JILA, National Institute of Standards and Technology and University of Colorado, Boulder, CO 80309.
JILA, National Institute of Standards and Technology and University of Colorado, Boulder, CO 80309;
Proc Natl Acad Sci U S A. 2021 Mar 30;118(13). doi: 10.1073/pnas.2020083118.
Single amino acid mutations provide quantitative insight into the energetics that underlie the dynamics and folding of membrane proteins. Chemical denaturation is the most widely used assay and yields the change in unfolding free energy (ΔΔ). It has been applied to >80 different residues of bacteriorhodopsin (bR), a model membrane protein. However, such experiments have several key limitations: 1) a nonnative lipid environment, 2) a denatured state with significant secondary structure, 3) error introduced by extrapolation to zero denaturant, and 4) the requirement of globally reversible refolding. We overcame these limitations by reversibly unfolding local regions of an individual protein with mechanical force using an atomic-force-microscope assay optimized for 2 μs time resolution and 1 pN force stability. In this assay, bR was unfolded from its native bilayer into a well-defined, stretched state. To measure ΔΔ, we introduced two alanine point mutations into an 8-amino-acid region at the C-terminal end of bR's G helix. For each, we reversibly unfolded and refolded this region hundreds of times while the rest of the protein remained folded. Our single-molecule-derived ΔΔ for mutant L223A (-2.3 ± 0.6 kcal/mol) quantitatively agreed with past chemical denaturation results while our ΔΔ for mutant V217A was 2.2-fold larger (-2.4 ± 0.6 kcal/mol). We attribute the latter result, in part, to contact between Val and a natively bound squalene lipid, highlighting the contribution of membrane protein-lipid contacts not present in chemical denaturation assays. More generally, we established a platform for determining ΔΔ for a fully folded membrane protein embedded in its native bilayer.
单个氨基酸突变提供了定量的见解，深入了解膜蛋白的动力学和折叠的基础能量学。化学变性是最广泛使用的测定方法，产生解折叠自由能的变化(ΔΔ)。它已经被应用于>80 个不同的细菌视紫红质（bR）残基，一个模型膜蛋白。然而，这样的实验有几个关键的限制：1）非天然的脂质环境，2）具有显著二级结构的变性状态，3）外推到零变性剂的误差，4）需要全局可逆的重折叠。我们通过使用原子力显微镜测定法克服了这些限制，该测定法针对 2 μs 时间分辨率和 1 pN 力稳定性进行了优化，该方法通过机械力可逆地展开单个蛋白质的局部区域。在这个测定中，bR 从其天然双层结构展开成一个定义明确的拉伸状态。为了测量ΔΔ，我们在 bR 的 G 螺旋的 C 末端的 8 个氨基酸区域引入了两个丙氨酸点突变。对于每个突变，我们可逆地展开和折叠这个区域数百次，而蛋白质的其余部分保持折叠状态。我们从单分子中得到的突变 L223A 的ΔΔ（-2.3±0.6 kcal/mol）与过去的化学变性结果定量一致，而我们突变 V217A 的ΔΔ则大了 2.2 倍（-2.4±0.6 kcal/mol）。我们将后者的结果归因于 Val 与天然结合的角鲨烯脂质之间的接触，这突出了膜蛋白-脂质接触的贡献，这些接触在化学变性测定中不存在。更一般地说，我们建立了一个平台，用于确定完全折叠的膜蛋白在其天然双层中的ΔΔ。
