Membrane-Protein Unfolding Intermediates Detected with Enhanced Precision Using a Zigzag Force Ramp
Membrane-Protein Unfolding Intermediates Detected with Enhanced Precision Using a Zigzag Force Ramp
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DOI：
10.1016/j.bpj.2019.12.003
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发表时间：
2020-02-04
影响因子：
3.4
通讯作者：
Perkins, Thomas T.
中科院分区：
文献类型：
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作者：
Jacobson, David R.;Uyetake, Lyle;Perkins, Thomas T.
精确定量的能量和相互作用，稳定膜蛋白在脂质双层是一个长期追求的目标。为此，原子力显微镜已被用于展开嵌入在其天然脂质双层中的单个膜蛋白，通常通过以恒定速度缩回悬臂。最近，使用聚焦离子束改性的超短光杠杆检测到由少至两个氨基酸分离的展开中间体。然而，明确区分这种紧密间隔的状态仍然具有挑战性，部分原因是任何单独的展开轨迹仅占据中间体总数的子集。此外，这些中间体通过蠕虫状链分析的结构分配受到短暂停留时间与热和仪器噪声的阻碍。为了克服这些问题，我们以6-12 nm的方向图移动悬臂，每个周期偏移0.25-1 nm，产生交替的正和负加载速率的“方向图”力斜坡。我们将此协议的模型膜蛋白细菌视紫红质（bR）。与提取bR的光活性视网膜沿着与第一跨膜螺旋的传统研究相反，我们在其视网膜的存在下展开bR。为此，我们在螺旋E和F之间引入了先前开发的酶切位点，并使用位点特异性共价连接从E螺旋的顶部拉出。由此产生的之字形展开轨迹占用40%以上的状态，每个轨迹和占用这些状态的时间比传统的等速记录。总之，我们确定了31个中间体在展开的EF切割的bR的5个螺旋。这些包括以前报道的，位于螺旋C和B之间的机械上坚固的中间体，随着我们的分辨率提高，现在显示为由三个氨基酸分开的两个不同的状态。有趣的是，另一种中间体直接与视黄醛相互作用，这种相互作用通过去除视黄醛来证实。
Precise quantification of the energetics and interactions that stabilize membrane proteins in a lipid bilayer is a long-sought goal. Toward this end, atomic force microscopy has been used to unfold individual membrane proteins embedded in their native lipid bilayer, typically by retracting the cantilever at a constant velocity. Recently, unfolding intermediates separated by as few as two amino acids were detected using focused-ion-beam-modified ultrashort cantilevers. However, unambiguously discriminating between such closely spaced states remains challenging, in part because any individual unfolding trajectory only occupies a subset of the total number of intermediates. Moreover, structural assignment of these intermediates via worm-like-chain analysis is hindered by brief dwell times compounded with thermal and instrumental noise. To overcome these issues, we moved the cantilever in a sawtooth pattern of 6-12 nm, offset by 0.25-1 nm per cycle, generating a "zigzag'' force ramp of alternating positive and negative loading rates. We applied this protocol to the model membrane protein bacteriorhodopsin (bR). In contrast to conventional studies that extract bR's photoactive retinal along with the first transmembrane helix, we unfolded bR in the presence of its retinal. To do so, we introduced a previously developed enzymatic-cleavage site between helices E and F and pulled from the top of the E helix using a site-specific, covalent attachment. The resulting zigzag unfolding trajectories occupied 40% more states per trajectory and occupied those states for longer times than traditional constant-velocity records. In total, we identified 31 intermediates during the unfolding of five helices of EF-cleaved bR. These included a previously reported, mechanically robust intermediate located between helices C and B that, with our enhanced resolution, is now shown to be two distinct states separated by three amino acids. Interestingly, another intermediate directly interacted with the retinal, an interaction confirmed by removing the retinal.