

Two-step counterdiffusion protocol for the crystallization of haemoglobin II from Lucina pectinata in the pH range 4-9.

Project description:Lucina pectinata haemoglobin II (HbII) transports oxygen in the presence of H(2)S to the symbiotic system in this bivalve mollusc. The composition of the haem pocket at the distal site includes TyrB10 and GlnE7, which are very common in other haem proteins. Obtaining crystals of oxyHbII at various pH values is required in order to elucidate the changes in the conformations of TyrB10 and GlnE7 and structural scenarios induced by changes in pH. Here, the growth of crystals of oxyHbII using the capillary counterdiffusion (CCD) technique at various pH values using a two-step protocol is reported. In the first step, a mini-screen was used to validate sodium formate as the best precipitating reagent for the growth of oxyHbII crystals. The second step, a pH screen typically used for optimization, was used to produce crystals in the pH range 4-9. Very well faceted prismatic ruby-red crystals were obtained at all pH values. X-ray data sets were acquired using synchrotron radiation of wavelength 0.886 A (for the crystals obtained at pH 5) and 0.908 A (for those obtained at pH 4, 8 and 9) to maximum resolutions of 3.30, 1.95, 1.85 and 2.00 A for the crystals obtained at pH 4, 5, 8 and 9, respectively. All of the crystals were isomorphous and belonged to space group P4(2)2(1)2.

| S-EPMC2833032 | biostudies-literature

Effects of active site mutations in haemoglobin I from Lucina pectinata: a molecular dynamic study.

Project description:Haemoglobin I from Lucina pectinata is a monomeric protein consisting of 142 amino acids. Its active site contains a peculiar arrangement of phenylalanine residues (PheB10, PheCD1 and PheE11) and a distal Gln at position E7. Active site mutations at positions B10, E7 and E11 were performed in deoxy haemoglobin I (HbI), followed by 10 ns molecular dynamic simulations. The results showed that the mutations induced changes in domains far from the active site producing more flexible structures than the native HbI. Distance analyses revealed that the heme pocket amino acids at positions E7 and B10 are extremely sensitive to any heme pocket residue mutation. The high flexibility observed by the E7 position suggests an important role in the ligand binding kinetics in ferrous HbI, while both positions play a major role in the ligand stabilisation processes. Furthermore, our results showed that E11Phe plays a pivotal role in protein stability.

| S-EPMC2657002 | biostudies-literature

Lucina pectinata oxyhemoglobin (II-III) heterodimer pH susceptibility.

Project description:Lucina pectinata live in high concentrations of hydrogen sulfide (H2S) and contains one hemoglobin, Hemoglobin I (HbI), transporting H2S and two hemoglobins, Hemoglobin II (HbII) and Hemoglobin (HbIII), transferring dioxygen to symbionts. HbII and HbIII contain B10 tyrosine (Tyr) and E7 glutamine (Gln) in the heme pocket generating an efficient hydrogen bonding network with the (HbII-HbIII)-O2 species, leading to very low ligand dissociation rates. The results indicate that the oxy-hemeprotein is susceptible to pH from 4 to 9, at acidic conditions, and as a function of the potassium ferricyanide concentration, 100% of the met-aquo derivative is produced. Without a strong oxidant, pH 5 generates a small concentration of the met-aquo complex. The process is accelerated by the presence of salts, as indicated by the crystallization structures and UV-Vis spectra. The results suggest that acidic pH generates conformational changes associated with B10 and E7 heme pocket amino acids, weakening the (HbII-HbIII)-O2 hydrogen bond network. The observation is supported by X-ray crystallography, since at pH 4 and 5, the heme-Fe tends to oxidize, while at pH 7, the oxy-heterodimer is present. Conformational changes also are observed at higher pH by the presence of a 605 nm transition associated with the iron heme-Tyr interaction. Therefore, pH is one crucial factor regulating the (HbII-HbIII)-O2 complex hydrogen-bonding network. Thus, it can be proposed that the hydrogen bonding adjustments between the heme bound O2 and the Tyr and Gln amino acids contribute to oxygen dissociation from the (HbII-HbIII)-O2 system.

| S-EPMC7247926 | biostudies-literature

Structure and ligand selection of hemoglobin II from Lucina pectinata.

Project description:Lucina pectinata ctenidia harbor three heme proteins: sulfide-reactive hemoglobin I (HbI(Lp)) and the oxygen transporting hemoglobins II and III (HbII(Lp) and HbIII(Lp)) that remain unaffected by the presence of H(2)S. The mechanisms used by these three proteins for their function, including ligand control, remain unknown. The crystal structure of oxygen-bound HbII(Lp) shows a dimeric oxyHbII(Lp) where oxygen is tightly anchored to the heme through hydrogen bonds with Tyr(30)(B10) and Gln(65)(E7). The heme group is buried farther within HbII(Lp) than in HbI(Lp). The proximal His(97)(F8) is hydrogen bonded to a water molecule, which interacts electrostatically with a propionate group, resulting in a Fe-His vibration at 211 cm(-1). The combined effects of the HbII(Lp) small heme pocket, the hydrogen bonding network, the His(97) trans-effect, and the orientation of the oxygen molecule confer stability to the oxy-HbII(Lp) complex. Oxidation of HbI(Lp) Phe(B10) --> Tyr and HbII(Lp) only occurs when the pH is decreased from pH 7.5 to 5.0. Structural and resonance Raman spectroscopy studies suggest that HbII(Lp) oxygen binding and transport to the host bacteria may be regulated by the dynamic displacements of the Gln(65)(E7) and Tyr(30)(B10) pair toward the heme to protect it from changes in the heme oxidation state from Fe(II) to Fe(III).

| S-EPMC2431033 | biostudies-literature

Crystallization and diffraction patterns of the oxy and cyano forms of the Lucina pectinata haemoglobins complex.

Project description:The native oxygen-carrier haemoglobins complex (HbII-III) is composed of haemoglobin II (HbII) and haemoglobin III (HbIII), which are found in the ctenidia tissue of the bivalve mollusc Lucina pectinata. This protein complex was isolated and purified from its natural source and crystallized using the vapour-diffusion and capillary counter-diffusion methods. Oxy and cyano derivatives of the complex crystallized using several conditions, but the best crystals in terms of quality and size were obtained from sodium formate pH 5 using the counter-diffusion method in a single capillary. Crystals of the oxy and cyano complexes, which showed a ruby-red colour and nonsingular prismatic shapes, scattered X-rays to resolution limits of 2.15 and 2.20 A, respectively, using a 0.886 A synchrotron-radiation source. The crystals belonged to the tetragonal system, space group P4(2)2(1)2, with unit-cell parameters a = b = 74.07, c = 152.07 and a = b = 73.83, c = 152.49 A for the oxy and cyano complexes, respectively. The asymmetric unit of both crystals is composed of a single copy of the heterodimer, with Matthew coefficients (V(M)) of 3.08 and 3.06 A(3) Da(-1) for the oxy and cyano complexes, respectively, which correspond to a solvent content of approximately 60.0% by volume.

| S-EPMC2628858 | biostudies-literature

Characterization of Histone Genes from the Bivalve Lucina Pectinata.

Project description:Lucina pectinata is a clam that lives in sulfide-rich environments and houses intracellular sulfide-oxidizing endosymbionts. To identify new Lucina pectinata proteins, we produced libraries for genome and transcriptome sequencing and assembled them de novo. We searched for histone-like sequences using the Lucina pectinata histone H3 partial nucleotide sequence against our previously described genome assembly to obtain the complete coding region and identify H3 coding sequences from mollusk sequences in Genbank. Solen marginatus histone nucleotide sequences were used as query sequences using the genome and transcriptome assemblies to identify the Lucina pectinata H1, H2A, H2B and H4 genes and mRNAs and obtained the complete coding regions of the five histone genes by RT-PCR combined with automated Sanger DNA sequencing. The amino acid sequence conservation between the Lucina pectinata and Solen marginatus histones was: 77%, 93%, 83%, 96% and 97% for H1, H2A, H2B, H3 and H4, respectively. As expected, the H3 and H4 proteins were the most conserved and the H1 proteins were most similar to H1's from aquatic organisms like Crassostrea gigas, Aplysia californica, Mytilus trossulus and Biomphalaria glabrata. The Lucina pectinata draft genome and transcriptome assemblies, obtained by semiconductor sequencing, were adequate for identification of conserved proteins as evidenced by our results for the histone genes.

| S-EPMC6210712 | biostudies-literature

SAXS structure of homodimeric oxyHemoglobin III from bivalve Lucina pectinata.

Project description:Hemoglobin III (HbIII) is one of the two oxygen reactive hemoproteins present in the bivalve, Lucina pectinata. The clam inhabits a sulfur-rich environment and HbIII is the only hemoprotein present in the system which does not yet have a structure described elsewhere. It is known that HbIII exists as a heterodimer with hemoglobin II (HbII) to generate the stable Oxy(HbII-HbIII) complex but it remains unknown if HbIII can form a homodimeric species. Here, a new chromatographic methodology to separate OxyHbIII from the HbII-HbIII dimer has been developed, employing a fast performance liquid chromatography and ionic exchange chromatography column. The nature of OxyHbIII in solution at concentrations from 1.6 mg/mL to 20.4 mg/mL was studied using small angle X-ray scattering (SAXS). The results show that at all concentrations, the Oxy(HbIII-HbIII) dimer dominates in solution. However, as the concentration increases to nonphysiological values, 20.4 mg/mL, HbIII forms a 30% tetrameric fraction. Thus, there is a direct relationship between the Oxy(HbIII-HbIII) oligomeric form and hemoglobin concentration. We suggest it is likely that the OxyHbIII dimer contributes to active oxygen transport in tissues of L pectinata, where the Oxy(HbII-HbIII) complex is not present.

| S-EPMC8265735 | biostudies-literature

Molecular Cloning and Characterization of a (Lys)6-Tagged Sulfide-Reactive Hemoglobin I from Lucina pectinata.

Project description:A poly-Lys tag was fused to the Lucina pectinata hemoglobin I (HbI) coding sequence and purified using an efficient and fast process. HbI is a hemeprotein that binds hydrogen sulfide (H2S) with high affinity and it has been used to understand physiologically relevant reactions of this signaling molecule. The (Lys)6-tagged rHbI construct was expressed in E. coli and purified by immobilization on a cation exchange matrix, followed by size-exclusion chromatography. The identity, structure, and function of the (Lys)6-tagged rHbI were assessed by mass spectrometry, small and wide X-ray scattering, optical spectroscopy, and kinetic analysis. The scattering and spectroscopic results showed that the (Lys)6-tagged rHbI is structurally and functionally analogous to the native protein as well as to the (His)6-tagged rHbI. Kinetics studies with H2S indicated that the association (k on) and dissociation (k off) rate constants were 1.4 × 10(5)/M/s and 0.1 × 10(-3)/s, respectively. This results confirmed that the (Lys)6-tagged rHbI binds H2S with the same high affinity as its homologue.

| S-EPMC4937220 | biostudies-literature

Purification, crystallization, preliminary X-ray diffraction and molecular-replacement studies of catfish (Clarias magur) haemoglobin.

Project description:Haemoglobin is an interesting physiologically significant protein composed of specific functional prosthetic haem and globin moieties. In recent decades, there has been substantial interest in attempting to understand the structural basis and functional diversity of fish haemoglobins (Hbs). Towards this end, purification, crystallization, preliminary X-ray diffraction and molecular-replacement studies have been carried out on Clarias magur Hb. Crystals were grown by the hanging-drop vapour-diffusion method using PEG 2000 and NaCl as precipitants. The crystals belonged to the primitive monoclinic system P2, with unit-cell parameters a=98.35, b=56.63, c=112.88?Å, ?=100.22°; a complete data set was collected to a resolution of 2.4?Å. The Matthews coefficient of 2.42?Å3?Da(-1) for the crystal indicated the presence of two ?2?2 tetramers in the asymmetric unit.

| S-EPMC3515384 | biostudies-literature

Characterization and Expression of the Lucina pectinata Oxygen and Sulfide Binding Hemoglobin Genes.

Project description:The clam Lucina pectinata lives in sulfide-rich muds and houses intracellular symbiotic bacteria that need to be supplied with hydrogen sulfide and oxygen. This clam possesses three hemoglobins: hemoglobin I (HbI), a sulfide-reactive protein, and hemoglobin II (HbII) and III (HbIII), which are oxygen-reactive. We characterized the complete gene sequence and promoter regions for the oxygen reactive hemoglobins and the partial structure and promoters of the HbI gene from Lucina pectinata. We show that HbI has two mRNA variants, where the 5'end had either a sequence of 96 bp (long variant) or 37 bp (short variant). The gene structure of the oxygen reactive Hbs is defined by having 4-exons/3-introns with conservation of intron location at B12.2 and G7.0 and the presence of pre-coding introns, while the partial gene structure of HbI has the same intron conservation but appears to have a 5-exon/ 4-intron structure. A search for putative transcription factor binding sites (TFBSs) was done with the promoters for HbII, HbIII, HbI short and HbI long. The HbII, HbIII and HbI long promoters showed similar predicted TFBSs. We also characterized MITE-like elements in the HbI and HbII gene promoters and intronic regions that are similar to sequences found in other mollusk genomes. The gene expression levels of the clam Hbs, from sulfide-rich and sulfide-poor environments showed a significant decrease of expression in the symbiont-containing tissue for those clams in a sulfide-poor environment, suggesting that the sulfide concentration may be involved in the regulation of these proteins. Gene expression evaluation of the two HbI mRNA variants indicated that the longer variant is expressed at higher levels than the shorter variant in both environments.

| S-EPMC4732748 | biostudies-literature

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