Abstract
Antifreeze glycoproteins (AFGPs) are able to bind to ice, halt its growth, and are the most potent inhibitors of ice recrystallization known. The structural basis for AFGP’s unique properties remains largely elusive. Here we determined the antifreeze activities of AFGP variants that we constructed by chemically modifying the hydroxyl groups of the disaccharide of natural AFGPs. Using nuclear magnetic resonance, two-dimensional infrared spectroscopy, and circular dichroism, the expected modifications were confirmed as well as their effect on AFGPs solution structure. We find that the presence of all the hydroxyls on the disaccharides is a requirement for the native AFGP hysteresis as well as the maximal inhibition of ice recrystallization. The saccharide hydroxyls are apparently as important as the acetyl group on the galactosamine, the α-linkage between the disaccharide and threonine, and the methyl groups on the threonine and alanine. We conclude that the use of hydrogen-bonding through the hydroxyl groups of the disaccharide and hydrophobic interactions through the polypeptide backbone are equally important in promoting the antifreeze activities observed in the native AFGPs. These important criteria should be considered when designing synthetic mimics.
Highlights
Antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs) are a unique class of macromolecules that inhibit ice growth in the body fluids of organisms, thereby enabling their survival in freezing environments.[1,2] These natural proteins, as well as synthetic mimics, are of tremendous interest for their use in the cold storage of biological tissues, food, and other water-based materials.[3,4] All AF(G)Ps have characteristic core activities that include the ability to depress the freezing point in a noncolligative manner without substantially affecting the melting point,[5] the ability to shape ice crystals into unusual morphologies,[6] and the ability to inhibit the recrystallization of ice (IRI).[7]
The modifications are (I) the AFGP-aldehyde (AFGP-ald) variant obtained by oxidizing the C-6 hydroxyl of the galactose moieties of the natural AFGP isoforms to an aldehyde using galactose oxidase;[24] (II) the AFGP-carboxyl (AFGP-car) variant obtained by oxidizing the AFGP-ald variant with bromine, and (III) the AFGP-isopropylidene (AFGP-ipp) variant obtained by adding an isopropylidene (IPP) group that replaced the hydroxyls of C-3 and C-4 of the galactose.[24] (IV) technically not a modification, the addition of borate at pH 9.0 results in the formation of a complex with the cis-hydroxyls of C-3 and C-4 of the galactose and eliminates most of the antifreeze activity (AFGP-bor).[1,8,37]
We confirmed the success of the AFGP-ald modification by observing a new NMR signal at ∼9.2 ppm, which we assigned to the aldehyde proton and an aldehyde specific IR signal at ∼1700 cm−1
Summary
Antifreeze proteins (AFPs) and antifreeze glycoproteins (AFGPs) are a unique class of macromolecules that inhibit ice growth in the body fluids of organisms, thereby enabling their survival in freezing environments.[1,2] These natural proteins, as well as synthetic mimics, are of tremendous interest for their use in the cold storage of biological tissues, food, and other water-based materials.[3,4] All AF(G)Ps have characteristic core activities that include the ability to depress the freezing point in a noncolligative manner without substantially affecting the melting point,[5] the ability to shape ice crystals into unusual morphologies,[6] and the ability to inhibit the recrystallization of ice (IRI).[7]. The relative magnitude of each effect varies between different types of antifreeze proteins with the AFGPs exhibiting a moderate hysteresis (1.5 °C) but being by far the most potent of the ice recrystallization inhibitors known.[6] The molecular details of how the AF(G)Ps achieve their unique antifreeze properties remain largely unknown.[8−11] It is generally accepted that all AF(G)Ps function by an adsorption-inhibition mechanism in which the proteins recognize and irreversibly bind to specific crystal faces of microscopic ice crystals, thereby preventing macroscopic ice growth.[12] A long-standing question concerning the mechanism of antifreeze activities of the AFGP is which part of the molecule binds to ice and what forces mediate the interaction. In the case of some fish and insect AFPs of known structure, the ice-binding sites (IBS) have been identified as combinations of flat hydrophobic surfaces and, in some cases, the presence of preordered interfacial water molecules associated with the hydrophobic faces.[13−17]
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