Native oligomerisation of a small type I antifreeze protein from winter flounder

Authors

Virgilio, L. L., Omar, A., Chang, X. J., Stolow, J., Lee, M., Hossain, A., Kiradziev, B., Ewart, K. V.

Abstract

Antifreeze proteins and peptides adhere to the surfaces of ice crystals and inhibit their growth, resulting in non-colligative freezing point depression. AFP6 is a predominant antifreeze peptide in the blood serum of winter flounder (Pseudopleuronectes americanus). This 37-residue amphiphilic -helical peptide can be dissolved in water; however, its ice-binding surface is the hydrophobic side of the helix. In this study, modelling of the peptide suggested the formation of an antiparallel dimer burying the hydrophobic faces of the helices, consistent with the behaviour of amphiphilic helices more generally. The oligomerisation of recombinant non-amidated and synthetic amidated forms of AFP6 were therefore examined in aqueous solutions using size-exclusion chromatography, chemical crosslinking, and in silico modelling. Crosslinking of non-amidated AFP6, followed by denaturing electrophoresis, resulted in more slowly migrating bands consistent with dimers, whereas the amidated form and a recombinant mutant also showed bands suggesting trimers and tetramers. Bands consistent with the presence of monomers and dimers were predominant following non-amidated AFP6 crosslinking under a variety of solution conditions, with smaller proportions of peptide assembled to tetrameric sizes; however, these larger oligomers appeared to be absent from AFP6 treated with sodium bicarbonate. Isolated oligomers of non-amidated AFP6 produced ice crystal morphologies and thermal hysteresis comparable to the unfractionated peptide suggesting similar interaction with ice. This is the first AFP oligomer shown to have no effect on ice interaction. Instead, the oligomerisation may allow greater solubility of this amphiphilic -helical peptide in aqueous solutions.

Preprint server: bioRxiv
The authors list and abstract were imported from bioRxiv on 29 Sep 2026.

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