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The effects of steric mutations on the structure of type III antifreeze protein and its interaction with ice

机译：空间突变对III型抗冻蛋白结构及其与冰的相互作用的影响

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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. (C) 1998 Academic Press Limited. [References: 26]
机译：蛋白质与冰的相互作用了解甚少且难以研究，部分原因是冰是短暂的，可呈现许多结合表面，部分原因是仅确定了两种冰结合蛋白的结构。本文重点介绍一种，一种来自鳗鱼out嘴的66残基抗冻蛋白（AFP）。这种鱼AFP的高分辨率X射线结构表明，对于这么小的蛋白质，拟议的冰结合表面非常平坦。认为与冰结合有关的平面上的残基受到氢键或侧链紧密堆积的限制。为了探查平坦结合表面的要求，将AFP平面中心的保守丙氨酸替换为较大的残基。设计了六种丙氨酸替代突变体（Ala16> Cys，Thr，Met，Arg，His和Tyr），旨在破坏表面的平面性并在空间上阻断与冰的结合，并通过X射线晶体学进行了表征，并与野生型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。取代，Ala16> His和Ala16> Tyr，足够大以屏蔽Gln44（一种假定的冰结合残基），从而导致其极低的热滞后活性。除了在空间上阻碍推定的冰结合位点外，由于侧链在平面上的紧密堆积，较大的残基还导致推定的冰结合残基发生位移。突变的这种意想不到的结果有助于解释抗冻活性的严重降低。对于某些突变体中剩余抗冻活性的一种解释是，AFP可能会在其结合的冰上形成位点。因此，侧链位错可能会被冰冻结，从而部分冻结。显然，通过在拟议的冰结合位点的中心引入较大的残留物来破坏平面性并不是造成防冻剂活性下降的唯一因素。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堵塞。 （C）1998 Academic Press Limited。 [参考：26]

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