Crystallization of hemoglobins II and III of the symbiont-harboring clam Lucina pectinata

Doyle, M. A.; Vitali, Jacqueline; Wittenberg, Jonathan B.; Vinogradov, Stanley N.; Walz, Daniel A.; Edwards, Brian F.P.; Martin, Philip D.

doi:10.1107/s0907444994002556

Cited by 4 publications

(4 citation statements)

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“…Initial crystallization conditions were screened using the 24-condition Crystallization Screening Kit (GCB-CSK; Triana Science and Technology) adapted from the mini-screen described by Kimber & Vallee (2003). As previously observed during crystallization experiments of HbII using vapour diffusion (Kemling et al, 1991;Doyle et al, 1994) and counterdiffusion techniques (Gavira et al, 2006) Representative crystals of oxyHbII obtained in sodium formate at (a) pH 4, (b) pH 5, (c) pH 8 and (d) pH 9. (Ruiz-Martínez et al, 2009) a large number of hits were obtained (11 conditions), but only those crystals grown in the presence of sulfate or formate were of good optical quality.…”

Section: Resultsmentioning

confidence: 99%

“…The first HbII crystallization condition was obtained using the hanging-drop vapour-diffusion method. The crystallization condition employed was 8% saturated ammonium sulfate solution and 50 mM Tris-HCl pH 7, using protein at 1.9 mM in 50 mM phosphate buffer pH 7.5 and 0.05 mM EDTA (Doyle et al, 1994;Kemling et al, 1991). Another crystallization condition was obtained in a 0.2 mm capillary containing 0.1%(w/v) agarose using a lyophilized HbII sample dissolved in 50 mM bis-tris propane buffer pH 7 with 0.5 mM EDTA.…”

Section: Introductionmentioning

confidence: 99%

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Two-step counterdiffusion protocol for the crystallization of haemoglobin II fromLucina pectinatain the pH range 4–9

et al. 2010

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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 Å (for the crystals obtained at pH 5) and 0.908 Å (for those obtained at pH 4, 8 and 9) to maximum resolutions of 3.30, 1.95, 1.85 and 2.00 Å 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.

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Two-step counterdiffusion protocol for the crystallization of haemoglobin II fromLucina pectinatain the pH range 4–9

et al. 2010

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“…The structure of haemoglobin I from L. pectinata was solved more than 10 y ago (Rizzi et al, 1994(Rizzi et al, , 1996, but the structures of haemoglobin II and III remain unsolved, although preliminary crystal-lization data were reported in 1991 (Kemling et al, 1991) and 1994 (Doyle et al, 1994). Here, we report the crystallization and the first crystallographic data of haemoglobin II from the clam L. pectinata.…”

Section: Introductionmentioning

confidence: 90%

Capillary crystallization and molecular-replacement solution of haemoglobin II from the clamLucina pectinata

et al. 2006

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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.

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“…[ 11 ] Later, crystallographic results of the HbII and HbIII homodimer complexes concluded that the crystals were isomorphous, diffracted to 2.7 Å, and contained a dimer in the asymmetric unit. [ 12 ] Gavira et al . employed a capillary counter‐diffusion technique for Oxy(HbII‐HbII) to obtained crystals and reported the crystallographic structure at 1.9 Å resolution [ 13 ] (PDB ID 2OLP).…”

Section: Introductionmentioning

confidence: 99%

SAXS structure of homodimeric oxyHemoglobin III from bivalve Lucina pectinata

et al. 2021

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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.

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Crystallization of hemoglobins II and III of the symbiont-harboring clam Lucina pectinata

Cited by 4 publications

References 4 publications

Two-step counterdiffusion protocol for the crystallization of haemoglobin II fromLucina pectinatain the pH range 4–9

Two-step counterdiffusion protocol for the crystallization of haemoglobin II fromLucina pectinatain the pH range 4–9

Capillary crystallization and molecular-replacement solution of haemoglobin II from the clamLucina pectinata

SAXS structure of homodimeric oxyHemoglobin III from bivalve Lucina pectinata

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