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The crystallization and X-ray investigation of one form of human carbonic anhydrase
PLATE
1. Crystals of human carbonic anhydrase, type CA V. (magnification>: 225).
J. Mol. Biol. (1962) 5, 583 -584
The Crystallization and X-ray Investigation of One Form of Human Carbonic Anhydrase The form of human carbonic anhydrase designated CA V was prepared by' the method of Nyman (1961). The protein solution from the final purification step was rc-dialysed against 0·005 sr-sodium phosphate, pH 6,0, and concentrated by adsorption on a column packed with sulphoethyldextran gel (obtained from AB Pharmacia, Uppsala) followed by elution with 0·1 M-Na2HP04 • This technique readily gives solutions of 8 to 10% concentration. The crystals used in thc X-ray examination (Plate I) were prepared by dialysing a 1% enzyme solution at 2°C against a 4 M-ammonium sulphate solution in 0·05 M-tris-HCI buffer at pH 8·5; the salt concentration was raised to this value in steps of about 0·6 moles/litre with intervals of about 2 hours between each increment. When concentrating the protein by freeze-drying no crystals can be obtained. The single crystals selected had the approximate dimensions 0·30 x 0·20 x 0·15 mm. They were mounted in sealed capillaries with some mother liquor at each end to maintain constant vapour pressure according to the standard method adopted for examining protein crystals. Diffraction patterns were obtained with a Buerger-Supper precession camera using copper K« radiation from a Philips microfocus tube. 9° photographs of the hOl, hll and hkO layers gave the following constants for the monoclinic cell: a = 43·1 A, b = 42·1 A, c = 73·6 A, f1 = 104'6°, V = 129,000 As. The space group is P2 1 • The density of the crystals was found to be 1·31 ± 0·04 g/cms using the flotation method. From these results it was concluded that the unit cell contains two asymmetric units each of which has a weight of 50,600 on the atomic mass scale. Since the molecular weight ofCA V is 34,000 (Nyman, 1961) the number of enzyme molecules per asymmetric unit cannot be greater than one. From the above, half an enzyme molecule could be present in the asymmetric unit but this possibility is excluded by the occurrence of several amino acids in odd numbers. Stockell (1961) points out that for a number of globular proteins the protein weight in the crystals is within the range 0·35 to 0·51 mass units per As. The value for CA V is 0·53. A 24° precession pattern of the hOllayer (about I·!) A resolution) was also taken. The intensities in this pattern were measured with a Nonius single-beam densitometer, corrected for Lorenz and polarization factors, and the resulting F2 values were then used as coefficients for an hOl Patterson projection. This calculation was numerically integrated and the resulting radial distribution was eompared with the theoretical radial distribution for a single ex-helix. The result is shown in Fig. 1. Pauling & Corey (1951) have made a similar comparison for haemoglobin using a Patterson projection to provide data for the experimental curve. While a detailed interpretation of the distribution curve is not possible until similar eurves have been established for several proteins with known structure, its shape in the region 4 to 7 A indicates some type of secondary structure and does not contradict the existence of ex-helix. This would be in agreement with results from electron microscope investigations (Levin, private communication). The occurrence of helices would simplify the interpretation of the electron density map particularly at low resolution. 583
584
STRA~DBERG.
TILAXDER, FRIDBORG. LINDSKOG AND NYMAN
It seems probable that a slightly different experimental curve would result from the use of three-dimensional X-ray data in an appropriate calculation. The maximum at 9·7 A may be explained by abundant occurrence of helices in contact. This is the case in the molecules of haemoglobin (Cullis, Muirhead, Perutz, Rossmann & North, 1962) and myoglobin (Kendrew et al., 1960). As expected the ,F2-distributions of these proteins have maxima at about 10 A (Crick, 1953; Bodo, Dintzis, Kendrew & Wyckoff,1959).
4
5
6
789
10
II
12
13
14
o
A-
FIG. 1. Comparison of the experimental radial distribution function for human carbonic anhydrase, type CA V, with the corresponding calculated function for a single a-helix according to Pauling & Corey. A .8-carbon is included in esoh residue. The scale is chosen arbitrarily.
We have recently prepared complexes between carbonic anhydrase and mercury derivatives of sulphonamide inhibitors and found that these crystallize isomorphously with the native protein. With the aid of such derivatives we hope ultimately to solve the structure of the enzyme. We wish to express our gratitude to Professor I. Lindqvist and Dr. B. Mahnstr6m for stimulating interest and valuable discussions. We also thank Professors A. Fredga, G. Hagg, A. Tiselius and Dr. J. Porath for their encouragement and the facilities placed at our disposal. This work has been supported by grants from the Swedish Natural Science Research Council (259--15, 17, 19 and 220 4), from the Magnus Bergwall fund and from t.he Division of General Medical Sciences, U.S. Public Health Service (RG-6542-C2). Institute of Chemistry University of Uppsala, Uppsala Sweden
B. B. K.
Institute of Biochemistry University of Uppsala, Uppsala Sweden Received 1 August 1962
S. LINDSKOG P. O. NYMAN
STRANDBERG TILAXDER FRIDBORG
RgFERENCES Bodo, G., Dintais, H., Kendrew, J. C. & Wyckoff, H. (1959). Proc. Roy. Soc. A, 253, 70. Crick, F. H. C. (1953). Acta Or/Jst. 6, 600. Cullis, A. F., Muirhead, H. , Perutz, M. F ., Rossmann, M. G. & North, A. C. T. (1962) . Proc, Roy. Soc. A, 265, 161. Kendrew, J. C., Dickerson, R. E., Strandberg, B. E., Hart, R . G., Davies, D. R ., Phillips, D. C. & Shore, V. C. (1960). Nature, 185, 1422. Nyman, P. O. (1961). Biochim, biophys. Acta, 52, 1. Pauling, L. & Corey, R. B. (1951). Proc. Nat. Acad. Sci., Wash. 37, 282. Stockell, A. (1961). J. Mol. Biol. 3,'112.
1. Crystals of human carbonic anhydrase, type CA V. (magnification>: 225).
J. Mol. Biol. (1962) 5, 583 -584
The Crystallization and X-ray Investigation of One Form of Human Carbonic Anhydrase The form of human carbonic anhydrase designated CA V was prepared by' the method of Nyman (1961). The protein solution from the final purification step was rc-dialysed against 0·005 sr-sodium phosphate, pH 6,0, and concentrated by adsorption on a column packed with sulphoethyldextran gel (obtained from AB Pharmacia, Uppsala) followed by elution with 0·1 M-Na2HP04 • This technique readily gives solutions of 8 to 10% concentration. The crystals used in thc X-ray examination (Plate I) were prepared by dialysing a 1% enzyme solution at 2°C against a 4 M-ammonium sulphate solution in 0·05 M-tris-HCI buffer at pH 8·5; the salt concentration was raised to this value in steps of about 0·6 moles/litre with intervals of about 2 hours between each increment. When concentrating the protein by freeze-drying no crystals can be obtained. The single crystals selected had the approximate dimensions 0·30 x 0·20 x 0·15 mm. They were mounted in sealed capillaries with some mother liquor at each end to maintain constant vapour pressure according to the standard method adopted for examining protein crystals. Diffraction patterns were obtained with a Buerger-Supper precession camera using copper K« radiation from a Philips microfocus tube. 9° photographs of the hOl, hll and hkO layers gave the following constants for the monoclinic cell: a = 43·1 A, b = 42·1 A, c = 73·6 A, f1 = 104'6°, V = 129,000 As. The space group is P2 1 • The density of the crystals was found to be 1·31 ± 0·04 g/cms using the flotation method. From these results it was concluded that the unit cell contains two asymmetric units each of which has a weight of 50,600 on the atomic mass scale. Since the molecular weight ofCA V is 34,000 (Nyman, 1961) the number of enzyme molecules per asymmetric unit cannot be greater than one. From the above, half an enzyme molecule could be present in the asymmetric unit but this possibility is excluded by the occurrence of several amino acids in odd numbers. Stockell (1961) points out that for a number of globular proteins the protein weight in the crystals is within the range 0·35 to 0·51 mass units per As. The value for CA V is 0·53. A 24° precession pattern of the hOllayer (about I·!) A resolution) was also taken. The intensities in this pattern were measured with a Nonius single-beam densitometer, corrected for Lorenz and polarization factors, and the resulting F2 values were then used as coefficients for an hOl Patterson projection. This calculation was numerically integrated and the resulting radial distribution was eompared with the theoretical radial distribution for a single ex-helix. The result is shown in Fig. 1. Pauling & Corey (1951) have made a similar comparison for haemoglobin using a Patterson projection to provide data for the experimental curve. While a detailed interpretation of the distribution curve is not possible until similar eurves have been established for several proteins with known structure, its shape in the region 4 to 7 A indicates some type of secondary structure and does not contradict the existence of ex-helix. This would be in agreement with results from electron microscope investigations (Levin, private communication). The occurrence of helices would simplify the interpretation of the electron density map particularly at low resolution. 583
584
STRA~DBERG.
TILAXDER, FRIDBORG. LINDSKOG AND NYMAN
It seems probable that a slightly different experimental curve would result from the use of three-dimensional X-ray data in an appropriate calculation. The maximum at 9·7 A may be explained by abundant occurrence of helices in contact. This is the case in the molecules of haemoglobin (Cullis, Muirhead, Perutz, Rossmann & North, 1962) and myoglobin (Kendrew et al., 1960). As expected the ,F2-distributions of these proteins have maxima at about 10 A (Crick, 1953; Bodo, Dintzis, Kendrew & Wyckoff,1959).
4
5
6
789
10
II
12
13
14
o
A-
FIG. 1. Comparison of the experimental radial distribution function for human carbonic anhydrase, type CA V, with the corresponding calculated function for a single a-helix according to Pauling & Corey. A .8-carbon is included in esoh residue. The scale is chosen arbitrarily.
We have recently prepared complexes between carbonic anhydrase and mercury derivatives of sulphonamide inhibitors and found that these crystallize isomorphously with the native protein. With the aid of such derivatives we hope ultimately to solve the structure of the enzyme. We wish to express our gratitude to Professor I. Lindqvist and Dr. B. Mahnstr6m for stimulating interest and valuable discussions. We also thank Professors A. Fredga, G. Hagg, A. Tiselius and Dr. J. Porath for their encouragement and the facilities placed at our disposal. This work has been supported by grants from the Swedish Natural Science Research Council (259--15, 17, 19 and 220 4), from the Magnus Bergwall fund and from t.he Division of General Medical Sciences, U.S. Public Health Service (RG-6542-C2). Institute of Chemistry University of Uppsala, Uppsala Sweden
B. B. K.
Institute of Biochemistry University of Uppsala, Uppsala Sweden Received 1 August 1962
S. LINDSKOG P. O. NYMAN
STRANDBERG TILAXDER FRIDBORG
RgFERENCES Bodo, G., Dintais, H., Kendrew, J. C. & Wyckoff, H. (1959). Proc. Roy. Soc. A, 253, 70. Crick, F. H. C. (1953). Acta Or/Jst. 6, 600. Cullis, A. F., Muirhead, H. , Perutz, M. F ., Rossmann, M. G. & North, A. C. T. (1962) . Proc, Roy. Soc. A, 265, 161. Kendrew, J. C., Dickerson, R. E., Strandberg, B. E., Hart, R . G., Davies, D. R ., Phillips, D. C. & Shore, V. C. (1960). Nature, 185, 1422. Nyman, P. O. (1961). Biochim, biophys. Acta, 52, 1. Pauling, L. & Corey, R. B. (1951). Proc. Nat. Acad. Sci., Wash. 37, 282. Stockell, A. (1961). J. Mol. Biol. 3,'112.