A Diminished Role for Hydrogen Bonds in Antifreeze Protein Binding to Ice

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 generally 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 ba...

Increased flexibility decreases antifreeze protein activity

“…29,[31][32][33][34][35][36]46 The effects of the mutations can be broadly classified into those that (a) disrupt favorable contacts between the protein and ice because of the substitution of a residue to one with a long side chain (e.g., Ala to Leu mutation), 35 (b) result in a loss of a favorable contact between the protein and ice because of the deletion of a functional group (e.g., Thr to Ser mutations), 31,32,37 or (c) disrupt the backbone structure of the protein (e.g., loss of the salt bridge). 29 In the cases involving the nonamidated rHPLC6-Arg 37 and rHPLC6-Ala 37 , none of these possibilities are fully consistent with the experimental observations made here.…”

“…35 In contrast, removal of the Thr hydroxyl groups by mutation of the central two Thr residues to Val resulted in a moderate loss of activity ($15% loss). 31,37 These results suggest that van der Waals interactions between the methyl groups of these residues and the ice surface keep the protein bound to ice. Why hydrogen bonds are not involved and how van der Waals interactions promote ice binding are still unclear.…”

“…The methyl groups of the Ala-rich face (Ala 17 and Ala 21 , and equivalent positions) and the methyl groups of the four Thr residues (Thr 2 , Thr 13 , Thr 24 , and Thr 35 ) were shown to be essential for full TH activity. Removal of the central two Thr methyl groups (by substitution with Ser) 31,32,37 or changing Ala residues on the Ala 17 face to Leu resulted in a >50% loss in activity. 35 In contrast, removal of the Thr hydroxyl groups by mutation of the central two Thr residues to Val resulted in a moderate loss of activity ($15% loss).…”

Polycarboxylates enhance beetle antifreeze protein activity

“…The details of the means by which AFPs bind to ice are somewhat controversial, and probably depend to some extent on the type of AFP. Proposed mechanisms involve hydrophobic and van der Waals interactions [18][19][20] and/or hydrogen bonding [21][22][23]. The adsorption of AFPs on the ice surface only allows ice crystal growth to take place in highly curved fronts, whose surface free energy is higher than that of the usual low radius of curvature fronts in the absence of AFPs.…”

Interactions of β-Helical Antifreeze Protein Mutants with Ice

“…[26][27][28] When Thr residues were replaced with Val, which is sterically equivalent to Thr but lacks the side chain hydroxyl and instead has another methyl group, only a minor loss of activity was observed. In contrast, substitution to Ser, which preserves the side chain hydroxyl, had a more detrimental effect on TH activity, [26][27][28] suggesting that hydrophobic interactions and van der Waals forces contribute significantly to ice recognition by AFPs. Further mutagenesis studies showed that the Ala residues adjacent to the four Thr residues are crucial for TH activity, leading to the conception that protein-ice interactions occur through this Alarich face and neighboring Thr residues.…”