Single crystals of a winter flounder antifreeze polypeptide

J. Mol. Biol. (1986) 189, 725

Single Crystals of a Winter Flounder Antifreeze Polypeptide Component 6 of the winter flounder’s antifreeze polypeptides has been crystallized. The space group is P2,, with cell parameters of a = 38.14 .&, b = 37.19 8, c = 21.82 A. fl = 101.5”. There are two molecules of 3300 M, per asymmetric unit.

Pseudopleuronectes winter flounder, a common inhabitant of the ice-laden region of the eastern Canadian coast and northeastern United States coast, produces antifreeze peptides in the winter, which are rich in alanine (60%). These macromolecules, which lower the freezing temperature of body fluids below that of the surrounding waters, are essential for the survival of this species during winter. The effects of these macromolecules on freezing temperature are non-colligative, i.e. they lower the freezing point much more than would be expected on the basis of the osmolality of their solution. On a weight basis, these antifreeze polypeptides and NaCl are approximately equally effective in lowering the freezing temperature of water; however, on a molal basis, these macromolecular antifreezes are up to two orders of magnitude more effective as “antifreeze” agents. These polypeptides depress the freezing point several hundred times more than do other proteins such as lysozyme and bovine serum albumin (Feeney & Yeh, 19’78; DeVries, 1984). Fractionation by high-pressure liquid chromatography on these antifreeze polypeptides yielded at least seven components, the two major components (nos 6 and 8) differ only by an aspartic acid to glut’amic acid replacement (Davies et al., 1982; Fourney et a,Z., 1984). Crystals were grown from a mixture of acetone and water. Portions of acetone were added to a solution of the protein (4 mg/ml) in 20 mMTris. HC1 (pH 7.3) to reach its supersaturation point (approximately 5 parts acetone to 1 part solution). Crystals were observed in a few days at’ 18°C and attained maximum size in several weeks. A ninhydrin test confirmed that the crystals grown were indeed protein. A 20” X-ray precession photograph shows the X-ray diffraction pattern of the crystal to at least 2.26 8. The crystal is very stable to X-ray The

americawus,

irradiation. The space group is P2, and the cell are a = 38.14 8, b = 37.19 A, parameters c = 21.82 A and /3 = 101.5”. Assuming two protein molecules per asymmetric unit and a molecular weight of 3300 M,, one finds a specific volume of 2.3 A3/dalton, which is within the range observed for most protein crystals (Matthews, 1968). Daniel S. C. Yang Yong Je Chung Ping Chen John P. Rose BioCrystallography Laboratory P.O. Box 12055 VA Medical Center Pittsburgh, PA 15240, U.S.A. and Department of Crystallography IJniversity of Pittsburgh Pittsburgh, PA 15260, U.S.A.

Choy L. Hew Department of Biochemistry Research Institute Hospital for Sick Children 555 University Avenue Toronto, Ontario, M5G 1X8, Canada

Received 31 January

1986

References Davies, P. L., Roach, A. H. & Hew, C. L. (1982). Proc. Nat. Acad. Sci., U.S.A. 79, 335-339. DeVries, A. (1984). Phil. Trans. Roy. Sot. ser. R. 304, 575-588. Feeney, R. E. & Yeh, Y. (1978). Adaan. Protein Chem. 32, 191--282. Fourney, R. M., Joshi, S. B., Kao. M. H. & Hew, C. L. (1984). Canad. J. 2001. 62, 28-33. Mat,thews, B. W. (1968). J. Mol. Biol. 33. 491-497.

Edited by A. Klug

0022%2836/86/12072541

$03.00/O

725

0

1986 Academic

Press Inc.

(London)

Ltd.