2 Results and Discussion

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2 ZHAO Ying et al: Crystallization of Nitrogenase MoFe Protein (NifB Av1) from a nifb Mutated Strain UW45 of Azotobacter vinelandii 821 In order to prevent the NifB Av1 from a damage resulted possibly from heat treatment at 52, the crude extract of UW was not heated. The crude extract ( mg) was applied to DE52 column, followed by developing with NaCl gradient ( mol/l) in 25 mmol/l Tris buffer (ph 7.4) containing 0.3 mg/ml Na 2 S 2 O 4 (DT). After chromatography again on the second DE52 columns, the partially purified NifB Av1 (1 922 mg) and NifB - Av2 (1 017 mg) were obtained. The NifB Av1 was further purified on Sephacryl S-300 column (2.5 cm 72.0 cm) and concentrated on a small DE52 column. Extraction of FeMoco from Av1 were performed according to the methods of Shah et al (1997) and Huang et al (1999). Determination of protein concentration and substratereducing activity, and SDS-PAGE were carried out according to Shah and Brill (1977) and Paustian et al (1990). Crystallization of NifB Av1 was performed according to Drenth et al (1991) and Huang et al (1999). Unless described elsewhere, the crystals were grown by vapor diffusion using the hanging drop method for 7 d at 20. All of the operations were done under Ar. All of the solutions used were rigorously degassed and filled with Ar. The concentrations of DT, Tris, and NaCl in the NifB Av1 solution were 0.3 g/l, 25 mmol/l and 250 mmol/l. The concentration of DT in precipitant solution was 12.0 mmol/l. 2 Results and Discussion 2.1 Identification of NifB Av1 The NifB Av1 (628 mg) from the last DE52 column was basically homogeneous as determined by Coomassie staining of SDS gels, and both the positions and amount ratio of two main bands were almost the same as those of and subunits from Av1 (Fig. 1). And a mixture of NifB Av1 with Av1 on the gel showed that the bands for the two proteins Fig.1. SDS-PAGE (6.0%) of NifB Av1. 1 and 5, NifB Av1 (5.1 and 2.5 µg); 2 and 4, mixture of NifB Av1 (2.5 µg) with Av1 (2.5 and 7.5 µg); 6 and 8, mixture of NifB Av1 (2.5 µg) with Av1 (2.5 and 5.0 µg); 3 and 7, Av1 (7.5 and 5.0 µg). well overlapped each other, respectively. This indicates that kind and composition of the NifB Av1 are almost the same as those of Av1. As reported previously in other laboratories (Shah and Brill, 1977; Christiansen et al, 1998; Huang et al, 1999), the crude extract of UW hardly exhibited any C 2 H 2 -reducing activity, but was able to be efficiently complemented with Av1 (Table 1); and either the crude extract or NifB Av1 could be significantly reactivated by FeMoco extracted from Av1 with NMF (Table 2). The activities of the Av1 complemented with the partially purified NifB Av2 were almost the same as that of the same batch Av1 complemented with Av2 (Huang et al, 1999). This indicates that NifB Av2 is similar to Av2 in the activity and FeMoco can activate the inactive NifB Av1 in vitro. 2.2 Crystallization of NifB Av1 Besides the property, purity and concentration of a protein, there are many factors affecting crystallization of the protein, such as the kind, concentration and ratios of all components in the precipitant solution, method of crystallization, ph of the solution, temperature and technical bias (Schindelin et al, 1997; McPherson, 1983; Drenth et al, 1991; Kim and Rees, 1992; Huang et al, 1999). Therefore, it is necessary to optimize the above conditions in order to obtain crystals of NifB Av1 suitable for X-ray Table 1 Complement of Av1 with the NifB crude extract and partially purified NifB Av2 Sample Total activity Specific activity of Av1 (4) (nmol C 2 H 4 /min) (nmol C 2 H 4.min 1.mg 1 pro.) NifB CE (1) 0 NifB CE (1) + Av1 (2) NifB Av2 (3) + Av1 (2) (1), UW45 crude extract (4.30 mg); (2), Av1 (0.075 mg); (3), partially purified NifB Av2 (0.62 mg); (4), specific activity of Av1 calculated after subtraction of activity induced by NifB - Av2 alone. Table 2 Activation of NifB Av1 by FeMoco Sample Total activity Specific activity (nmol H 2 /min) NifB Av1 FeMoco (nmol H 2.min 1.mg 1) (nmol H 2.min 1.nmol 1) NifB CE (1) 0 0 NifB CE (1) FeMoco (2) FeMoco (2) (4) 847 (4) 310 (4) NifB Av1 (3) (1), UW45 crude extract (4.300 mg); (2), NifB CE (4.300 mg) was incubated with FeMoco extract (0.340 ng Mo atoms) from Av1 for 70 min at 30 ; (3), NifB Av1 (0.042 mg) was incubated with FeMoco extract (0.115 ng Mo atoms) from Av1 for 70 min at 30 ; (4), complemented with NifB Av2 (0.620 mg), the activity was calculated after subtraction of activity induced by NifB Av2 incubated with NMF.

3 diffraction analysis Effect of precipitant solution on the crystallization Hepes buffer Within the Hepes concentrations shown in Tables 3 and 4 (ph 8.2), the possibility of nucleation of NifB Av1 in the first week increased obviously with the increase of Hepes concentration. In the precipitant solution #1 containing 51 mmol/l Hepes shown in Table 3, a crystal formed in the second week could gradually grow. After incubation for four months, the longest two sizes and diagonals of the crystal were 0.10 mm 0.10 mm and 0.17 mm 0.09 mm, respectively (Fig.2). It is indicated that the concentration of the buffer in the precipitant solution could affect the electric charge and conformation of protein, resulting in a change of the possibility and rate of nucleation. PEG 8000 Under the conditions shown in Table 4, the concentration of PEG 8000 had also a significant effect on the crystal number and size of NifB Av1. In comparison with nifz Av1 (Huang et al, 1999), the optimal PEG concentration for crystallization of NifB Av1 at which the crystals were bigger in size and fewer in number was slightly higher. The protein crystals could not be formed or a large amount of small crystals were formed when the PEG concentration was out of this range. PEG could absorb water from the environment around the protein molecules, resulting in decreasing protein dissolution (Drenth et al, 1991). When the PEG concentration was lower than a critical value, its crystals could not be formed. When the concentration was too high, the acceleration or disturbance of crystal formation was resulted from the acceleration of loss of water from the protein, leading to the following possibilities that the crystalline nuclei could not be formed or were too much. Only under the optimal concentration of PEG, the rate of losing water could benefit a formation of a few nuclei and their slow growth, leading to formation of the single and big crystals of good quality. MgCl 2 and NaCl The concentration of MgCl 2 could significantly affect the number and size of crystals from the mutant protein (Table 5). In comparison with nifz Av1 (Huang et al, 1999), the optimal concentration (about 250 Fig.2. Crystal of NifB Av1 observed in a hanging drop two weeks later ( 250). The protein and crystalline condition are the same as those of crystalline solution #1 containing 51 mmol/l Hepes in Table 3. Table 3 Effect of Hepes concentration on crystallization of NifB Av1 by vapor diffusion method in the hanging drop Hepes concentration (mmol/l) Solution (1) Crystal number Crystal size Crystal number Crystal size Crystal number Crystal size Crystal number Crystal size # Small #2 0 1 Small (2) 0 5 Small #3 0 2 Small (2) 13 Small 8 Middle # Small 3 Small (1), the concentrations of NifB - Av1 and MgCl 2 were 8.48 mg/ml and mmol/l, respectively, and the concentrations of PEG 8000 and NaCl in solution #1, #2, #3 and #4 were 5.50% and mmol/l, 5.00% and mmol/l, 4.75% and mmol/l, and 4.30% and mmol/l, respectively; (2), crystal of bad quality (sides of crystals were not sharp). Table 4 Effect of the concentration of PEG 8000 on crystallization of NifB Av1 by vapor diffusion method in the hanging drop Exp.#1 Exp.#2 (1) PEG % Crystal number Crystal size Crystal number Crystal size Small Small Middle 5 Middle Small 5 Small Small 5 Small Small 20 Small (1), concentrations of Hepes in Exp.#1 and #2 are and mmol/l (ph 8.2), respectively; and concentrations of NifB Av1, NaCl and MgCl 2 in Exp. #1 and Exp. #2 are 8.48 mg/ml, and mmol/l, respectively.

4 ZHAO Ying et al: Crystallization of Nitrogenase MoFe Protein (NifB Av1) from a nifb Mutated Strain UW45 of Azotobacter vinelandii 823 Table 5 Effect of MgCl 2 concentration on crystallization of NifB Av1 by vapor diffusion method in the hanging drop (1) MgCl 2 Drop #1 (2) Drop #2 (2) (mmol/l) Crystal number Crystal size Crystal number Crystal size Middle 3 Small Middle 20 Small Middle 20 Small Small 1 Small Small (1), concentrations of protein, PEG 8000, Hepes and NaCl were 8.48 mg/ml, 4.30%, and mmol/l, respectively; (2), two drops containing the same solutions on one slide. Fig. 3 Crystals of NifB Av1 observed in a small glass tube one week later ( 150). Fifteen µl of the solution #4 containing 51 mmol/l Hepes and 15 µl of the protein shown in Table 3 were added to a glass tube (internal diameter was mm). mmol/l) for its crystallization was slightly lower. Like PEG, both MgCl 2 and NaCl could not only absorb water molecules from the environment around the protein, but also release cations (Mg 2+ and Na +, respectively), leading to an effect of the crystalline process by a change of the electric charges on proteins. In general, the effect increases with the increase of the ion concentration and square charge number of cations (McPherson, 1983; Tal et al, 1991; Huang et al, 1999). A body of experiments in our laboratory exhibited that the effect of MgCl 2 was obviously higher than that of NaCl Effect of technical bias on the crystallization Under the same conditions for crystallization by the vapor diffusion using the hanging drop method, the crystals formed in two drops sited on one slide were obviously different from each other in both the number and size, although their crystal qualities were not easy to be differentiated (Table 5). Two technical biases exhibited: (1) the error of the very small sample volume which led to a significant change in the concentration; and (2) it was possible to let the protein be differently mixed with the precipitant solution when the slide was turned over and put against the reservoir. Therefore, it could make the water molecules lose with different rates from the environment around protein molecules, leading to a different process of crystallization Effect of crystallization method on the crystallization Under the same conditions including the same protein and precipitant solutions, the results obtained by the vapor diffusion was different from those by the liquid/ liquid diffusion, and sometimes the differences were very obvious. In one week, the crystals were not formed by the vapor diffusion, but more than 10 middle crystals were observed by the liquid/liquid diffusion using the same protein and precipitant solution #4 containing 51 mmol/l Hepes shown in Table 3 (Fig.3). In comparison with the crystals shown in Fig.2, the crystals exhibited thicker. Both the rate and route for diffusion of water molecules from the environment around protein molecules could be different when the protein was crystallized by the different diffusion method, resulting in different processes for crystallization. The observation by the optical differential microscope showed that the crystals formed from the solution containing NifB - Av1 could be crystals formed from a protein, not from compounds in the precipitant solution. The dark brown color of crystals from both Av1 and nifz Av1 were related to the large amount of iron in the proteins (Huang et al, 1999). Since NifB Av1 still has P-cluster of which iron content is about a half of Av1, the color of the crystal is reasonable to be brown. Since the NifB Av1 used for crystallization was basically homogeneous judged by SDS-PAGE, it was almost impossible that the crystals were from other contamination proteins. Finally, like nifz Av1 (Huang et al, 1999), NifB Av1 could be crystallized only under an anaerobic condition and the formed crystals disappeared after an exposure to air. Besides the protein, only DT can be oxidized in the crystalline system while DT crystal can not be brown in color. Therefore, it seems to be reasonable to suppose that the formed crystals were those of the protein. However, the final conclusion could be made only after X-ray diffraction analysis of the crystals. The research work still goes on the way. Acknowledgements: The authors wish to thank Mr. ZHANG Hua-Feng for his help in some experiments.

5 References: Allen R M, Chartterjee R, Ludden P W, Shah V K Incorporation of iron and sulfur from NifB cofactor into the iron-molybdenum cofactor of nitrogenase. J Biol Chem, 270: Christiansen J, Goodwin P J, Lanzilotta W N, Seefeldt L C, Dean D R Catalytic and biophysical properties of a Nitrogenase Apo-MoFe protein produced by a nifbdeleted mutant of Azotobacter vinelandii. Biochemistry, 37: Drenth J, Helliwell J R, Little W The Crystal Growth of Biological Materials. Walter H U, Ge P-W, Wang J-T, Xu Z-Y, Li G-D, Hu W-R, Zhang X-M, Zhong X-R, Tang L-S, Gu B-Y, Xu S-C, Pan H-R, Huo C-R trans. Fluid Science and Material Science in Space. Beijing: Chinese Science and Technology Press (in Chinese) Huang J-F Wang D-Y, Dong Z-G, Wang Z-P, Li J-G Relationship between nifz and the synthesis of P-cluster in nitrogenase MoFe protein of Azotobacter vinelandii. Acta Bot Sin, 41: (in Chinese with English abstract) Kim J, Rees D C Structure models for the metal centers in the nitrogenase molybdenum-iron protein. Science, 257: Shah V K, Brill W G Isolation of an iron-molybdenum cofactor from nitrogenase. Proc Natl Acad Sci USA, 74: Paustian T D, Shah V K, Rorbert G P Apodinitrogenase: Purification, association with a 20-kilodalton protein, and activation by the iron-molybdenum cofactor in the absence of dinitrogenase reductase. Biochemistry, 29: McPherson A J Group of Crystalline Growth, Institute of Biophysics, the Chinese Academy of Sciences trans. The growth and preliminary investigation of protein and nuclei acid crystals for X-ray diffraction analysis. Glick D. Methods of Biochemical Analysis. Vol. l23. Beijing: Chinese Science and Technology Press (in Chinese) Rangaraj P, Ruttimann-Johnson C, Shah V K, Ludden P W Accumulation of 55 Fe-labeled precursors of the iron-molybdenum cofactor of nitrogenase on NifH and NifX of Azotobacter vinelandii. J Biol Chem, 276: Schindelin H, Kisker C, Schiessman J L, Howard J B, Rees D C Structure of ADP AlF4-stabilized nitrogenase complex and its implications for signal transduction. Nature, 387: Tal S, Chun T W, Gavini N, Burgess B K The nifb (or nife) FeMo cofactor-deficient MoFe protein is different from the nifh protein. J Biol Chem, 266: (Managing editor: HE Ping) ( 001CB ); ;