De novo DESIGN AND SYNTHESIS OF AN ICE-BINDING, DENDRIMERIC, POLYPEPTIDE BASED ON INSECT ANTIFREEZE PROTEINS

A new strategy is presented for the design

and synthesis of peptides that exhibit

ice-binding and antifreeze activity. A

pennant-type dendrimer polypeptide

scaffold combining an α-helical backbone

with four short β-strand branches was

synthesized in solid phase using Fmoc

chemistry in a divergent approach. The

51-residue dendrimer was characterized

by reverse phase high performance liquid

chromatography, mass spectrometry and

circular dichroism. Each β-strand branch

contained three overlapping TXT amino

acid repeats, an ice-binding motif found

in the ice-binding face of the spruce

budworm (Choristoneura fumiferana)

and beetle (Tenebrio molitor) antifreeze

proteins. Ice crystals in the presence of

the polypeptide monomer displayed flat,

hexagonal plate morphology, similar to

that produced by weakly active antifreeze

proteins. An oxidized dimeric form of the

dendrimer polypeptide also produced flat

hexagonal ice crystals and was capable

of inhibiting ice crystal growth upon

temperature reduction, a phenomenon

termed thermal hysteresis, a defining

property of antifreeze proteins. Linkage

of the pennant-type dendrimer to a trifunctional

cascade-type polypeptide

produced a trimeric macromolecule that

gave flat hexagonal ice crystals with

higher thermal hysteresis activity than

the dimer or monomer and an ice crystal burst pattern similar to that produced

by samples containing insect antifreeze

proteins. This macromolecule was also

capable of inhibiting ice recrystallization.