No abstract

Antifreeze proteins (AFP) inhibit ice growth by surface adsorption that results in a depression of the freezing point below the melting point. The maximum level of this thermal hysteresis shown by the four structurally unrelated fish AFP is approximately 1.5 degrees C. In contrast, hemolymph and crude extracts from insects can have 5 degrees to 10 degrees C of thermal hysteresis. Based on the isolation, cloning, and expression of a thermal hysteresis protein (THP) from spruce budworm (Choristoneura fumiferana), the vastly greater activity is attributable to a 9 kDa protein. This novel, threonine- and cysteine-rich THP has striking effects on ice crystal morphology, both before and during freezing. It is also 10 to 30 times more active than any known fish AFP, offering the prospect of superior antifreeze properties in cryoprotective applications.

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.

The gene encoding the glycolytic enzyme triose-phosphate isomerase (TPI; EC 5.3.1.1) has been central to the long-standing controversy on the origin and evolutionary significance of spliceosomal introns by virtue of its pivotal support for the introns-early view, or exon theory of genes.Putative correlations between intron positions and TPI protein structure have led to the conjecture that the gene was assembled by exon shuffling, and five TPI intron positions are old by the criterion of being conserved between animals and plants. We have sequenced TPI genes from three diverse eukaryotes-the basidiomycete Coprinus cinereus, the nematode Caenorhabditis elegans, and the insect Heliothis virescens and have found introns at seven novel positions that disrupt previously recognized gene/protein structure correlations.The set of 21 TPI introns now known is consistent with a random model of intron insertion. Twelve of the 21 TPI introns appear to be of recent origin since each is present in but a single examined species. These results, together with their implication that as more TPI genes are sequenced more intron positions will be found, render TPI untenable as a paradigm for the introns-early theory and, instead, support the introns-late view that spliceosomal introns have been inserted into preexisting genes during eukaryotic evolution.The surprising discovery of spliceosomal introns in 1977 was soon followed by considerable speculation about their origin, evolution, and significance (1-3). Nearly 20 years later, the issue is very much alive and has long since become polarized into two opposing theories. The introns-early theory, or exon theory of genes, posits the presence of many introns in the common ancestor of all life, followed by massive, often complete, intron loss in many independent lineages (4, 5). Introns are thought to have functioned in the primordial assembly of protein genes by promoting the recombinational shuffling of short exons, each encoding 15-20 amino acid units of protein structure (6-8). The other theory, termed introns-late, posits that spliceosomal introns were not present in the common ancestor of life but, instead, arose and spread within eukaryotic evolution (9-11); therefore, these introns could not have played any role in ancient gene and protein assembly.A major part of the evidence in favor of the introns-early theory has been supplied by the ancient gene encoding the glycolytic enzyme triose-phosphate isomerase (TPI; EC 5.3.1.1; refs. 6-8 and 12). As soon as the first eukaryotic TPI gene was sequenced, a correspondence was noted between exons and secondary structural elements, with all six chicken introns falling at or near the ends of a-helices and (3-strands (13). More TPI intron positions were discovered in 1986 from a plant and a fungus (6). Five introns are located in the same positions in plant and animal TPI genes, indicating that these introns were in place prior to the presumably ancient divergence of these taxa (6). Since these new data did not support a straightf...

The spruce budworm, Choristoneura fumiferana, produces antifreeze protein (AFP) to assist in the protection of the overwintering larval stage. AFPs are thought to lower the freezing point of the hemolymph, noncolligatively, by interaction with the surface of ice crystals. Previously, we had identified a cDNA encoding a 9-kDa AFP with 10±30 times the thermal hysteresis activity, on a molar basis, than that shown by fish AFPs. To identify important residues for ice interaction and to investigate the basis for the hyperactivity of the insect AFPs, six new spruce budworm AFP cDNA isoforms were isolated and sequenced. They differ in amino-acid identity as much as 36% from the originally characterized AFP and can be divided into three classes according to the length of their 3 H untranslated regions (UTRs). The new isoforms have at least five putative`Thr-X-Thr' ice-binding motifs and three of the new isoforms encode larger, 12-kDa proteins. These appear to be a result of a 30 amino-acid insertion bearing two additional ice-binding motifs spaced 15 residues apart. Molecular modeling, based on the NMR structure of a short isoform, suggests that the insertion folds into two additional b-helix loops with their Thr-XThr motifs in perfect alignment with the others. The first Thr of the motifs are often substituted by Val, Ile or Arg and a recombinantly expressed isoform with both Val and Arg substitutions, showed wild-type thermal hysteresis activity. The analysis of these AFP isoforms suggests therefore that specific substitutions at the first Thr in the ice binding motif can be tolerated, and have no discernible effect on activity, but the second Thr appears to be conserved. The second Thr is thus likely important for the dynamics of initial ice contact and interaction by these hyperactive antifreezes.