Fourier transform infrared spectroscopy and site-directed isotope labeling as a probe of local secondary structure in the transmembrane domain of phospholamban.
Fourier transform infrared spectroscopy and site-directed isotope labeling as a probe of local secondary structure in the transmembrane domain of phospholamban.
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傅里叶变换红外光谱和定点同位素标记作为受磷蛋白跨膜域局部二级结构的探针。
DOI：
10.1016/s0006-3495(96)79735-7
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发表时间：
1996
影响因子：
3.4
通讯作者：
Rothschild,KJ
中科院分区：
文献类型：
--
作者：
Ludlam,CF;Arkin,IT;Liu,XM;Rothman,MS;Rath,P;Aimoto,S;Smith,SO;Engelman,DM;Rothschild,KJ
受磷蛋白是一种由52个氨基酸残基组成的膜蛋白，调节心肌细胞肌浆网中Ca（2+）-ATP酶的活性。受磷蛋白（hPLB）的疏水性C-末端28个氨基酸片段将蛋白质锚定在膜中，并可能形成Ca（2+）选择性离子通道的一部分。我们使用偏振衰减全反射-傅里叶变换红外（ATR-FTIR）光谱沿着与定点同位素标记来探测hPLB的局部结构。分别出现在1658 cm-1和1544 cm-1处的酰胺I和II带的频率和二色性表明，重构成二肉豆蔻酰磷脂酰胆碱双层膜的脱水和水合hPLB主要是α-螺旋的，并且具有净跨膜取向。通过在蛋白质骨架中的两个位置掺入13 C来探测hPLB的特定局部二级结构。在1614 cm-1附近观察到的小条带归属于13 C标记酰胺羰基的酰胺I模式。该带的频率和二色性表明残基39和46是α-螺旋的，具有相对于膜法线大约30度的轴向取向。在暴露于2 H2O（D2 O）时，hPLB中30%的肽酰胺基团经历缓慢的氘/氢交换。蛋白质的其余部分，包括Leu-39和Leu-42的肽基团，似乎无法交换，表明大部分hPLB片段嵌入脂质双层中。通过扩展PLB的光谱表征，包括水合，氘代以及定点同位素标记的hPLB膜，我们的研究结果强烈支持预测存在的α-螺旋疏水区域跨越膜结构域的PLB模型。
Phospholamban is a 52-amino acid residue membrane protein that regulates Ca(2+)-ATPase activity in the sarcoplasmic reticulum of cardiac muscle cells. The hydrophobic C-terminal 28 amino acid fragment of phospholamban (hPLB) anchors the protein in the membrane and may form part of a Ca(2+)-selective ion channel. We have used polarized attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectroscopy along with site-directed isotope labeling to probe the local structure of hPLB. The frequency and dichroism of the amide I and II bands appearing at 1658 cm-1 and 1544 cm-1, respectively, show that dehydrated and hydrated hPLB reconstituted into dimyristoylphosphatidycholine bilayer membranes is predominantly alpha-helical and has a net transmembrane orientation. Specific local secondary structure of hPLB was probed by incorporating 13C at two positions in the protein backbone. A small band seen near 1614 cm-1 is assigned to the amide I mode of the 13C-labeled amide carbonyl group(s). The frequency and dichroism of this band indicate that residues 39 and 46 are alpha-helical, with an axial orientation that is approximately 30 degrees relative to the membrane normal. Upon exposure to 2H2O (D2O), 30% of the peptide amide groups in hPLB undergo a slow deuterium/hydrogen exchange. The remainder of the protein, including the peptide groups of Leu-39 and Leu-42, appear inaccessible to exchange, indicating that most of the hPLB fragment is embedded in the lipid bilayer. By extending spectroscopic characterization of PLB to include hydrated, deuterated as well as site-directed isotope-labeled hPLB films, our results strongly support models of PLB that predict the existence of an alpha-helical hydrophobic region spanning the membrane domain.