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Crystallization and preliminary diffraction data for iso-1-cytochrome c from yeast
,I. Mol. Biol. (1985) 185. 209-210
LETTERS TO THEEDITOR
Crystallization and Preliminary Diffraction Iso-1-Cytochrome c from Yeast
Data for
Deep red crystals of the electron transfer protein, iso- 1-cytochrome c from yeast cerevisiae), have been obtained from a 90% saturated solut)ion of (NH,),SO, containing 2 mg protein/ml, 0.1 M-sodium phosphate and adjusted to pH 6.7. The space group is P412,2 (or P4,2,2) with a = b = 36.4 A, c = 136.8 A and Z = 8. Crystals are stable for at least ten days in the X-ray beam and diffract to better than 2-O A resolution. Comparable and morphologically similar crystal forms of three iso-1-cytochrome c mutants at Phe87, a pivotal residue in the electron transport chain, have also been obtained. (Saccharomyces
Cytochrome c is a component of the respiratory electron transport chain of eukaryotic organisms, acting as an electron shuttle between multi-enzyme complexes embedded in the inner mitochondrial & membrane (Boyer et al., 1977; Rackovsky Goldstein, 1984). Although a component of mitochondrion, yeast cytochrome c is specified by a nuclear gene (Sherman et al., 1966) and synthesized in t’he cytoplasm (Clark-Walker & Linnane, 1967). Newly synthesized yeast iso-1-cytochrome c is modified by the covalent attachment of the heme group and trimethylation of Lys77 (Margoliash & Schejter, 1966; Sherman & Stewart, 1974; Delange et al., 1970). Yeast iso-1-cytochrome c (M, 12,700) consists of a single polypeptide chain of 108 amino acids (Smith et al., 1979; Lederer et al., 1972) covalently attached to a heme group. The heme ligands include two covalent thioether bridges from cysteine residues 19 and 22. Both Met85 and His23 provide additional ligands to the heme iron atom; they are on opposite sides of, and perpendicular to, the plane of the heme group. Extensive genetic and biochemical analyses related to the expression and function of yeast iso-1-cytochrome c have been carried out (Sherman & Stewart, 1971; Sherman et al., 1974). We now report the crystallization of the iso-lrytochrome c of yeast. The protein preparation used was obtained commercially (Sigma Co,) and its purity checked against standards obtained locally (G. Pielak. personal communication). Crystals were grown using the hanging drop vapour diffusion technique as follows. Yeast iso-1-cytochrome c was dissolved in a solution 75% saturated in (NH,),SO, to a final concentration of 2 mg/ml in the presence of reducing agent. This solution was buffered with 0.1 N-sodium phosphate and the pH adjusted to 6.7 with either NH,OH or sodium acetate. The protein solution was then filtered through a 0.22 pm HPLC filter (Micron Separation Inc.) and allowed to equilibrate against a reservoir solution containing 90% saturated (NH,)#O,. Crystals appeared within 3 to 14 days and generally grew to 0.75 mm x 0.4 mm x 0.4 mm in size. Larger crystals were commonly observed in those hanging drops 0032%2836/85/
170209-02
$03.00/0
which contained only a few nucleation sites. The resultant deep red crystals have the overall morphology of tetragonal bipyramids. X-ray diffraction precession photography indicates iso-lcytochrome c crystals are of the tetragonal space group P4,2,2 (or its enantiomorph P4,2,2) with a = b = 36.4 I%, c = 136.8 A and Z = 8. Crystals are stable in the X-ray beam for up to ten days and diffract to at least 2.0 A resolution. The volume per unit mass, V,, for these crystals is 1.78 A3/dalton (Matthews, 1968), indicating a solvent content of - 30%. These preliminary diffraction studies represent the first step in the elucidation of the full threedimensional structure of yeast iso-1-cytochrome c. The goal of these structural studies is to gain further insight into the electron transfer mechanism mediated by this protein and to clearly define the role of Phe87 in this process. Primary sequence comparisons show this residue is invariably found in all the cytochrome c sequences thus far studied and it has been proposed to play a pivotal role in the electron transfer process. In the tertiary structures of cytochrome c from other organisms (Takano & Dickerson, 1981), this residue is located at the entrance to the heme pocket and in part determines t’he dielectric environment of the heme group (Kassner, 1972, 1973). Furthermore, this phenylalanine appears to form part of the aromatic electron-transfer conduit between the heme groups of cytochrome c and cytochrome c peroxidase during the catalytic cycle of the latter protein (Poulos & Kraut, 1980). We have chosen to study the functional role of Phe87 in iso-l -cytochrome c, since Saccharomyces cerevisiae is amenable to site-directed mutagenesis techniques (Zoller & Smith, 1983) and therefore the mutation of Phe87 or any other residue of yeast cytochrome c to another amino acid residue can be accomplished easily. Three such mutations of Phe87, to a tyrosine, a serine and a glycine residue, have been produced and their functional properties characterized (Pielak et al., 1985). Preliminary crystallization trials with these mutated proteins indicate that crystals can be grown under the same
209
0 1985 Academic Press Inc. (London)
Ltd.
C. Sherwood and G. D. Brayer
210
conditions as those used for the native protein. Both native and mutant protein crystal forms have similar morphologies. The application of X-ray diffraction techniques to obtain the high resolution, three-dimensional structures of native and mutant forms of yeast iso-1-cytochrome c, in conjunction with solution biochemical analyses, holds the greatest promise for a more complete understanding of the phenomena of electron transport.
Clark-Walker, G. D. & Linnane, A. W. (1967). J. Cell. Biol. 34, 1-14. Delange, R. J., Glazer, A. N. & Smith, E. L. (1970). J. Biol. Chem. 245, 3325-3327. Kassner, R. J. (1972). Proc. Nut. Acad. Sci.. ~‘.&“.A. 69. 2263. Kassner, R. J. (1973). J. Amer. Chem. Sot. 95. 2674. Lederer, F.. Simon, A. & Verdiere, J. (1972). Biochem. Biophys. Res. Comma. 47, 55-58. Margoliash, E. & Schejter, A. (1966). Arch. Protein Chem.
The authors thank W. Hutcheon, G. Louie and G. Pielak for helpful discussions. This research was supported by the Medical Research Council of Canada.
Matthews, B. W. (1968). J. Mol. Biol. 33, 491-497. Pielak. G. J., Mauk, A. G. & Smith, M. (1985). Nature
21, 113-286. (London),
313, 152-153.
Poulos, T. L. & Kraut. J. (1980). J. Biol. C’hem. 225. 10322. Rackovsky, S. & Goldstein, D. A. (1984). Proc. Nut. Acud. hi., U.S.A. 81, 5901-5905. Sherman, F. & St,ewart, J. W. (1971). Annu. Kea. Genet.
Chris Sherwood Gary D. Brayer? Department of Biochemistry 2146 Health Sciences Mall The University of British Columbia Vancouver, B.C. Canada V6T 1W5
5, 257-296.
Received 14 December 1984, and in revised form 24 April 1985
References Boyer, P. D., Chance, B., Ernster, L., Mitchell! P., Racker, E. $ Slater, E. C. (1977). Annu. Rev. Biochem. 46, 955-1026. Edited
t Author to whom all correspondence should be sent.
Sherman, F. & Stewart, J. W. (1974). Genetics, 78, 97113. Sherman, F., Stewart, J. W., Margoliash, E., Parker, J. & Campbell, W. (1966). PTOC. iVat. Acad. Ski.. U.S.A. 55. 1498-1504. Sherman, F., Stewart, J. W.! Jackson, M., Gilmore. R. A. & Parker, J. H. (1974). Genetics, 77, 2555284. Smith, M., Leung, D. W., Gillam, 6. & Astell, C. R. (1979). Cell, 16, 753-761. Takano. T. & Dickerson. R. E. (1981). J. :%IoZ.Hiol. 153, 79-94. Zoller, M. J. & Smith, M. (1983). Methods Enzymol. 100, 468-500. by A. Klug
LETTERS TO THEEDITOR
Crystallization and Preliminary Diffraction Iso-1-Cytochrome c from Yeast
Data for
Deep red crystals of the electron transfer protein, iso- 1-cytochrome c from yeast cerevisiae), have been obtained from a 90% saturated solut)ion of (NH,),SO, containing 2 mg protein/ml, 0.1 M-sodium phosphate and adjusted to pH 6.7. The space group is P412,2 (or P4,2,2) with a = b = 36.4 A, c = 136.8 A and Z = 8. Crystals are stable for at least ten days in the X-ray beam and diffract to better than 2-O A resolution. Comparable and morphologically similar crystal forms of three iso-1-cytochrome c mutants at Phe87, a pivotal residue in the electron transport chain, have also been obtained. (Saccharomyces
Cytochrome c is a component of the respiratory electron transport chain of eukaryotic organisms, acting as an electron shuttle between multi-enzyme complexes embedded in the inner mitochondrial & membrane (Boyer et al., 1977; Rackovsky Goldstein, 1984). Although a component of mitochondrion, yeast cytochrome c is specified by a nuclear gene (Sherman et al., 1966) and synthesized in t’he cytoplasm (Clark-Walker & Linnane, 1967). Newly synthesized yeast iso-1-cytochrome c is modified by the covalent attachment of the heme group and trimethylation of Lys77 (Margoliash & Schejter, 1966; Sherman & Stewart, 1974; Delange et al., 1970). Yeast iso-1-cytochrome c (M, 12,700) consists of a single polypeptide chain of 108 amino acids (Smith et al., 1979; Lederer et al., 1972) covalently attached to a heme group. The heme ligands include two covalent thioether bridges from cysteine residues 19 and 22. Both Met85 and His23 provide additional ligands to the heme iron atom; they are on opposite sides of, and perpendicular to, the plane of the heme group. Extensive genetic and biochemical analyses related to the expression and function of yeast iso-1-cytochrome c have been carried out (Sherman & Stewart, 1971; Sherman et al., 1974). We now report the crystallization of the iso-lrytochrome c of yeast. The protein preparation used was obtained commercially (Sigma Co,) and its purity checked against standards obtained locally (G. Pielak. personal communication). Crystals were grown using the hanging drop vapour diffusion technique as follows. Yeast iso-1-cytochrome c was dissolved in a solution 75% saturated in (NH,),SO, to a final concentration of 2 mg/ml in the presence of reducing agent. This solution was buffered with 0.1 N-sodium phosphate and the pH adjusted to 6.7 with either NH,OH or sodium acetate. The protein solution was then filtered through a 0.22 pm HPLC filter (Micron Separation Inc.) and allowed to equilibrate against a reservoir solution containing 90% saturated (NH,)#O,. Crystals appeared within 3 to 14 days and generally grew to 0.75 mm x 0.4 mm x 0.4 mm in size. Larger crystals were commonly observed in those hanging drops 0032%2836/85/
170209-02
$03.00/0
which contained only a few nucleation sites. The resultant deep red crystals have the overall morphology of tetragonal bipyramids. X-ray diffraction precession photography indicates iso-lcytochrome c crystals are of the tetragonal space group P4,2,2 (or its enantiomorph P4,2,2) with a = b = 36.4 I%, c = 136.8 A and Z = 8. Crystals are stable in the X-ray beam for up to ten days and diffract to at least 2.0 A resolution. The volume per unit mass, V,, for these crystals is 1.78 A3/dalton (Matthews, 1968), indicating a solvent content of - 30%. These preliminary diffraction studies represent the first step in the elucidation of the full threedimensional structure of yeast iso-1-cytochrome c. The goal of these structural studies is to gain further insight into the electron transfer mechanism mediated by this protein and to clearly define the role of Phe87 in this process. Primary sequence comparisons show this residue is invariably found in all the cytochrome c sequences thus far studied and it has been proposed to play a pivotal role in the electron transfer process. In the tertiary structures of cytochrome c from other organisms (Takano & Dickerson, 1981), this residue is located at the entrance to the heme pocket and in part determines t’he dielectric environment of the heme group (Kassner, 1972, 1973). Furthermore, this phenylalanine appears to form part of the aromatic electron-transfer conduit between the heme groups of cytochrome c and cytochrome c peroxidase during the catalytic cycle of the latter protein (Poulos & Kraut, 1980). We have chosen to study the functional role of Phe87 in iso-l -cytochrome c, since Saccharomyces cerevisiae is amenable to site-directed mutagenesis techniques (Zoller & Smith, 1983) and therefore the mutation of Phe87 or any other residue of yeast cytochrome c to another amino acid residue can be accomplished easily. Three such mutations of Phe87, to a tyrosine, a serine and a glycine residue, have been produced and their functional properties characterized (Pielak et al., 1985). Preliminary crystallization trials with these mutated proteins indicate that crystals can be grown under the same
209
0 1985 Academic Press Inc. (London)
Ltd.
C. Sherwood and G. D. Brayer
210
conditions as those used for the native protein. Both native and mutant protein crystal forms have similar morphologies. The application of X-ray diffraction techniques to obtain the high resolution, three-dimensional structures of native and mutant forms of yeast iso-1-cytochrome c, in conjunction with solution biochemical analyses, holds the greatest promise for a more complete understanding of the phenomena of electron transport.
Clark-Walker, G. D. & Linnane, A. W. (1967). J. Cell. Biol. 34, 1-14. Delange, R. J., Glazer, A. N. & Smith, E. L. (1970). J. Biol. Chem. 245, 3325-3327. Kassner, R. J. (1972). Proc. Nut. Acad. Sci.. ~‘.&“.A. 69. 2263. Kassner, R. J. (1973). J. Amer. Chem. Sot. 95. 2674. Lederer, F.. Simon, A. & Verdiere, J. (1972). Biochem. Biophys. Res. Comma. 47, 55-58. Margoliash, E. & Schejter, A. (1966). Arch. Protein Chem.
The authors thank W. Hutcheon, G. Louie and G. Pielak for helpful discussions. This research was supported by the Medical Research Council of Canada.
Matthews, B. W. (1968). J. Mol. Biol. 33, 491-497. Pielak. G. J., Mauk, A. G. & Smith, M. (1985). Nature
21, 113-286. (London),
313, 152-153.
Poulos, T. L. & Kraut. J. (1980). J. Biol. C’hem. 225. 10322. Rackovsky, S. & Goldstein, D. A. (1984). Proc. Nut. Acud. hi., U.S.A. 81, 5901-5905. Sherman, F. & St,ewart, J. W. (1971). Annu. Kea. Genet.
Chris Sherwood Gary D. Brayer? Department of Biochemistry 2146 Health Sciences Mall The University of British Columbia Vancouver, B.C. Canada V6T 1W5
5, 257-296.
Received 14 December 1984, and in revised form 24 April 1985
References Boyer, P. D., Chance, B., Ernster, L., Mitchell! P., Racker, E. $ Slater, E. C. (1977). Annu. Rev. Biochem. 46, 955-1026. Edited
t Author to whom all correspondence should be sent.
Sherman, F. & Stewart, J. W. (1974). Genetics, 78, 97113. Sherman, F., Stewart, J. W., Margoliash, E., Parker, J. & Campbell, W. (1966). PTOC. iVat. Acad. Ski.. U.S.A. 55. 1498-1504. Sherman, F., Stewart, J. W.! Jackson, M., Gilmore. R. A. & Parker, J. H. (1974). Genetics, 77, 2555284. Smith, M., Leung, D. W., Gillam, 6. & Astell, C. R. (1979). Cell, 16, 753-761. Takano. T. & Dickerson. R. E. (1981). J. :%IoZ.Hiol. 153, 79-94. Zoller, M. J. & Smith, M. (1983). Methods Enzymol. 100, 468-500. by A. Klug