Alternative roles for putative ice-binding residues in type I antifreeze protein.
Alternative roles for putative ice-binding residues in type I antifreeze protein.
复制标题
DOI：
10.1021/bi982602p
复制
发表时间：
1999-04
期刊：
影响因子：
2.9
通讯作者：
M. Loewen;H. Chao;M. Houston;J. Baardsnes;R. Hodges;C. Kay;B. Sykes;F. Sönnichsen;P. Davies
中科院分区：
文献类型：
--
作者：
M. Loewen;H. Chao;M. Houston;J. Baardsnes;R. Hodges;C. Kay;B. Sykes;F. Sönnichsen;P. Davies
合成了两组I型抗冻蛋白的变异体，以研究Leu和Asn在这个37个残基的α-螺旋活性中的作用。Leu和ASN分别位于I-1和I+3位置上四个规则间隔的冰结合Thr的中央两个侧翼。所有这三个残基都从螺旋的同一侧突出，形成蛋白质假定的冰吸附位，在一些模型中被认为是共同作用的“冰结合基序”。用具有较短侧链的残基取代天冬氨酸会导致抗冻剂活性的小幅损失(ALA)或增加(Thr)。然而，天冬氨酸被其稍大的同系物(Gln)取代后，热滞活性消失。含有谷氨酰胺的多肽是非常易溶的，主要是单体，并且完全螺旋。在Ala取代Leu的三个变异体中，三个变异体中的两个比含Leu的变异体更具活性，但随着肽浓度的增加，所有三个变异体都开始沉淀。在测试的七种变种中，没有一种在冰晶形态上与野生种的冰晶形态有显著差异。这些结果与亮氨酸在鱼血清中通常存在的抗冻蛋白浓度(10毫克/毫升)下防止多肽聚集的主要作用是一致的。类似地，ASN的作用可能更多地与提高这些相当疏水的多肽的溶解度有关，而不是像传统认为的那样，使立体特异性氢键与冰晶格相匹配。然而，在天冬氨酸到谷氨酰胺的替换中活性的急剧丧失表明了在多肽的冰结合位置或附近残基的空间限制。
Two sets of variants of type I antifreeze protein have been synthesized to investigate the role of Leu and Asn in the activity of this 37-residue alpha-helix. Leu and Asn flank the central two of four regularly spaced ice-binding Thr in the i-1 and i + 3 positions, respectively. All three residues project from the same side of the helix to form the protein's putative ice-adsorption site and are considered in some models to act together as an "ice-binding motif". Replacement of Asn by residues with shorter side chains resulted in either a small loss (Ala) or gain (Thr) of antifreeze activity. However, substitution of Asn by its slightly larger homologue (Gln) abolished thermal hysteresis activity. The Gln-containing peptide was very soluble, largely monomeric, and fully helical. Of the three variants in which Leu was replaced by Ala, two of the three were more active than their Leu-containing counterparts, but all three variants began to precipitate as the peptide concentration increased. None of the seven variants tested showed dramatic differences in ice crystal morphology from that established by the wild type. These results are consistent with a primary role for Leu in preventing peptide aggregation at the antifreeze protein concentrations (10 mg/mL) normally present in fish serum. Similarly the role for Asn may have more to do with enhancing the solubility of these rather hydrophobic peptides than of making a stereospecific hydrogen-bonding match to the ice lattice as traditionally thought. Nevertheless, the dramatic loss of activity in the Asn-to-Gln replacement demonstrates the steric restriction on residues in or near the ice-binding site of the peptide.