Fully active QAE isoform confers thermal hysteresis activity on a defective SP isoform of type III antifreeze protein

Abstract

Type III antifreeze protein is naturally expressed as a mixture of sulfopropyl‐Sephadex (SP) and quaternary aminoethyl‐Sephadex (QAE)‐binding isoforms, whose sequence identity is approximately 55%. We studied the ice‐binding properties of a SP isoform (nfeAFP6) and the differences from those of a QAE isoform (nfeAFP8); both of these isoforms have been identified from the Japanese fish Zoarces elongatus Kner. The two isoforms possessed ice‐shaping ability, such as the creation of an ice bipyramid, but nfeAFP6 was unable to halt crystal growth and exhibited no thermal hysteresis activity. For example, the ice growth rate for nfeAFP6 was 1000‐fold higher than that for nfeAFP8 when measured for 0.1 mm protein solution at 0.25 °C below the melting point. Nevertheless, nfeAFP6 exhibited full thermal hysteresis activity in the presence of only 1% nfeAFP8 (i.e. [nfeAFP8]/[nfeAFP6] = 0.01), the effectiveness of which was indistinguishable from that of nfeAFP8 alone. We also observed a burst of ice crystal growth from the tip of the ice bipyramid for both isoforms on lowering the temperature. These results suggest that the ice growth inhibitory activity of an antifreeze protein isoform lacking the active component is restored by the addition of a minute amount of the active isoform.

Engineering a naturally inactive isoform of type III antifreeze protein into one that can stop the growth of ice

“…All proteins assayed in this study (nfeAFP8 and nfeAFP11 (wild type and mutants)) were produced recombinantly in Escherichia coli BL21 (DE3) and purified via ion-exchange chromatography as previously described [5]. For NMR measurements, E. coli cells were transformed with a pKK223-3UC-based expression plasmid containing a synthetic gene encoding either nfeAFP11 or nfeAFP11-V9Q/V19L/G20V.…”

“…Like nfeAFP6, nfeAFP11 (a QAE2 isoform) is also unable to prevent ice crystal growth at undercooling temperatures, despite its ability to shape ice into hexagonal trapezohedrons [4,5]. NfeAFP11 differs in its ice-binding residues in having Val, Val, Gly, and Ile at positions 9, 19, 20, and 41, respectively (Fig.…”

“…QAE1-isoforms are fully active as antifreezes and completely arrest ice crystal growth at temperatures below the equilibrium freezing point of a solution. QAE2-and SP-isoforms have the ability to shape ice crystals into hexagonal trapezohedrons, but are incapable of preventing their growth unless supplemented with some QAE1-isoform [4,5].…”

NMR structure note: a defective isoform and its activity-improved variant of a type III antifreeze protein from Zoarces elongates Kner

“…AFPs also modify the shape of an ice crystal uniquely, hexagonal bipyramid for example, in the temperature range of TH. These two activities have been assumed to be common for all AFPs; however, it has recently been found that they are not always the rule for every species, such as the AFP type III (denoted AFPIII) found in fish (Takamichi et al 2008).…”

“…AFPs also modify the shape of an ice crystal uniquely, hexagonal bipyramid for example, in the temperature range of TH. These two activities have been assumed to be common for all AFPs; however, it has recently been found that they are not always the rule for every species, such as the AFP type III (denoted AFPIII) found in fish (Takamichi et al 2008).Fish AFPIII is a globular protein made up of many short β-strands and one helical turn. AFPIII is generally produced in vivo as a mixture of quaternary-amino-ethyl (QAE)-Sephadex-and sulfopropyl (SP)-Sephadex-binding isoforms.…”