Golbek Thaddeus W, Otto Shauna C, Roeters Steven J, Weidner Tobias, Johnson Colin P, Baio Joe E
Department of Chemistry, Aarhus University, 8000 Aarhus C, Denmark.
Department of Biochemistry and Biophysics, Oregon State University, Corvallis, Oregon 97331, United States.
J Phys Chem B. 2021 Jan 14;125(1):148-157. doi: 10.1021/acs.jpcb.0c07143. Epub 2020 Dec 23.
Mechanical stress on sarcolemma can create small tears in the muscle cell membrane. Within the sarcolemma resides the multidomain dysferlin protein. Mutations in this protein render it unable to repair the sarcolemma and have been linked to muscular dystrophy. A key step in dysferlin-regulated repair is the binding of the C2A domain to the lipid membrane upon increased intracellular calcium. Mutations mapped to this domain cause loss of binding ability of the C2A domain. There is a crucial need to understand the geometry of dysferlin C2A at a membrane interface as well as cell membrane lipid reorientation when compared to that of a mutant. Here, we describe a comparison between the wild-type dysferlin C2A and a mutation to the conserved aspartic acids in the domain binding loops. To identify both the geometry and the cell membrane lipid reorientation, we applied sum frequency generation (SFG) vibrational spectroscopy and coupled it with simulated SFG spectra to observe and quantify the interaction with a model cell membrane composed of phosphotidylserine and phosphotidylcholine. Observed changes in surface pressure demonstrate that calcium-bridged electrostatic interactions govern the initial interaction of the C2A domains docking with a lipid membrane. SFG spectra taken from the amide-I region for the wild type and variant contain features near 1642, 1663, and 1675 cm related to the C2A domain β-sandwich secondary structure, indicating that the domain binds in a specific orientation. Mapping simulated SFG spectra to the experimentally collected spectra indicated that both wild-type and variant domains have nearly the same orientation to the membrane surface. However, examining the ordering of the lipids that make up a model membrane using SFG, we find that the wild type clusters the lipids as seen by the increase in the ratio of the CD and CD symmetric intensities by 170% for the wild type and by 120% for the variant. This study highlights the capabilities of SFG to probe with great detail biological mutations in proteins at cell membrane interfaces.
肌膜上的机械应力可在肌细胞膜上造成小的撕裂。肌膜内存在多结构域的dysferlin蛋白。该蛋白的突变使其无法修复肌膜，并与肌肉萎缩症有关。dysferlin调节修复的一个关键步骤是在细胞内钙增加时，C2A结构域与脂质膜结合。定位到该结构域的突变会导致C2A结构域结合能力丧失。与突变体相比，迫切需要了解dysferlin C2A在膜界面的几何结构以及细胞膜脂质重排情况。在此，我们描述了野生型dysferlin C2A与该结构域结合环中保守天冬氨酸突变体之间的比较。为了确定几何结构和细胞膜脂质重排，我们应用和频振动光谱（SFG），并将其与模拟SFG光谱相结合，以观察和量化与由磷脂酰丝氨酸和磷脂酰胆碱组成的模型细胞膜的相互作用。观察到的表面压力变化表明，钙桥接的静电相互作用控制着C2A结构域与脂质膜对接的初始相互作用。从野生型和变体的酰胺-I区域获取的SFG光谱在1642、1663和1675 cm附近具有与C2A结构域β-三明治二级结构相关的特征，表明该结构域以特定方向结合。将模拟SFG光谱映射到实验收集的光谱上表明，野生型和变体结构域与膜表面的方向几乎相同。然而，使用SFG检查构成模型膜的脂质的有序性时，我们发现野生型使脂质聚集，野生型的CD和CD对称强度之比增加了170%，变体增加了120%。这项研究突出了SFG在细胞膜界面详细探测蛋白质生物学突变的能力。
