Department of Physiology, University of Texas Southwestern Medical Center at Dallas, Texas, United States of America.
PLoS Biol. 2010 Dec 7;8(12):e1000555. doi: 10.1371/journal.pbio.1000555.
While the bacterial mechanosensitive channel of large conductance (MscL) is the best studied biological mechanosensor and serves as a paradigm for how a protein can sense and respond to membrane tension, the simple matter of its oligomeric state has led to debate, with models ranging from tetramers to hexamers. Indeed, two different oligomeric states of the bacterial mechanosensitive channel MscL have been resolved by X-ray crystallography: The M. tuberculosis channel (MtMscL) is a pentamer, while the S. aureus protein (SaMscL) forms a tetramer. Because several studies suggest that, like MtMscL, the E. coli MscL (EcoMscL) is a pentamer, we re-investigated the oligomeric state of SaMscL. To determine the structural organization of MscL in the cell membrane we developed a disulfide-trapping approach. Surprisingly, we found that virtually all SaMscL channels in vivo are pentameric, indicating this as the physiologically relevant and functional oligomeric state. Complementing our in vivo results, we purified SaMscL and assessed its oligomeric state using three independent approaches (sedimentation equilibrium centrifugation, crosslinking, and light scattering) and established that SaMscL is a pentamer when solubilized in Triton X-100 and C(8)E(5) detergents. However, performing similar experiments on SaMscL solubilized in LDAO, the detergent used in the crystallographic study, confirmed the tetrameric oligomerization resolved by X-ray crystallography. We further demonstrate that this stoichiometric shift is reversible by conventional detergent exchange experiments. Our results firmly establish the pentameric organization of SaMscL in vivo. Furthermore they demonstrate that detergents can alter the subunit stoichiometry of membrane protein complexes in vitro; thus, in vivo assays are necessary to firmly establish a membrane protein's true functionally relevant oligomeric state.
虽然细菌大电导机械敏感通道（MscL）是研究最为透彻的生物机械感受器，并且为蛋白质如何感知和响应膜张力提供了范例，但它的寡聚状态简单问题却引发了争议，模型范围从四聚体到六聚体。事实上，通过 X 射线晶体学已经解析了细菌机械敏感通道 MscL 的两种不同寡聚状态：结核分枝杆菌通道（MtMscL）是五聚体，而金黄色葡萄球菌蛋白（SaMscL）形成四聚体。由于几项研究表明，与 MtMscL 一样，大肠杆菌 MscL（EcoMscL）也是五聚体，我们重新研究了 SaMscL 的寡聚状态。为了确定 MscL 在细胞膜中的结构组织，我们开发了一种二硫键捕获方法。令人惊讶的是，我们发现体内几乎所有的 SaMscL 通道都是五聚体，表明这是生理相关和功能上的寡聚状态。补充我们的体内结果，我们纯化了 SaMscL，并使用三种独立的方法（沉降平衡离心、交联和光散射）评估了其寡聚状态，并确定当在 Triton X-100 和 C（8）E（5）去污剂中溶解时，SaMscL 是五聚体。然而，在用于晶体学研究的 LDAO 去污剂中溶解的 SaMscL 上进行类似的实验，证实了 X 射线晶体学解析的四聚体寡聚化。我们进一步证明，这种化学计量的转变可以通过常规去污剂交换实验来逆转。我们的结果牢固地确立了 SaMscL 在体内的五聚体组织。此外，它们表明去污剂可以改变体外膜蛋白复合物的亚基化学计量；因此，体内测定对于牢固确立膜蛋白的真实功能相关寡聚状态是必要的。
