Structures and ice-binding faces of the alanine-rich type I antifreeze proteinsThis paper is one of a selection of papers published in this special issue entitled “Canadian Society of Biochemistry, Molecular & Cellular Biology 52nd Annual Meeting — Protein Folding: Principles and Diseases” and has undergone the Journal's usual peer review process.

Abstract

Antifreeze proteins (AFPs) protect cold-blooded organisms from the damage caused by freezing through their ability to inhibit ice growth. The type I AFP family, found in several fish species, contains proteins that have a high alanine content (>60% of the sequence) and structures that are almost all α-helical. We examine the structure of the type I AFP isoforms HPLC6 from winter flounder, shorthorn sculpin 3, and the winter flounder hyperactive type I AFP. The HPLC6 isoform structure consists of a single α-helix that is 37 residues long, whereas the shorthorn sculpin 3 isoform consists of two helical regions separated by a kink. The high-resolution structure of the hyperactive type I AFP has yet to be determined, but circular dichroism data and analytical ultracentrifugation suggest that the 195 residue protein is a side-by-side dimer of two α-helices. The alanine-rich ice-binding faces of HPLC6 and hyperactive type I AFP are discussed, and we propose that the ice-binding face of the shorthorn sculpin 3 AFP contains Ala14, Ala19, and Ala25. We also propose that the denaturation of hyperactive type I AFP at room temperature is explained by the stabilization of the dimerization interface through hydrogen bonds.

Bacterial Ice Crystal Controlling Proteins

“…However, the adsorption inhibition mechanism has one major assumption: irreversible binding of antifreeze proteins [76]. The problem with reversible binding is that transient desorption of antifreeze can reveal new sites for energetically favourable additions of water molecules; this can lead to uncontrollable ice growth [74].…”

“…These repeats are non-specific but are structured so that ice binding residues are aligned on one side to create an ice binding face [26, 28, 29, 76]. Furthermore, regularity of repeats creates order in the spacing between ice binding residues, which is essential to complement ice crystal planes.…”

“…However, this postulated mechanism was insufficient in explaining the high adsorption rate explicitly to ice crystals instead of excess water molecules in the system. Furthermore, one study found that the loss of a methyl group had a greater detrimental impact on thermal hysteresis activity than the loss of a hydroxyl group [76]. Regarding the loss of a hydroxyl group, a mutation from Thr to Ser led to loss of 90% of activity, while the latter condition, a mutation from Thr to Val, reported only a loss of 15% of activity.…”

Antifreeze Peptides and Glycopeptides, and Their Derivatives: Potential Uses in Biotechnology

“…It has three repeated sequences, which contain 11 amino acid repeats; Thr-Ala-Ala-X-Ala-X-X-Ala-Ala-X-X, where X can be any amino acid. The crystal structure of the HPLC6 AFP (PDB code 1WFA) [36] revealed that this has an amphipathic right-handed α-helical conformation with N - and C -terminal cap structures to ensure its stability in solution. The N - and C -terminal cap structures are stabilized by a hydrogen bond network that involves structurally ordered water molecules, and help the protein maintain its helicity (Figure 1A).…”

“…Type I AFPs are alanine-rich peptides found in blood samples from winter flounder ( Pseudopleuronectes americanus ) and shorthorn sculpin ( Myoxocephalus scorpius ). To date, three-dimensional (3D) structures of the AFPs HPLC6 and ss3 have been solved [36,37] and their ice-binding residues have been identified using structure-based mutagenesis studies. Accumulated structure-function relationship studies on Type I AFPs have revealed that common Ala-rich hydrophobic regions are essential for potent antifreeze activity [36].…”