A β-Helical Antifreeze Protein Isoform with Increased Activity

Abstract

The insect spruce budworm (Choristoneura fumiferana)(Cf) produces a number of isoforms of its highly active antifreeze protein (CfAFP). Although most of the CfAFP isoforms are in the 9-kDa range, isoforms containing a 30-or 31-amino acid insertion have also been identified. Here we describe the functional and structural analysis of a selected long isoform, CfAFP-501. Xray crystal structure determination reveals that the 31-amino acid insertion found in CfAFP-501 forms two additional loops within its highly regular ␤-helical structure. This effectively extends the area of the twodimensional Thr array and ice-binding surface of the protein. The larger isoform has 3 times the thermal hysteresis activity of the 9-kDa CfAFP-337. As well, a deletion of the 31-amino acid insertion within CfAFP-501 to form CfAFP-501-⌬-2-loop, results in a protein with reduced activity similar to the shorter CfAFP isoforms. Thus, the enhanced antifreeze activity of CfAFP-501 is directly correlated to the length of its ␤-helical structure and hence the size of its ice-binding face.

Antifreeze Protein Dimer

“…Another example of increasing antifreeze activity with increasing ice-binding site area is the spruce budworm AFP. A 12-kDa isoform (501) with two additional coils of ␤-helix is more than twice as active as the shorter 9-kDa isoform (36). As the disulfide-bonded A65C dimer has demonstrated, there is little activity gained from simple dimerization of the AFP domains.…”

Hyperactive Antifreeze Protein from Winter Flounder Is a Very Long Rod-like Dimer of α-Helices

“…In trying to account for the superior TH activity of this protein, it is clear that specific activity increases with the size of the AFP (26), particularly when it also increases the surface area of the ice binding site (11,20,42,43). This is the general trend in both insect and fish AFPs.…”

“…This is the general trend in both insect and fish AFPs. Among insect AFPs, a natural isoform of the spruce budworm AFP with 8 ␤-helical loops is three times more active than the abundant 6-loop isoform (42), and an engineered variant of the T. molitor AFP with two added coils is twice as active as the wild-type AFP from which it was derived (20). In fish, the larger antifreeze glycoproteins (AFGPs 1-5) are considerably more active than the shorter ones (AFGPs 6 -8) (44).…”

Modeling repetitive, non‐globular proteins

“…Many of the IBPs with known structures have been determined experimentally to have sequences that are composed of multiple short repeats (3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19) residues in length) that in turn form repeating structural motifs. These structural motifs include a-helices and polyproline type II helices, but the majority of repetitive IBPs form a b-solenoid structure.…”

“…18 Spruce budworm AFP (SbwAFP) has a well-characterized isoform (501) that is 30 residues longer than most, with the extra sequence making exactly two 15-residue repeats that each makes a coil of the b-solenoid structure. 19 While detection of repeats is often not especially difficult, finding the correct "frame" of the repeats is important for establishing the repeating structure, or coil. Frequently the repeats near the amino-and carboxy-termini of the domain are less like those in the middle of the domain, so definition of the actual repeat boundaries can be more difficult than the initial identification of a repeating sequence.…”