Probing the oligomeric state of phospholamban variants in phospholipid bilayers from solid-state NMR measurements of rotational diffusion rates.
Probing the oligomeric state of phospholamban variants in phospholipid bilayers from solid-state NMR measurements of rotational diffusion rates.
复制标题
通过旋转扩散速率的固态核磁共振测量来探测磷脂双层中受磷蛋白变体的寡聚状态。
作者：
E. Hughes;J. Clayton;D. Middleton
受磷蛋白（PLB）是一种小的跨膜蛋白，可调节心肌细胞肌浆网（SR）中的钙转运。PLB在十二烷基硫酸钠（SDS）胶束中自缔合成五聚体，但PLB在SR膜中的低聚状态尚不清楚。这项工作已经表明，PLB的突变体，其中所有天然半胱氨酸残基被丙氨酸取代（Ala-PLB），在SDS-PAGE凝胶上作为单体运行，与先前的研究一致[Karim等人（2000）Biochemistry 39，10892-10897]。相比之下，代表无半胱氨酸突变体（TM-Ala-PLB）的跨膜结构域的肽在凝胶上以五聚体、二聚体和单体共存。用固体核磁共振方法研究了Ala-PLB和TM-Ala-PLB的大小和异质性，并在跨膜结构域中标记了（13）C和（2）H，并掺入到二肉豆蔻酰磷脂酰胆碱（DMPC）双层中。Ala-PLB和TM-Ala-PLB的宽谱线（2）H NMR和（13）C交叉极化魔角旋转（CP-MAS）NMR谱揭示了膜中每种蛋白质的两种不同种类。在Ala-PLB的情况下，最初存在一个物种，12小时后出现第二个物种。对两种Ala-PLB的（1）H-（13）C偶极耦合的测量表明，一种物质的旋转扩散相对较快，由小于10 μ s的相关时间（τ（R））定义，而另一种物质的旋转相对较慢（τ（R）约60 μ s）。这些结果表明，尽管Ala-PLB在凝胶上作为单体运行，但DMPC双层中存在不同寡聚形式的蛋白质的混合物，可能是单体和五聚体。因此，在使用SDS-PAGE得出关于脂质双层中PLB变体的寡聚状态的结论时必须谨慎。
Phospholamban (PLB) is a small transmembrane protein that regulates calcium transport across the sarcoplasmic reticulum (SR) of cardiac cells. PLB self-associates into pentamers within sodium dodecyl sulfate (SDS) micelles, but the oligomeric status of PLB in SR membranes is not known. This work has shown that a mutant of PLB, with all native cysteine residues replaced by alanine (Ala-PLB), runs as a monomer on SDS-PAGE gels, in agreement with previous studies [Karim et al. (2000) Biochemistry 39, 10892-10897]. By contrast, a peptide representing the transmembrane domain of the cysteine-free mutant (TM-Ala-PLB) coexists as pentamers, dimers, and monomers on gels. Solid-state NMR methods were used to examine the size and heterogeneity of Ala-PLB and TM-Ala-PLB labeled with (13)C and (2)H in the transmembrane domain and incorporated into dimyristoylphosphatidylcholine (DMPC) bilayers. Wide line (2)H NMR and (13)C cross-polarization magic-angle spinning (CP-MAS) NMR spectra of Ala-PLB and TM-Ala-PLB revealed two distinct species of each of the proteins in the membranes. In the case of Ala-PLB one species was present initially and a second species emerged after 12 h. Measurements of (1)H-(13)C dipolar couplings for the two species of Ala-PLB showed that the rotational diffusion of one species was relatively rapid, defined by a correlation time (tau(R)) of less than 10 micros, whereas the rotation of the other species was comparatively slow (tau(R) approximately 60 micros). These results suggest that although Ala-PLB runs as a monomer on gels, a mixture of different oligomeric forms of the protein, possibly monomers and pentamers, is present in DMPC bilayers. Caution must therefore be exercised in using SDS-PAGE to draw conclusions about the oligomeric state of PLB variants in lipid bilayers.