1 Institut für Organische Chemie und Biochemie, Albertstrasse 21,D-79104 Freiburg im Breisgau and 2 Institut für Organische Chemie, Lehrstuhl Biochemie der Universität, Richard-Willstätter-Allee, D-76128 Karlsruhe, Germany
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Abstract |
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Keywords: disulfide engineering/Ellman's reaction/histidine ammonia-lyase/unspecific aggregation/X-ray crystallography
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Introduction |
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Materials and methods |
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The histidase gene from P.putida was expressed in Escherichia coli BL21(DE3-pT7-7 hutH) cells following Langer et al. (1994). Purification was performed by ammonium sulfate fractionation (2.13.0 M), gel permeation chromatography (Superdex 200, 26/60) and ion-exchange chromatography (Q-Sepharose FF) using throughout a buffer of 50 mM KH2PO4, pH 7.2, and 1 mM MgSO4. Typically 120180 mg of pure histidase were obtained from a 1 l culture. Crystallization conditions were initially found by screening in hanging drops (Jancarik and Kim, 1991). Crystallization occurred only at acidic pH and was accompanied by protein precipitation (Teo et al., 1998
). The elaborated droplet composition was a 1:1 mixture of a 12 mg/ml protein solution with the reservoir buffer (1.6 M sodium potassium phosphate, 0.1 M HEPES, 1.5 mM EDTA, 3% dioxane, pH 3.85).
Protein characterization
The protein melting temperature was determined by monitoring the tryptophan fluorescence (Hitachi Perkin-Elmer MPF-2A, ex = 282 nm,
em = 334 nm) during thermal denaturation (rate 1°C/min). The oligomeric state was determined by gel permeation chromatography (Superdex 200, 16/60). Thiol content was determined using a modification of the method of Ellman (1959). After complete denaturation and reduction for 30 min at 75°C (8 M urea, 10 mM DTT, 150 mM NaCl, 1 mM EDTA, 20 mM K2HPO4, pH 8.0), excess DTT was removed by multiple ultrafiltration (Centricon 30, Millipore). The protein concentration was determined spectrophotometrically and the reaction of 5,5'-dithiobis-2-nitrobenzoate with sulfhydryl groups was monitored at
= 412 nm (
TNB = 13 600 M1 cm1) and scaled on a molar basis. Blinds were subtracted; all handling was carried out in the dark.
Mutagenesis and preliminary X-ray analysis
The gene of histidase was subcloned into M13mp18/19 in two fragments using an internal SalI restriction site (Langer et al., 1994). Site directed mutagenesis was performed using the Eckstein method (Sayers et al., 1988
) with the primer 5'-GCACAAGAACGCCGACAAGGTCC-3'. The new crystallization conditions for the mutant Cys273
Ala were a 1:1 mixture of 14 mg/ml protein with the reservoir [2.0 M (NH4)2SO4, 1% glycerol, 2% PEG-400, 0.1 M HEPES, pH 8.1] as sitting drops.
X-ray diffraction data were collected at room temperature on a 30 cm MAR-Research imaging plate and processed with the program MOSFLM (CCP4, 1994). Data from mutant Cys273
Ala were collected using synchrotron radiation at beamline X11 (
= 0.907 Å, EMBL Outstation at DESY, Hamburg). Self-rotation functions were calculated with the program POLARRFN (CCP4, 1994
) including data in the range between 10 and 5 Å. A heavy atom derivative was produced by adding 20 mM methylmercury acetate to the crystals in the droplet. The derivative data were collected with Cu K
radiation from a rotating anode source (RU200B, Rigaku).
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Results and discussion |
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Consequently, we produced mutant Cys273Ala without the `exposed' cysteine and checked it for its thiol content using a modified version of the method of Ellman (1959). The removal of one of the cysteines could be confirmed reproducibly (Figure 2B
). Note that the exact values of the Ellman reaction were obtained only after about 7 h. In gel permeation chromatography (Figure 2A
), the protein ran in a single peak, indicating that there are no higher aggregates. The melting temperature was not affected by the mutation. The specific activity (Langer et al., 1994
) dropped to 18% of the wild-type activity (31.4 U/mg) indicating that Cys273 influences catalysis.
Mutant Cys273Ala yielded readily rectangular crystals with average dimensions of 300x300x800 µm3 that diffracted isotropically to 1.8 Å resolution. Similar crystallization improvements in conjunction with protein homogenization have been reported for HIV-1 integrase by Jenkins et al. (1995), who had to produce 28 mutants, however, before they succeeded. The crystals belong to space group P21; the unit cell parameters are given in Table I
. The unit cell contains most likely one histidase tetramer per asymmetric unit with a solvent content of 57%. A self-rotation function yielded strong peaks in the section
= 180° at (
,
)-values of (0.9°, 0°) and (90.9°, 0°). These amounted to about 99% of those at the crystallographic twofold screw axis at (
,
) = (90°, 90°). This indicates that the tetramer has D2 symmetry and that one of its twofold axes runs nearly parallel to the crystallographic twofold screw axis. Diffraction data for a methylmercury acetate derivative were collected (Table I
) and four major heavy atom sites were located in difference-Patterson maps using the program SHELXS-96 (Sheldrick et al., 1993
). Further minor sites were identified in difference-Fourier maps. Given the extensive non-crystallographic symmetry, we expect that a single derivative will suffice for phasing.
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Cysteine residues at the surface of oligomeric proteins can cause unspecific aggregations and thereby complicate all handling and virtually inhibit the formation of highly ordered crystals. It has been shown that the identification of such residues by predicting solvent accessibility and their removal by site-directed mutagenesis is a powerful tool for improving protein homogeneity and crystallization.
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Acknowledgments |
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Notes |
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References |
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Received August 12, 1998; revised October 14, 1998; accepted October 22, 1998.