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Desulforedoxin: Preliminary X-ray diffraction study of a new iron-containing protein
J. Mol. Biol. (1980) 144, 593-594
Desulforedoxin: Preliminary X-ray Diffraction of a New Iron-containing Protein
Study
Desulforedoxin, a two-iron, two-chain protein from the bacterium Desulfovibrio gigas, has been crystallized. X-ray diffraction photos show symmetry consistent with space group P3i21 or the enantiomorph P3,21. Cell parameters are a = b = 42.28 A, c = 72.46 A. Crystals develop sufficiently well for an X-ray diffraction analysis of at least 2.0 L\ resolution to be performed.
Desulforedoxin is a new type of non-heme iron protein isolated from Desulfovibrio gigas (Moura et al., 1977). Desulforedoxin bears some resemblance to rubredoxin in size, iron to cysteine content, spectral characteristics, and oxidation-reduction potential (Moura et al., 1978; Bruschi et al., 1979). Subsequent studies of this protein have revealed that the molecule is composed of two iron atoms and two identical polypeptide chains of 36 amino acid residues each, for a total of 72 and a molecular weight of 7740 (Bruschi et al., 1979). Each chain contains four cysteine residues, two of these being arranged in the N-terminal region of a chain in similar fashion to that of rubredoxin and the other two cysteine residues of the same chain adjoining each other at positions 28 and 29 (Bruschi et al., 1979). Shown below is the amino acid sequence of one of the desulforedoxin polypeptide chains : Ala-Asn-Glu-Gly-Asp-Val-Tyr-Lys-Cys-G1u-Leu-Cys-Gly-Gln-Val-Val-Lys-ValLeu-Glu-Glu-Gly-Gly-Gly-Thr-Leu-Val-Cys-Cys-Gly-Glu-Asp-Met-Val-Lys-Gln. The polypeptide
chain of rubredoxin
from D. gigas is also shown for comparison :
Met-Asp-Ile-Tyr-Val-Cys-Thr-Val-Cys-Gly-Tyr-Glu-Tyr-Asp-Pro-Ala-Lys-Gly.~sp-Pro-Asp-Ser-Gly-Ile-Lys-Pro-Gly-Thr-Lys-Phe-Glu-Asp-Leu-Pro-Asp-AspTrp-Ala-Cys-Pro-Val-Cys-Gly-Ala-Ser-Lys-Asp-Ala-Phe-Glu-Lys-Gln.
Desulforedoxin was isolated and purified as previously reported (Moura et al., 1977). Although multitudes of tiny crystals could be grown from solution by the addition of ammonium sulfate at pH 7.6, it was found necessary to explore crystallization conditions to attain crystals sufficiently large for X-ray diffraction studies. Good quality crystals develop from a 65 to 1.0% (w/v) protein solution containing 03 M-NaCl, 0.1 M-Tris-maleate (pH 5.0 to 5.4), 0.8 M-(NH&SO, by vapor equilibration (hanging drop technique) against buffered 2.7 to 3.9 M(NHJ2S0, solution where the range of (NH&SO4 concentration depends upon the pH. The best crystals seem to develop at 23°C rather than at 4°C. 593 0022-2S36/80/360593-02
$02.00/O
r) 1980 Academic Press Inc. (London) Ltd.
594
L. C. SIEKER
E7’ AL.
X-ray diffraction studies of these crystals show symmetry consistent with space group P3,21 or its enantiomorph P3,21. Cell parameters determined are: a = b = 42.28 ( + 0.02) -4, c = 7246 ( 5 0.03) ii and y = 120”. The cell volume is calculated to be 112,190 A3 and. if one assumes 1 molecule of M, 7740 per, asymmetric unit, the crystal specific volume is 242 If\3/dalton. This value falls in the normal range for protein crystals (Matthews, 1968). Friedel related reflection intensities have been measured to 2.0 .k resolution from a native crystal, and an attempt is being made to locate the iron positions using the anomalous scattering information from the two iron atoms in a Bijvoet difference Patterson map (Kraut et al., 1968). Experiments to find useful heavy-atom derivatives are in progress.
L. C. SIEKER L. H. JENSEN
Department of Biological Structure University of Washington Seattle, Washington 98195, U.S.A.
M. BRUSCHI
Laboratoire de Chimie Bacterienne CNRS, 13274 Marseille Cedex 2, France Centro de Quimica Estructural I.S.T.. Lisboa I, Portugal
de Universidade
J. LEGALL de Lisboa
I. MOURA A. 1’. XAVIER
Received 7 July 1980 REFERENCES Bruschi, M., Moura, I., LeGall, J., Xavier, A. V. & Sieker, L. C. (1979). Biochem. Biophys. Res. Commun. 90, 596-605. Kraut, J., Strahs, G. & Freer, S. T. (1968). In Structural Chemistry and Molecular Biology (Rich, A. & Davidson, N., eds), pp. 55-64, W. H. Freeman and Co., San Francisco and London. Matthews, B. W. (1968). J. Mol. Biol. 33, 491-497. Moura, I., Bruschi, M., LeGall, J., Moura, J. J. G. & Xavier, A. V. (1977). Hiochem. Biophys. Res. Commun. 75, 103771043. Moura, I., Xavier, A. V., Cammack, R., Bruschi, ,1/I.$ LeGall, J. (1978). Biochim. Hiophys. Acta, 533, 156-162.
Desulforedoxin: Preliminary X-ray Diffraction of a New Iron-containing Protein
Study
Desulforedoxin, a two-iron, two-chain protein from the bacterium Desulfovibrio gigas, has been crystallized. X-ray diffraction photos show symmetry consistent with space group P3i21 or the enantiomorph P3,21. Cell parameters are a = b = 42.28 A, c = 72.46 A. Crystals develop sufficiently well for an X-ray diffraction analysis of at least 2.0 L\ resolution to be performed.
Desulforedoxin is a new type of non-heme iron protein isolated from Desulfovibrio gigas (Moura et al., 1977). Desulforedoxin bears some resemblance to rubredoxin in size, iron to cysteine content, spectral characteristics, and oxidation-reduction potential (Moura et al., 1978; Bruschi et al., 1979). Subsequent studies of this protein have revealed that the molecule is composed of two iron atoms and two identical polypeptide chains of 36 amino acid residues each, for a total of 72 and a molecular weight of 7740 (Bruschi et al., 1979). Each chain contains four cysteine residues, two of these being arranged in the N-terminal region of a chain in similar fashion to that of rubredoxin and the other two cysteine residues of the same chain adjoining each other at positions 28 and 29 (Bruschi et al., 1979). Shown below is the amino acid sequence of one of the desulforedoxin polypeptide chains : Ala-Asn-Glu-Gly-Asp-Val-Tyr-Lys-Cys-G1u-Leu-Cys-Gly-Gln-Val-Val-Lys-ValLeu-Glu-Glu-Gly-Gly-Gly-Thr-Leu-Val-Cys-Cys-Gly-Glu-Asp-Met-Val-Lys-Gln. The polypeptide
chain of rubredoxin
from D. gigas is also shown for comparison :
Met-Asp-Ile-Tyr-Val-Cys-Thr-Val-Cys-Gly-Tyr-Glu-Tyr-Asp-Pro-Ala-Lys-Gly.~sp-Pro-Asp-Ser-Gly-Ile-Lys-Pro-Gly-Thr-Lys-Phe-Glu-Asp-Leu-Pro-Asp-AspTrp-Ala-Cys-Pro-Val-Cys-Gly-Ala-Ser-Lys-Asp-Ala-Phe-Glu-Lys-Gln.
Desulforedoxin was isolated and purified as previously reported (Moura et al., 1977). Although multitudes of tiny crystals could be grown from solution by the addition of ammonium sulfate at pH 7.6, it was found necessary to explore crystallization conditions to attain crystals sufficiently large for X-ray diffraction studies. Good quality crystals develop from a 65 to 1.0% (w/v) protein solution containing 03 M-NaCl, 0.1 M-Tris-maleate (pH 5.0 to 5.4), 0.8 M-(NH&SO, by vapor equilibration (hanging drop technique) against buffered 2.7 to 3.9 M(NHJ2S0, solution where the range of (NH&SO4 concentration depends upon the pH. The best crystals seem to develop at 23°C rather than at 4°C. 593 0022-2S36/80/360593-02
$02.00/O
r) 1980 Academic Press Inc. (London) Ltd.
594
L. C. SIEKER
E7’ AL.
X-ray diffraction studies of these crystals show symmetry consistent with space group P3,21 or its enantiomorph P3,21. Cell parameters determined are: a = b = 42.28 ( + 0.02) -4, c = 7246 ( 5 0.03) ii and y = 120”. The cell volume is calculated to be 112,190 A3 and. if one assumes 1 molecule of M, 7740 per, asymmetric unit, the crystal specific volume is 242 If\3/dalton. This value falls in the normal range for protein crystals (Matthews, 1968). Friedel related reflection intensities have been measured to 2.0 .k resolution from a native crystal, and an attempt is being made to locate the iron positions using the anomalous scattering information from the two iron atoms in a Bijvoet difference Patterson map (Kraut et al., 1968). Experiments to find useful heavy-atom derivatives are in progress.
L. C. SIEKER L. H. JENSEN
Department of Biological Structure University of Washington Seattle, Washington 98195, U.S.A.
M. BRUSCHI
Laboratoire de Chimie Bacterienne CNRS, 13274 Marseille Cedex 2, France Centro de Quimica Estructural I.S.T.. Lisboa I, Portugal
de Universidade
J. LEGALL de Lisboa
I. MOURA A. 1’. XAVIER
Received 7 July 1980 REFERENCES Bruschi, M., Moura, I., LeGall, J., Xavier, A. V. & Sieker, L. C. (1979). Biochem. Biophys. Res. Commun. 90, 596-605. Kraut, J., Strahs, G. & Freer, S. T. (1968). In Structural Chemistry and Molecular Biology (Rich, A. & Davidson, N., eds), pp. 55-64, W. H. Freeman and Co., San Francisco and London. Matthews, B. W. (1968). J. Mol. Biol. 33, 491-497. Moura, I., Bruschi, M., LeGall, J., Moura, J. J. G. & Xavier, A. V. (1977). Hiochem. Biophys. Res. Commun. 75, 103771043. Moura, I., Xavier, A. V., Cammack, R., Bruschi, ,1/I.$ LeGall, J. (1978). Biochim. Hiophys. Acta, 533, 156-162.