A Diminished Role for Hydrogen Bonds in Antifreeze Protein Binding to Ice ² Heman Chao, ‡ Michael E. Houston, Jr., ‡ Robert S. Hodges, ‡ Cyril M. Kay, ‡ Brian D. Sykes, ‡ Miche `le C. Loewen, § Peter L. Davies,* ,§ and Frank D. So ¨nnichsen | Protein Engineering Network of Centres of Excellence and Department of Biochemistry, UniVersity of Alberta, Edmonton, Alberta T6G 2S2, Canada, Department of Biochemistry, Queen’s UniVersity, Kingston, Ontario K7L 3N6, Canada, and Department of Physiology and Biophysics, Case Western ReserVe UniVersity, CleVeland, Ohio 44106-4970 ReceiVed April 8, 1997; ReVised Manuscript ReceiVed August 11, 1997 X ABSTRACT: The most abundant isoform (HPLC-6) of type I antifreeze protein (AFP 1 ) in winter flounder is a 37-amino-acid-long, alanine-rich, R-helical peptide, containing four Thr spaced 11 amino acids apart. It is generally assumed that HPLC-6 binds ice through a hydrogen-bonding match between the Thr and neighboring Asx residues to oxygens atoms on the {202 h1} plane of the ice lattice. The result is a lowering of the nonequilibrium freezing point below the melting point (thermal hysteresis). HPLC-6, and two variants in which the central two Thr were replaced with either Ser or Val, were synthesized. The Ser variant was virtually inactive, while only a minor loss of activity was observed in the Val variant. CD, ultracentrifugation, and NMR studies indicated no significant structural changes or aggregation of the variants compared to HPLC-6. These results call into question the role of hydrogen bonds and suggest a much more significant role for entropic effects and van der Waals interactions in binding AFP to ice. Type I AFP 1 is the smallest and arguably the simplest of the four macromolecular antifreeze types characterized to date (1). It is in effect a single, long R-helix and therefore lacks tertiary structure (2). The most abundant isoform of this AFP (HPLC-6) from winter flounder (Pleuronectes americanus) is 37 amino acids long, contains three complete 11-amino-acid repeats of Thr-X 2 -Asx-X 7 , where X is gener- ally alanine, and ends with the start of a fourth repeat. This helical periodicity that places the Thr and Asx residues on the same face of the helix, suggested a mechanism for adsorption of the AFP to ice in which these regularly spaced hydrophilic groups would hydrogen bond to oxygen atoms in the ice lattice (3). Adsorption leads to inhibition of ice crystal growth (4) because ice is forced to grow with a surface curvature between the bound AFP, which in turn results in a lowering of the nonequilibrium freezing point below the melting point (5, 6). The difference in these two temperatures is termed thermal hysteresis and is used as a measure of antifreeze activity. At very low concentrations, AFP bind to ice but do not stop its growth. Under these conditions bound AFP is frozen into the ice rather than excluded by the advancing ice front. The protein binding planes in these crystals have been made visable by sublimation (ice etching) and determined to be the {202 h1} pyramidal plane of hexagonal ice (I h ) for type I AFP (5). Moreover, because this antifreeze is a nonglobular, extended molecule it was possible to establish a direction 〈011 h2〉 of binding on the plane. An elegant proof of this resulted from the synthesis of an all D-type I AFP, which was shown by the ice etching method to bind to the same plane but in the mirror image direction (7). This information was used to suggest a hydrogen-bonding match between the i, i + 11 threonines spaced 16.5 Å apart along the helix and accessible ice lattice oxygens spaced 16.7 Å apart along the 〈011 h2〉 direction of the {202 h1} binding plane. On the basis of this match and the structure of the R-helix, a number of models have been proposed to account for the selective and specific binding of type I AFP to ice that is the foundation of the adsorption-inhibition mechanism. Although all of these models share the Thr hydrogen-bonding match, there is no consensus about the role of Asx. In two reports these residues were not featured in the ice-binding model (8, 9); in others they were hydrogen bonded to different ranks of oxygen atoms in the lattice (10, 11) or they occupied a cage position within the lattice without hydrogen bonding (12). Throughout all of these deliberations there has been concern that the number and strength of the available hydrogen bonds (with or without a contribution from the Asx residues) may be inadequate to explain tight binding of the antifreeze to ice. At least two imaginative ideas have been put forward to reinforce the hydrogen-bonding hypothesis by suggesting ways in which more hydrogen bonds can be brought to bear on the binding interaction. One suggestion, that the hydroxyl groups of the ice-binding Thr occupy lattice oxygen positions to form three hydrogen bonds instead of one (13), is, however, not compatible with the R-helical structure of the AFP (11). The other suggestion is that type I AFP forms patches on the ice surface through interpeptide interactions resulting from side-by-side alignment of the helices. Thus a cluster of n R-helices would be the operative unit, which would have a multiple of n hydrogen bonds. ² This work is supported by the Protein Engineering Network of Centres of Excellence of Canada and A/F Protein, Inc. F.D.S. acknowledges support from NIH Grant GM55362. * Author to whom correspondence should be addressed: Peter L. Davies, Department of Biochemistry, Queen’s University, Kingston, Ontario K7L 3N6, Canada. Tel: (613) 545-2983. Fax: (613) 545-2497. E-mail: daviesp@post.queensu.ca. ‡ University of Alberta. § Queen’s University. | Case Western Reserve University. X Abstract published in AdVance ACS Abstracts, November 15, 1997. 1 Abbreviations: AFP, antifreeze protein; HPLC, high-performance liquid chromatography; CD, circular dichroism; NMR, nuclear magnetic resonance; DQF-COSY, double-quantum-filtered correlation spectros- copy; PE-COSY, primitive exclusive correlation spectroscopy; TOCSY, total correlated spectroscopy; NOE, nuclear Overhauser effect; NOESY, nuclear Overhauser enhanced spectropscpy. 14652 Biochemistry 1997, 36, 14652-14660 S0006-2960(97)00817-9 CCC: $14.00 © 1997 American Chemical Society