Under a suitable condition of crystallization, dark brown short rhombohedron crystals could be obtained from nitrogenase MnFe protein purified from a mutant UW3 of Azotobacter vinelandii Lipmann grown in Mn-containing...Under a suitable condition of crystallization, dark brown short rhombohedron crystals could be obtained from nitrogenase MnFe protein purified from a mutant UW3 of Azotobacter vinelandii Lipmann grown in Mn-containing but Mo- and NH3-free medium. The possibility of crystallization, and number, size and quality of crystals were obviously dependent on concentrations of NaCl, MgCl2, PEG 8000,Tris and Hepes buffer and on methods for crystallization. PEG concentration affected on the shape of the crystals. The optimal, concentrations of the chemicals for crystallization of MnFe protein were slightly different from those for crystallization of Delta nifZ MoFe protein from a nifZ deleted strain of Azotobacter vinelandii. SDS-PAGE showed that the protein from the dissolved crystals was almost the same as MnFe protein before crystallization, indicating that the crystal was formed from MnFe protein.展开更多
By using the liquid/liquid diffusion method at a suitable crystallization conditions, large single and dark brown crystals (the sides of the largest crystals were 0.20 mm x 0.20 mm x 0.07 min and 0.18 mm x 0.18 mm x 0...By using the liquid/liquid diffusion method at a suitable crystallization conditions, large single and dark brown crystals (the sides of the largest crystals were 0.20 mm x 0.20 mm x 0.07 min and 0.18 mm x 0.18 mm x 0.05 mm, respectively) could be obtained from the solutions of nitrogenase CrFe protein and MnFe protein purified from a mutant UW3 of Azotobacter vinelandii Lipmarm grown in Cr- or Mn-containing but NH3-free medium. The time of crystal formation, as well as the number, size, shape and quality of crystals obviously depended on the concentrations of PEG, MgCl2 and NaCl. The liquid/liquid diffusion method seems to benefit CrFe protein and MnFe protein for the growth of large single crystals for X-ray diffraction analysis.展开更多
Nitrogenase CrFe protein and MnFe protein were purified from a mutant strain UW3 of Azotobacter vinelandii Lipmann grown on a medium containing Cr and Mn, respectively. In order to meet the requirement for crystal gro...Nitrogenase CrFe protein and MnFe protein were purified from a mutant strain UW3 of Azotobacter vinelandii Lipmann grown on a medium containing Cr and Mn, respectively. In order to meet the requirement for crystal growth Of O-2-susceptible proteins including nitrogenase in space, crystallization conditions were optimized for the proteins using a simple and suitable device, as a replacement for the cumbersome anaerobic box (dry box), for anaerobic addition of the protein samples. In all used precipitant and protein solutions added in the simplified plexi glass box, CrFe protein and MnFe protein could be crystallized on the spacecraft in one week by the liquid/liquid diffusion method and vapor diffusion by the sitting drop method, respectively. All formed crystals were single on the spacecraft, but under the same condition twin crystals appeared on the ground. The size of the largest crystal grown in space from CrFe protein was 2-fold larger than that on the ground. But the size of the largest crystal grown in space from MnFe protein was not larger than that on the ground. The difference in crystal growth in space between CrFe protein and MnFe protein could be resulted from the crystallization method, rather than the kind of protein.展开更多
Under a suitable condition of crystallization, dark brown rhombohedron crystals (the lengths of the longest two diagonals were 0.25 and 0.12 mm, respectively) could be obtained from nitrogenase CrFe protein purified f...Under a suitable condition of crystallization, dark brown rhombohedron crystals (the lengths of the longest two diagonals were 0.25 and 0.12 mm, respectively) could be obtained from nitrogenase CrFe protein purified from a mutant UW3 of Azotobacter vinelandii Lipmann grown in Cr-containing but NH3-free Medium, The possibility of crystallization, as well as the. number, size and quality of crystals obviously depended on the concentrations of PEG 8000, MgCl2, NaCl, Tris and Hepes buffer, and methods of crystallization. The optimum concentrations of the chemicals for crystallization of CrFe protein were slightly different from those for crystallization of MnFe protein from UW3 grown in Mn and DeltanifZ MoFe protein from a nifZ deleted strain of A. vinelandii. The crystal seemed to be formed from CrFe protein.展开更多
A mutant UW 3, which is unable to fix N 2 in the presence of Mo (Nif -) but undergo phenotypic reversal to Nif + under Mo deficiency, was able to grow in Mo- and NH 3-deficient medium containing Mn, and the growt...A mutant UW 3, which is unable to fix N 2 in the presence of Mo (Nif -) but undergo phenotypic reversal to Nif + under Mo deficiency, was able to grow in Mo- and NH 3-deficient medium containing Mn, and the growth was accelerated by Mn at low concentration. A partly purified nitrogenase component Ⅰ protein separated from UW 3 grown in the Mn-containing medium was shown to contain Fe and Mn atoms (ratio of Fe/Mo/Mn: 10.41/0.19/1.00) with C 2H 2- and H +-reducing activity which almost equal to half of that of MoFe protein purified from wild-type mutant of Azotobacter vinelandii Lipmann. This protein was obviously different from MoFe protein in both absorption spectrum and circular dichroism, and the molecular weight of subunits in Mn-containing protein was close to that of α subunit in MoFe protein. The preliminary results indicated that the protein containing Mn might be a nitrogenase component Ⅰ protein.展开更多
A mutant UW 3, which is unable to fix N 2 in the presence of Mo (Nif -) but can undergo phenotypic reversal to Nif + under Mo_deficient conditions, was able to grow in Cr_containing but Mo_ and NH 3_deficient...A mutant UW 3, which is unable to fix N 2 in the presence of Mo (Nif -) but can undergo phenotypic reversal to Nif + under Mo_deficient conditions, was able to grow in Cr_containing but Mo_ and NH 3_deficient medium. A partly purified nitrogenase component Ⅰ protein obtained from UW 3 grown on the Cr_containing medium was shown to contain Fe and Cr (atom ratio of Fe to Cr and Mo to Cr: 11.60 and 0.41) and to have 70% of the C 2H 2_ and H +_reduction activity of MoFe protein from the wild_type strain of Azotobacter vinelandii Lipmann. The Cr_containing protein was different in subunit composition from that of MnFe protein purified from the mutant strain grown in the presence of Mn, but similar to that of MoFe protein, that is, it was a tetramer composed of two different subunits (α 2β 2). The preliminary results indicated that the Cr_containing protein might be a nitrogenase component Ⅰ protein.展开更多
基金supported by the State Key Basic Research and Development Plan of China(Grant No.001CB1089-06)the National Science Foundation for Distinguished Young Scholars of China(Grant No.20403024)the National Natural Science Foundation of China(Grant No.30270296).
文摘Under a suitable condition of crystallization, dark brown short rhombohedron crystals could be obtained from nitrogenase MnFe protein purified from a mutant UW3 of Azotobacter vinelandii Lipmann grown in Mn-containing but Mo- and NH3-free medium. The possibility of crystallization, and number, size and quality of crystals were obviously dependent on concentrations of NaCl, MgCl2, PEG 8000,Tris and Hepes buffer and on methods for crystallization. PEG concentration affected on the shape of the crystals. The optimal, concentrations of the chemicals for crystallization of MnFe protein were slightly different from those for crystallization of Delta nifZ MoFe protein from a nifZ deleted strain of Azotobacter vinelandii. SDS-PAGE showed that the protein from the dissolved crystals was almost the same as MnFe protein before crystallization, indicating that the crystal was formed from MnFe protein.
文摘By using the liquid/liquid diffusion method at a suitable crystallization conditions, large single and dark brown crystals (the sides of the largest crystals were 0.20 mm x 0.20 mm x 0.07 min and 0.18 mm x 0.18 mm x 0.05 mm, respectively) could be obtained from the solutions of nitrogenase CrFe protein and MnFe protein purified from a mutant UW3 of Azotobacter vinelandii Lipmarm grown in Cr- or Mn-containing but NH3-free medium. The time of crystal formation, as well as the number, size, shape and quality of crystals obviously depended on the concentrations of PEG, MgCl2 and NaCl. The liquid/liquid diffusion method seems to benefit CrFe protein and MnFe protein for the growth of large single crystals for X-ray diffraction analysis.
文摘Nitrogenase CrFe protein and MnFe protein were purified from a mutant strain UW3 of Azotobacter vinelandii Lipmann grown on a medium containing Cr and Mn, respectively. In order to meet the requirement for crystal growth Of O-2-susceptible proteins including nitrogenase in space, crystallization conditions were optimized for the proteins using a simple and suitable device, as a replacement for the cumbersome anaerobic box (dry box), for anaerobic addition of the protein samples. In all used precipitant and protein solutions added in the simplified plexi glass box, CrFe protein and MnFe protein could be crystallized on the spacecraft in one week by the liquid/liquid diffusion method and vapor diffusion by the sitting drop method, respectively. All formed crystals were single on the spacecraft, but under the same condition twin crystals appeared on the ground. The size of the largest crystal grown in space from CrFe protein was 2-fold larger than that on the ground. But the size of the largest crystal grown in space from MnFe protein was not larger than that on the ground. The difference in crystal growth in space between CrFe protein and MnFe protein could be resulted from the crystallization method, rather than the kind of protein.
文摘Under a suitable condition of crystallization, dark brown rhombohedron crystals (the lengths of the longest two diagonals were 0.25 and 0.12 mm, respectively) could be obtained from nitrogenase CrFe protein purified from a mutant UW3 of Azotobacter vinelandii Lipmann grown in Cr-containing but NH3-free Medium, The possibility of crystallization, as well as the. number, size and quality of crystals obviously depended on the concentrations of PEG 8000, MgCl2, NaCl, Tris and Hepes buffer, and methods of crystallization. The optimum concentrations of the chemicals for crystallization of CrFe protein were slightly different from those for crystallization of MnFe protein from UW3 grown in Mn and DeltanifZ MoFe protein from a nifZ deleted strain of A. vinelandii. The crystal seemed to be formed from CrFe protein.
文摘A mutant UW 3, which is unable to fix N 2 in the presence of Mo (Nif -) but undergo phenotypic reversal to Nif + under Mo deficiency, was able to grow in Mo- and NH 3-deficient medium containing Mn, and the growth was accelerated by Mn at low concentration. A partly purified nitrogenase component Ⅰ protein separated from UW 3 grown in the Mn-containing medium was shown to contain Fe and Mn atoms (ratio of Fe/Mo/Mn: 10.41/0.19/1.00) with C 2H 2- and H +-reducing activity which almost equal to half of that of MoFe protein purified from wild-type mutant of Azotobacter vinelandii Lipmann. This protein was obviously different from MoFe protein in both absorption spectrum and circular dichroism, and the molecular weight of subunits in Mn-containing protein was close to that of α subunit in MoFe protein. The preliminary results indicated that the protein containing Mn might be a nitrogenase component Ⅰ protein.
文摘A mutant UW 3, which is unable to fix N 2 in the presence of Mo (Nif -) but can undergo phenotypic reversal to Nif + under Mo_deficient conditions, was able to grow in Cr_containing but Mo_ and NH 3_deficient medium. A partly purified nitrogenase component Ⅰ protein obtained from UW 3 grown on the Cr_containing medium was shown to contain Fe and Cr (atom ratio of Fe to Cr and Mo to Cr: 11.60 and 0.41) and to have 70% of the C 2H 2_ and H +_reduction activity of MoFe protein from the wild_type strain of Azotobacter vinelandii Lipmann. The Cr_containing protein was different in subunit composition from that of MnFe protein purified from the mutant strain grown in the presence of Mn, but similar to that of MoFe protein, that is, it was a tetramer composed of two different subunits (α 2β 2). The preliminary results indicated that the Cr_containing protein might be a nitrogenase component Ⅰ protein.
