The transmembrane domain of caveolin-1 exhibits a helix-break-helix structure.
The transmembrane domain of caveolin-1 exhibits a helix-break-helix structure.
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DOI：
10.1016/j.bbamem.2011.12.033
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发表时间：
2012-05
影响因子：
3.4
通讯作者：
Glover, Kerney Jebrell
中科院分区：
文献类型：
--
作者：
Lee, Jinwoo;Glover, Kerney Jebrell
关键词：
小窝蛋白是一种完整的膜蛋白，在小窝中含量很高。N端和C端都位于膜的同一侧，跨膜结构域被假定为形成不寻常的膜内马蹄形结构。为了探索跨膜结构域的结构，我们制备了一个包含残基96-136（整个完整的跨膜结构域）的caveolin-1结构。Caveolin-1（96-136）在大肠杆菌中过表达和同位素标记，纯化至均匀性，并纳入溶肉豆肉酰基磷脂酰甘油胶束。圆二色性和核磁共振光谱显示，小洞蛋白-1的跨膜结构域主要是α-螺旋结构域（57-65%）。此外，化学位移指数显示，跨膜结构域具有螺旋-断裂-螺旋结构，这可能是预测caveolin-1形成膜内马蹄形的关键。螺旋断裂横跨108至110个残基，并进行丙氨酸扫描诱变以探索这些残基的结构意义。我们的研究结果表明，甘氨酸108突变为丙氨酸不会破坏结构，但异亮氨酸109和脯氨酸110突变为丙氨酸会显著改变螺旋断裂-螺旋结构。为了进一步探索结构决定因素，进行了额外的诱变。甘氨酸108可以被其他小侧链氨基酸（即丙氨酸）取代，亮氨酸109可以被其他β-支链氨基酸（即缬氨酸）取代，脯氨酸110在不破坏螺旋-断裂-螺旋结构的情况下不能被取代。
Caveolin is an integral membrane protein that is found in high abundance in caveolae. Both the N- and C- termini lie on the same side of the membrane, and the transmembrane domain has been postulated to form an unusual intra-membrane horseshoe configuration. To probe the structure of the transmembrane domain, we have prepared a construct of caveolin-1 that encompasses residues 96-136 (the entire intact transmembrane domain). Caveolin-1(96-136) was over-expressed and isotopically labeled in E.coli, purified to homogeneity, and incorporated into lyso-myristoylphosphatidylglycerol micelles. Circular dichroism and NMR spectroscopy reveal that the transmembrane domain of caveolin-1 is primarily α-helical (57–65%). Furthermore, chemical shift indexing reveals that the transmembrane domain has a helix-break-helix structure which could be critical for the formation of the intra-membrane horseshoe conformation predicted for caveolin-1. The break in the helix spans residues 108 to 110, and alanine scanning mutagenesis was carried out to probe the structural significance of these residues. Our results indicate that mutation of glycine 108 to alanine does not disrupt the structure, but mutation of isoleucine 109 and proline 110 to alanine dramatically alters the helix-break-helix structure. To explore the structural determinants further, additional mutagenesis was performed. Glycine 108 can be substituted with other small side chain amino acids (i.e. alanine), leucine 109 can be substituted with other β-branched amino acids (i.e. valine), and proline 110 cannot be substituted without disrupting the helix-break-helix structure.
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