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.

中文翻译：

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使用锯齿形力斜坡以更高的精度检测膜蛋白展开中间体

对稳定脂双层中膜蛋白的能量和相互作用进行精确量化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其天然脂质双层中的单个膜蛋白，通常是通过以恒定速度收回悬臂。最近，使用聚焦离子束修饰的超短悬臂梁检测到由少至两个氨基酸分隔的展开中间体。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一个子集。此外，通过蠕虫状链分析对这些中间体进行结构分配受到短暂停留时间以及热和仪器噪声的阻碍。为了克服这些问题，我们以 6-12 nm 的锯齿图案移动悬臂，每个周期偏移 0.25-1 nm，产生交替正负负载率的“之字形”力斜坡。我们将此协议应用于模型膜蛋白细菌视紫红质 (bR)。与提取 bR 的光活性视网膜以及第一个跨膜螺旋的传统研究相比，我们在其视网膜存在的情况下展开了 br。为此，我们在螺旋 E 和 F 之间引入了先前开发的酶裂解位点，并使用位点特异性共价连接从 E 螺旋的顶部拉出。与传统的恒速记录相比，由此产生的锯齿形展开轨迹在每个轨迹上占据的状态多 40%，并且占据这些状态的时间更长。总共，我们在 EF 切割的 bR 的五个螺旋的展开过程中鉴定了 31 个中间体。其中包括先前报道的位于螺旋 C 和螺旋 B 之间的机械稳定中间体，随着我们的分辨率提高，现在显示为由三个氨基酸分隔的两个不同状态。有趣的是，另一种中间体直接与视网膜相互作用，通过去除视网膜证实了这种相互作用。