Modulation of the structural integrity of helix F in apomyoglobin by single amino acid replacements
Modulation of the structural integrity of helix F in apomyoglobin by single amino acid replacements
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DOI：
10.1110/ps.04635304
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发表时间：
2004-06-01
期刊：
影响因子：
8
通讯作者：
Fontana, A
中科院分区：
文献类型：
--
作者：
Picotti, P;Marabotti, A;Fontana, A
通过使用多达八种不同底物特异性的蛋白酶进行有限的蛋白水解实验，探究了抹香鲸脱辅基肌红蛋白 (apoMb) 在中性 pH 下的天然和突变形式的构象特征。研究表明，所有蛋白酶都在链段 82-94 (HEAELKPLAQSHA) 水平上选择性切割 apoMb，该链段包含天然蛋白全息形式 X 射线结构中的螺旋 F；例如，嗜热菌蛋白酶可裂解 Pro 88-Leu 89 肽键。这些结果表明，apoMb 中的螺旋 F 高度灵活或很大程度上被破坏。由于螺旋 F 含有破坏螺旋的 Pro 88 残基，因此我们建议螺旋 F 通过各种螺旋-血红素稳定相互作用保持在天然全息蛋白中。为了调节螺旋 F 的稳定性，通过定点诱变制备了 Pro88Ala 和 Pro88Gly 突变体，并通过远紫外圆二色光谱和有限蛋白水解研究了它们的构象特性。 Pro88Ala 突变体的螺旋含量相对于天然和 Pro88Gly 突变体有所增强，正如从 Ala 是 20 个氨基酸残基中最强的螺旋诱导剂这一事实所预期的那样。嗜热菌蛋白酶和蛋白酶 K 对三种 apoMb 变体的有限蛋白水解速率的顺序为天然 > Pro88Gly >> Pro88Ala，与 Ala、Gly 和 Pro 的螺旋倾向程度一致。讨论了柔性/未折叠链段82-94对于apoMb在细胞水平上的功能和命运的可能作用。
The conformational features of native and mutant forms of sperm-whale apomyoglobin (apoMb) at neutral pH were probed by limited proteolysis experiments utilizing up to eight proteases of different substrate specificities. It was shown that all proteases selectively cleave apoMb at the level of chain segment 82-94 (HEAELKPLAQSHA), encompassing helix F in the X-ray structure of the holo form of the native protein; for example, thermolysin cleaves the Pro 88-Leu 89 peptide bond. These results indicate that helix F is highly flexible or largely disrupted in apoMb. Because helix F contains the helix-breaking Pro 88 residue, we propose that helix F is kept in place in the native holo protein by a variety of helix-heme stabilizing interactions. To modulate the stability of helix F, the Pro88Ala and Pro88Gly mutants were prepared by site-directed mutagenesis, and their conformational properties investigated by both far-UV circular dichroism spectroscopy and limited proteolysis. The helix content of the Pro88Ala mutant was somewhat enhanced with respect to that of both native and Pro88Gly mutant, as expected from the fact that Ala is the strongest helix inducer among the 20 amino acid residues. The rate of limited proteolysis of the three apoMb variants by thermolysin and proteinase K was in the order native > Pro88Gly >> Pro88Ala, in agreement with the scale of helix propensity of Ala, Gly, and Pro. The possible role of the flexible/unfolded chain segment 82-94 for the function and fate of apoMb at the cellular level is discussed.