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Four crystalline tuna myoglobins
J. Mol. Biol. (1968) 38, 141-143
Four Crystalline
Tuna Myoglobins
I have begun an investigation of the crystal structure of tuna myoglobin with an ultimate goal of comparing its tertiary structure with those of other members of the myoglobin-hemoglobin family currently under investigation-sperm whale ferrimyoglobin (Kendrew et al., 1961), seal ferrimyoglobin (Scouloudi, 1960), porpoise ferrimyoglobin (Kretsinger & Diamond, manuscript in preparation), human deoxyhemoglobin (Muirhead, Cox, Mazzarella & Perutz,, 1967), horse oxy- and ferrihemoglobin (Perutz, Muirhead, Cox & Goaman, 1968), lamprey oxy- and deoxyhemoglobin (Hendrickson, Love & Murray, 1968) and ferrierythrocruorin (Huber, Formanek & Epp, 1968). Tuna myoglobin was chosen because it is phylogenetioally distant from the mammalian myoglobins, and because it has a single cysteine (Brown, Martinez & Olcott, 1961) as does the p-chain of hemoglobin, whereas no known mammalian myoglobin has cysteine. The four tuna myoglobins to be described were supplied by Drs D. Brown and B. Cobb of the Institute of Marine Resources, University of California at Berkeley. Even though the samples were quite pure, each was chromatographed on DEAE Sephadex, A50. Following dialysis against the starting buffer, up to 20 ml. of myoglobin solution at a concentration up to 50 mg/ml. were applied to a 85 cm x 2.4 cm column and eluted with a linear gradient formed from 700 ml. each of 0.025 M-TrisCl (pH 8.7) and O-050 i%i-Tris-Cl (pH 7.2). The myoglobin was eluted as a single symmetric peak at 350 ml. Small amounts of protein trailed behind the myoglobin, with some brown material stuck to the top of the column. The chromatographed myoglobins had absorbance peaks at 407 and 278 m/l characteristic of tuna ferrimyoglobin (Brown, Martinez, Johnstone BEOlcott, 1962). Yellow fin (Neothunnus mucropterus) myoglobin crystallized readily at concentrations from 10 to 30 mg protein/ml., 60 to 80% saturated ammonium sulfate and pH from 5.5 to 7.0. Crystals isomorphous with those grown from ammonium sulfate were grown from 2.7 rvr-sodium hydrogen phosphate (pH 7.2). The other myoglobins to be described did not crystallize from phosphate; none of these myoglobins crystallized from sodium citrate or from sodium chloride. The unit cell dimensions (Table 1) did not change significantly as a function of ammonium sulfate concentration. The intensity of reflections declined significantly beyond the 2.2 A spacing observable in a 21” precession photograph. Such a photograph required a 36-hour exposure of a crystal about O-3 mm on one side, irradiated with Ni-filtered Cu radiation from a Picker fine-focus (10 mm x 0.4 mm focal spot) tube operated at 13ma, 40 kv. Only after 100 hours of exposure at 20°C did the diffraction pattern show any change in intensity distribution. Blue fin (Thunnus thynnus) myoglobin formed crystals with poorly defined faces from ammonium sulfate. Their diffraction pattern showed disorder and the diffracted intensity did not extend to spacings beyond 3 A. Even though the unit cell dimensions (Table 1) differ significantly from those of yellow fin, the two do seem to be roughly isomorphous as judged by the distribution of inter&es in their diffraction patterns. 141
142
R.
H.
KRETSINGER
1
TABLE
Types of tuna myoglobin crystals
Myoglobin
Yellow
fin
60% s&;&m. sulf. 70% sat. am. sulf. 80% sat. am. sulf.
P212121
Blue fin Albacore Skip-jack
P 2121% P P:; (9)
44*65 44.44 44.45 42.1 42.6 61-8
72.4, 72.1, 72.2, 72.4 79.9 34.7
52*37 52-35 52,21 51.5 52.0 26.5
103O50’ 91°5’
The estimated error is based upon the comparison of dimensions obtained tals. The Supper precession oamera, film shrinkage and mean wavelength radiation were calibrated from precession photographs of beryllium (Tulinsky, Worthington & Pignataro, 1959).
0.2 0.2 o-2 o-5 o-2 0.5
41,950
A3
39,240 42,970 28,410
(x 2)
from different orysfor Ni-filtered CuKa acetate, a = 15.74 A
Albacore (Thunnus germo) myoglobin crystallized from ammonium sulfate over the range 65 to 75% saturated (pH 5.5 to 7.0). The crystals were generally elongated along the 2-fold screw axis b, and had well-developed faces, though these could not be indexed with certainty. Frequently crystals were twinned about c*. This might be related to the fact that there are two myoglobin molecules per crystallographic asymmetric unit. Skipjack (Katsuwonus pelamis) myoglobin formed irregular crystalline masses in ammonium sulfate. These crystals are disordered in the a b plane end unsuitable for detailed study, but it is noteworthy that the molecules in them must be relatively tightly packed, since the crystal volume per molecule is only 28,400 A3. Kendrew, Parrish, Marrack & Orlans (1954) reported the unit cell dimensions of 16 different crystalline myoglobins; the volume per molecule ranged from 33,100 A3 for carp to 47,000 A3 for penguin. After completing this preliminary survey of these four tuna myoglobins, I have selected yellow iln myoglobin for further study and am now collecting film data to 2-O A resolution. This Virginia,
research has been and by a National
Department University Charlottesville, Received
of Biology of Virginia Virginia 13 August
supported Institute
by the Center for Advanced of Health grant no. I-ROl-GM
Studies, University 1678B-01. R. H.
22903,
of
KRETSINOER
U.S.A.
1968 REFERENCES
Brown, Brown,
W. D., Martinez, W. D., Martinez,
M. M.,
t Olcott, John&one,
H.
S. (1961). J. Biol. C&m. 286, 92. & Olcott, H. S. (1962). J. Bid. Chem. 287,81. Hendrickson, W. A., Love, W. E. t Murray, G. C. (1968). J. Mol. Biol. .38, 829. Huber, R., Forrnanek, H. t Epp, 0. (1968). N&07&.9. 55, 75. Kendrew, J. C., Parrish, R. G., Marraok, J. R. & Orlans, E. S. (1954). Nature, 174, 946. M.
LETTERS Kendrew, Shore, Muirhead, Perutz, M. Scouloudi, Tulinsky,
TO
THE
EDITOR
J. E., Watson, H. C., Strandberg, B. E., Dickerson, R. E., Phillips, V. C. (1961). Nature, 190, 666. H., Cox, J. M., Mazzarella, L. & Perutz, M. F. (1967). J. Mol. Biol. F., Muirhead, H., Cox, J. M. & Goaman, L. C. G. (1968). Nature, H. (1960). Proc. Roy. Sot. A, 258, 181. A., Worthington, C. R. & Pignataro, E. (1959). Acta Cry&. 12, 623.
143 D.
C. &
28, 117. 219,
131.
Four Crystalline
Tuna Myoglobins
I have begun an investigation of the crystal structure of tuna myoglobin with an ultimate goal of comparing its tertiary structure with those of other members of the myoglobin-hemoglobin family currently under investigation-sperm whale ferrimyoglobin (Kendrew et al., 1961), seal ferrimyoglobin (Scouloudi, 1960), porpoise ferrimyoglobin (Kretsinger & Diamond, manuscript in preparation), human deoxyhemoglobin (Muirhead, Cox, Mazzarella & Perutz,, 1967), horse oxy- and ferrihemoglobin (Perutz, Muirhead, Cox & Goaman, 1968), lamprey oxy- and deoxyhemoglobin (Hendrickson, Love & Murray, 1968) and ferrierythrocruorin (Huber, Formanek & Epp, 1968). Tuna myoglobin was chosen because it is phylogenetioally distant from the mammalian myoglobins, and because it has a single cysteine (Brown, Martinez & Olcott, 1961) as does the p-chain of hemoglobin, whereas no known mammalian myoglobin has cysteine. The four tuna myoglobins to be described were supplied by Drs D. Brown and B. Cobb of the Institute of Marine Resources, University of California at Berkeley. Even though the samples were quite pure, each was chromatographed on DEAE Sephadex, A50. Following dialysis against the starting buffer, up to 20 ml. of myoglobin solution at a concentration up to 50 mg/ml. were applied to a 85 cm x 2.4 cm column and eluted with a linear gradient formed from 700 ml. each of 0.025 M-TrisCl (pH 8.7) and O-050 i%i-Tris-Cl (pH 7.2). The myoglobin was eluted as a single symmetric peak at 350 ml. Small amounts of protein trailed behind the myoglobin, with some brown material stuck to the top of the column. The chromatographed myoglobins had absorbance peaks at 407 and 278 m/l characteristic of tuna ferrimyoglobin (Brown, Martinez, Johnstone BEOlcott, 1962). Yellow fin (Neothunnus mucropterus) myoglobin crystallized readily at concentrations from 10 to 30 mg protein/ml., 60 to 80% saturated ammonium sulfate and pH from 5.5 to 7.0. Crystals isomorphous with those grown from ammonium sulfate were grown from 2.7 rvr-sodium hydrogen phosphate (pH 7.2). The other myoglobins to be described did not crystallize from phosphate; none of these myoglobins crystallized from sodium citrate or from sodium chloride. The unit cell dimensions (Table 1) did not change significantly as a function of ammonium sulfate concentration. The intensity of reflections declined significantly beyond the 2.2 A spacing observable in a 21” precession photograph. Such a photograph required a 36-hour exposure of a crystal about O-3 mm on one side, irradiated with Ni-filtered Cu radiation from a Picker fine-focus (10 mm x 0.4 mm focal spot) tube operated at 13ma, 40 kv. Only after 100 hours of exposure at 20°C did the diffraction pattern show any change in intensity distribution. Blue fin (Thunnus thynnus) myoglobin formed crystals with poorly defined faces from ammonium sulfate. Their diffraction pattern showed disorder and the diffracted intensity did not extend to spacings beyond 3 A. Even though the unit cell dimensions (Table 1) differ significantly from those of yellow fin, the two do seem to be roughly isomorphous as judged by the distribution of inter&es in their diffraction patterns. 141
142
R.
H.
KRETSINGER
1
TABLE
Types of tuna myoglobin crystals
Myoglobin
Yellow
fin
60% s&;&m. sulf. 70% sat. am. sulf. 80% sat. am. sulf.
P212121
Blue fin Albacore Skip-jack
P 2121% P P:; (9)
44*65 44.44 44.45 42.1 42.6 61-8
72.4, 72.1, 72.2, 72.4 79.9 34.7
52*37 52-35 52,21 51.5 52.0 26.5
103O50’ 91°5’
The estimated error is based upon the comparison of dimensions obtained tals. The Supper precession oamera, film shrinkage and mean wavelength radiation were calibrated from precession photographs of beryllium (Tulinsky, Worthington & Pignataro, 1959).
0.2 0.2 o-2 o-5 o-2 0.5
41,950
A3
39,240 42,970 28,410
(x 2)
from different orysfor Ni-filtered CuKa acetate, a = 15.74 A
Albacore (Thunnus germo) myoglobin crystallized from ammonium sulfate over the range 65 to 75% saturated (pH 5.5 to 7.0). The crystals were generally elongated along the 2-fold screw axis b, and had well-developed faces, though these could not be indexed with certainty. Frequently crystals were twinned about c*. This might be related to the fact that there are two myoglobin molecules per crystallographic asymmetric unit. Skipjack (Katsuwonus pelamis) myoglobin formed irregular crystalline masses in ammonium sulfate. These crystals are disordered in the a b plane end unsuitable for detailed study, but it is noteworthy that the molecules in them must be relatively tightly packed, since the crystal volume per molecule is only 28,400 A3. Kendrew, Parrish, Marrack & Orlans (1954) reported the unit cell dimensions of 16 different crystalline myoglobins; the volume per molecule ranged from 33,100 A3 for carp to 47,000 A3 for penguin. After completing this preliminary survey of these four tuna myoglobins, I have selected yellow iln myoglobin for further study and am now collecting film data to 2-O A resolution. This Virginia,
research has been and by a National
Department University Charlottesville, Received
of Biology of Virginia Virginia 13 August
supported Institute
by the Center for Advanced of Health grant no. I-ROl-GM
Studies, University 1678B-01. R. H.
22903,
of
KRETSINOER
U.S.A.
1968 REFERENCES
Brown, Brown,
W. D., Martinez, W. D., Martinez,
M. M.,
t Olcott, John&one,
H.
S. (1961). J. Biol. C&m. 286, 92. & Olcott, H. S. (1962). J. Bid. Chem. 287,81. Hendrickson, W. A., Love, W. E. t Murray, G. C. (1968). J. Mol. Biol. .38, 829. Huber, R., Forrnanek, H. t Epp, 0. (1968). N&07&.9. 55, 75. Kendrew, J. C., Parrish, R. G., Marraok, J. R. & Orlans, E. S. (1954). Nature, 174, 946. M.
LETTERS Kendrew, Shore, Muirhead, Perutz, M. Scouloudi, Tulinsky,
TO
THE
EDITOR
J. E., Watson, H. C., Strandberg, B. E., Dickerson, R. E., Phillips, V. C. (1961). Nature, 190, 666. H., Cox, J. M., Mazzarella, L. & Perutz, M. F. (1967). J. Mol. Biol. F., Muirhead, H., Cox, J. M. & Goaman, L. C. G. (1968). Nature, H. (1960). Proc. Roy. Sot. A, 258, 181. A., Worthington, C. R. & Pignataro, E. (1959). Acta Cry&. 12, 623.
143 D.
C. &
28, 117. 219,
131.