Concentration-dependent oligomerization of an alpha-helical antifreeze polypeptide makes it hyperactive

Abstract

A supersoluble 40-residue type I antifreeze protein (AFP) was discovered in a righteye flounder, the barfin plaice (bp). Unlike all other AFPs characterized to date, bpAFP transitions from moderately-active to hyperactive with increasing concentration. At sub-mM concentrations, bpAFP bound to pyramidal planes of ice to shape it into a bi-pyramidal hexagonal trapezohedron, similarly to the other moderately-active AFPs. At mM concentrations, bpAFP uniquely underwent further binding to the whole ice crystal surface including the basal planes. The latter caused a bursting ice crystal growth normal to c-axis, 3 °C of high thermal hysteresis, and alteration of an ice crystal into a smaller lemon-shaped morphology, all of which are well-known properties of hyperactive AFPs. Analytical ultracentrifugation showed this activity transition is associated with oligomerization to form tetramer, which might be the forerunner of a naturally occurring four-helix-bundle AFP in other flounders.

Fish-Derived Antifreeze Proteins and Antifreeze Glycoprotein Exhibit a Different Ice-Binding Property with Increasing Concentration

“…The fish species barfin plaice (Liposetta pinnifasciata), longsnout poacher (Brachyopsis rostratus), notched-fin eelpout (Zoarces elongatus Kner), and saffron cod (Eleginus gracilis) were collected by Nichirei Corporation. Size exclusion chromatography and anion exchange chromatography were successively performed for the prepared crude sample powders with a Sephadex G-25 size-exclusion column (XK 50/30, 500 mL; GE Healthcare Life Sciences, Pittsburgh, PA, USA) and DEAE Sepharose anion-exchange column (XK 50/20, 80 mL; GE Healthcare), respectively [7]. The purified samples were dialyzed against Milli-Q water for three overnights, and then lyophilized for frozen storage.…”

“…Biomolecules 2019, 9, x 4 of 14 powder according to previously described procedures [7][8][9][10][11], which yielded approximately 1500, 200, 130, and 1600 mg of lyophilized powders of AFP I-III and AFGP, respectively. Then, chromatography was performed again to obtain the gram order of the samples to be analyzed with high-performance liquid chromatography, which gave us several isoform fractions.…”

“…Sample powders of AFP I-III and AFGP of 20-30% purity were obtained from Nichirei Corporation (Tokyo, Japan), and were extracted from the muscle homogenates of barfin plaice, longsnout poacher, notched-fin eelpout, and saffron cod, respectively [11]. Size exclusion chromatography followed by anion exchange chromatography were performed on 6 g of each crude powder according to previously described procedures [7][8][9][10][11], which yielded approximately 1500, 200, 130, and 1600 mg of lyophilized powders of AFP I-III and AFGP, respectively. Then, chromatography was performed again to obtain the gram order of the samples to be analyzed with high-performance liquid chromatography, which gave us several isoform fractions.…”

“…Note that 100-1000 mg of the purified samples were necessary for these experiments (Figure 1). To obtain such large amounts of AFP and AFGP, a method was developed that includes AFPs into a growing ice crystal with ice-affinity protocol [42,43], as well as a method that utilizes fish muscle homogenates [7][8][9][10][11]. The latter method was used to obtain the present samples.…”

Ice Crystal Growth in Sucrose Solutions Containing Kappa‐ and Iota‐Carrageenans

“…Therefore, preliminary experiments have been carried out to reveal whether TH activities of AFP I and AFGP can be detected with this system. AFP I is a low-molecular-weight protein (< 30 kDa) obtained from the barfin plaice (bp), also called the native bpAFP [19] with a molecular weight of 3.5 kDa [20]. AFGP was provided by Nichirei Co. Ltd. as a purified sample of AFGP from Eleginus gracilis [21] with an assumed molecular weight of 15 kDa [20].…”