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Loop sequence dictates the secondary structure of a human membrane protein hairpin

机译：环序列决定了人膜蛋白发夹的二级结构

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Membrane proteins adopt two fundamental types of folds in nature: membranes in all organisms harbor α-helical bundles linked by extramembranous loops of varying length, while β-barrel structures are found in the outer membrane of Gram-negative bacteria, mitochondria, and chloroplasts. Here we report that turn-inducing loop mutations in a transmembrane hairpin induce the conversion of an α-helical hairpin to β-sheet oligomers in membrane environments. On the basis of an observation of a sequence bias toward Pro and Gly in the turns of native β-barrel membrane proteins, we characterized in sodium dodecyl sulfate (SDS) micelles and 1-palmitoyl-2-oleoyl-sn-glycero-3- phosphocholine (POPC) bilayers several "hairpin" constructs of cystic fibrosis transmembrane conductance regulator transmembrane segments 3 and 4 (TM3-loop-TM4; loop region being ~(215)IWELLQASA~(223)) in which Pro-Gly residues were either inserted or substituted at several positions. Remarkably, suitable positioning of the Pro-Gly doublet caused the adoption of stable β-sheet structures by several mutants in SDS micelles, as shown by circular dichroism spectroscopy, concurrent with a ladder of discrete oligomers observed via SDS-polyacrylamide gel electrophoresis. Reconstitution of wild-type (WT) TM3/4 into POPC vesicles studied by Trp fluorescence, in conjunction with positional quenchers in brominated phospholipids, indicated a transbilayer position for helical WT TM3/4, but likely a largely surface-embedded conformation for the β-sheet mutant with loop region IWPGELLQASA. To the best of our knowledge, such a complete change in the fold with a minimal number of mutations has not been previously observed for a membrane protein. These facile α-helix to β-sheet conversions highlight the contribution of loops to membrane protein structure.
机译：膜蛋白在自然界中具有两种基本的折叠形式：所有生物体的膜都具有由长度不同的膜外环连接的α-螺旋束，而革兰氏阴性细菌，线粒体和叶绿体的外膜中则存在β-桶状结构。在这里，我们报告说，跨膜发夹中的转导诱导环突变诱导了膜环境中的α-螺旋发夹向β-片层低聚物的转化。基于观察到天然β-桶状膜蛋白依次偏向Pro和Gly的序列，我们表征了十二烷基硫酸钠（SDS）胶束和1-palmitoyl-2-oleoyl-sn-glycero-3-磷酸胆碱（POPC）双层包裹囊性纤维化跨膜电导调节剂跨膜片段3和4（TM3-loop-TM4;环路区域为〜（215）IWELLQASA〜（223））的多个“发夹”结构，其中插入了Pro-Gly残基或在多个位置替换。值得注意的是，Pro-Gly双峰的适当定位导致SDS胶束中的几个突变体采用了稳定的β-折叠结构，如圆二色性光谱所示，同时通过SDS-聚丙烯酰胺凝胶电泳观察到了一系列的低聚物。通过Trp荧光研究野生型（WT）TM3 / 4重构为POPC囊泡，结合溴化磷脂中的位置猝灭剂，表明螺旋WT TM3 / 4的跨双分子层位置，但可能是β的很大程度上是表面包埋的构象环状区域IWPGELLQASA的双表突变体。据我们所知，以前从未观察到膜蛋白具有最小数目的突变的这种完全折叠。这些容易的从α-螺旋到β-折叠的转化突出了环对膜蛋白结构的贡献。

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