DeLuca C I, Davies P L, Ye Q, Jia Z
Department of Biochemistry, Queen's University, Kingston, Ontario, Canada.
J Mol Biol. 1998 Jan 23;275(3):515-25. doi: 10.1006/jmbi.1997.1482.
The interaction of proteins with ice is poorly understood and difficult to study, partly because ice is transitory and can present many binding surfaces, and partly because structures have been determined for only 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-binding surface is remarkably flat for such a small protein. The residues on the planar surface thought to be involved in ice binding are restrained by hydrogen bonds or by tight packing of their side-chains. To probe the requirement for a flat binding surface, a conserved alanine in the center of the AFP planar surface was substituted with larger residues. Six alanine replacement mutants (Ala16 > Cys, Thr, Met, Arg, His and Tyr), designed to disrupt the planarity of the surface and sterically block binding to ice, were characterized by X-ray crystallography and compared with the wild-type AFP. In each case, the detail provided by these crystal structures has helped explain the effects of the mutation on antifreeze activity. The substitutions, Ala16 > His and Ala16 > Tyr, were large enough to shield Gln44, one of the putative ice-binding residues, contributing to their very low thermal hysteresis activity. In addition to sterically hindering the putative ice-binding site, the bulkier residues also caused shifts in the putative ice-binding residues owing to the tight packing of side-chains on the planar surface. This unexpected consequence of the mutations helps account for the severely reduced antifreeze activity. One explanation for residual antifreeze activity in some of the mutants lies in the possibility that 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 that can freeze around them. It is evident that the disruption of the planarity, 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 change and steric blockage of some putative ice-binding residues.
蛋白质与冰的相互作用目前还知之甚少，且难以研究，部分原因是冰是短暂存在的，并且可能呈现出许多结合表面，部分原因是仅确定了两种冰结合蛋白的结构。本文聚焦于其中一种，即来自长绵鳚的一种由66个残基组成的抗冻蛋白（AFP）。这种鱼类AFP的高分辨率X射线结构表明，对于如此小的一种蛋白质而言，其假定的冰结合表面非常平坦。平面表面上被认为参与冰结合的残基通过氢键或其侧链的紧密堆积而受到限制。为了探究对平坦结合表面的需求，在AFP平面表面中心的一个保守丙氨酸被替换为更大的残基。通过X射线晶体学对六个丙氨酸替代突变体（Ala16 > Cys、Thr、Met、Arg、His和Tyr）进行了表征，这些突变体旨在破坏表面的平面性并在空间上阻碍与冰的结合，并将其与野生型AFP进行了比较。在每种情况下，这些晶体结构提供的细节都有助于解释突变对抗冻活性的影响。Ala16 > His和Ala16 > Tyr这两种替换足够大，能够屏蔽假定的冰结合残基之一Gln44，这导致它们的热滞活性非常低。除了在空间上阻碍假定的冰结合位点外，体积更大的残基还由于平面表面上侧链的紧密堆积而导致假定的冰结合残基发生位移。这些突变的这一意外结果有助于解释抗冻活性的严重降低。对一些突变体中残留抗冻活性的一种解释是，抗冻蛋白可能在塑造它们所结合的冰表面位点方面发挥作用。因此，侧链的错位可能会被能够在其周围冻结的冰部分容纳。很明显，通过在假定的冰结合位点中心引入更大的残基来破坏平面性，并不是导致抗冻活性丧失的唯一因素。还有多种原因，包括一些假定的冰结合残基的位置变化和空间阻碍。
