Marassi F M, Ma C, Gratkowski H, Straus S K, Strebel K, Oblatt-Montal M, Montal M, Opella S J
Department of Chemistry, University of Pennsylvania, Philadelphia, PA 19104, USA.
Proc Natl Acad Sci U S A. 1999 Dec 7;96(25):14336-41. doi: 10.1073/pnas.96.25.14336.
Vpu is an 81-residue membrane protein encoded by the HIV-1 genome. NMR experiments show that the protein folds into two distinct domains, a transmembrane hydrophobic helix and a cytoplasmic domain with two in-plane amphipathic alpha-helices separated by a linker region. Resonances in one-dimensional solid-state NMR spectra of uniformly (15)N labeled Vpu are clearly segregated into two bands at chemical shift frequencies associated with NH bonds in a transmembrane alpha-helix, perpendicular to the membrane surface, and with NH bonds in the cytoplasmic helices parallel to the membrane surface. Solid-state NMR spectra of truncated Vpu(2-51) (residues 2-51), which contains the transmembrane alpha-helix and the first amphipathic helix of the cytoplasmic domain, and of a construct Vpu(28-81) (residues 28-81), which contains only the cytoplasmic domain, support this structural model of Vpu in the membrane. Full-length Vpu (residues 2-81) forms discrete ion-conducting channels of heterogeneous conductance in lipid bilayers. The most frequent conductances were 22 +/- 3 pS and 12 +/- 3 pS in 0.5 M KCl and 29 +/- 3 pS and 12 +/- 3 pS in 0.5 M NaCl. In agreement with the structural model, truncated Vpu(2-51), which has the transmembrane helix, forms discrete channels in lipid bilayers, whereas the cytoplasmic domain Vpu(28-81), which lacks the transmembrane helix, does not. This finding shows that the channel activity is associated with the transmembrane helical domain. The pattern of channel activity is characteristic of the self-assembly of conductive oligomers in the membrane and is compatible with the structural and functional findings.
Vpu是一种由HIV-1基因组编码的含81个氨基酸残基的膜蛋白。核磁共振实验表明，该蛋白折叠成两个不同的结构域，一个跨膜疏水螺旋和一个细胞质结构域，其中有两个平面内两亲性α螺旋，由一个连接区隔开。在均匀（15）N标记的Vpu的一维固态核磁共振谱中，共振信号在化学位移频率处明显分为两条带，一条与垂直于膜表面的跨膜α螺旋中的NH键相关，另一条与平行于膜表面的细胞质螺旋中的NH键相关。截短的Vpu（2-51）（第2至51位氨基酸残基）包含跨膜α螺旋和细胞质结构域的第一个两亲性螺旋，以及构建体Vpu（28-81）（第28至81位氨基酸残基），其仅包含细胞质结构域，它们的固态核磁共振谱支持了Vpu在膜中的这种结构模型。全长Vpu（第2至81位氨基酸残基）在脂质双层中形成具有异质电导的离散离子传导通道。在0.5 M KCl中，最常见的电导为22±3 pS和12±3 pS；在0.5 M NaCl中，最常见的电导为29±3 pS和12±3 pS。与结构模型一致，具有跨膜螺旋的截短的Vpu（2-51）在脂质双层中形成离散通道，而缺乏跨膜螺旋的细胞质结构域Vpu（28-81）则不能。这一发现表明通道活性与跨膜螺旋结构域相关。通道活性模式是膜中导电寡聚体自组装的特征，并且与结构和功能研究结果相符。
