THE EFFECTS OF STERIC MUTATIONS ON THE STRUCTURE OF TYPE-III ANTIFREEZE PROTEIN AND ITS INTERACTION WITH ICE

The interaction of proteins with ice is poorly understood and difficul t to study, partly because ice is transitory and can present many bind ing surfaces, and partly because structures have been determined for o nly two ice-binding proteins. This paper focuses on one of these, a 66 -residue antifreeze protein (AFP) from eel pout. The high resolution X -ray structure of this fish AFP demonstrated that the proposed ice-bin ding surface is remarkably flat for such a small protein. The residues on the planar surface thought to be involved in ice binding are restr ained by hydrogen bonds or by tight packing of their side-chains. To p robe the requirement for a flat binding surface, a conserved alanine i n the center of the AFP planar surface was substituted with larger res idues. Six alanine replacement mutants (Ala16 > Cys, Thr, Met, Arg, Hi s and Tyr), designed to disrupt the planarity of the surface and steri cally block binding to ice, were characterized by X-ray crystallograph y and compared with the wild-type AFP. In each case, the detail provid ed by these crystal structures has helped explain the effects of the m utation on antifreeze activity. The substitutions, Ala16 > His and Ala 16 > Tyr, were large enough to shield Gln44, one of the putative ice-b inding residues, contributing to their very low thermal hysteresis act ivity. In addition to sterically hindering the putative ice-binding si te, the bulkier residues also caused shifts in the putative ice-bindin g residues owing to the tight packing of side-chains on the planar sur face. This unexpected consequence of the mutations helps account for t he severely reduced antifreeze activity. One explanation for residual antifreeze activity in some of the mutants lies in the possibility tha t AFPs have a role in shaping the site on the ice to which they bind. Thus, side-chain dislocations might be partially accommodated by ice t hat can freeze around them. It is evident that the disruption of the p lanarity, by introducing larger residues at the center of the proposed ice-binding site, is not the only factor responsible for the loss of antifreeze activity. There are multiple causes including positional ch ange and steric blockage of some putative ice-binding residues. (C) 19 98 Academic Press Limited.