Structure-function relationships in spruce budworm antifreeze protein revealed by isoform diversity.
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' 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 beta-helix loops with their Thr-X-Thr 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.
Doucet
D
D
Department of Biology, Queen's University, Kingston, Ontario, Canada.
Tyshenko
M G
MG
Kuiper
M J
MJ
Graether
S P
SP
Sykes
B D
BD
Daugulis
A J
AJ
Davies
P L
PL
Walker
V K
VK
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D021301:Antifreeze Proteins / Q000737:chemistry* / Q000235:genetics; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D019476:Insect Proteins / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009036:Moths / Q000737:chemistry* / Q000235:genetics; D010802:Phylogeny; D011487:Protein Conformation; D020033:Protein Isoforms / Q000737:chemistry* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship
D021301:Antifreeze Proteins; D018076:DNA, Complementary; D019476:Insect Proteins; D020033:Protein Isoforms; D011993:Recombinant Fusion Proteins