School of Physics and Astronomy, University of Nottingham, University Park, Nottingham, NG7 2RD, UK.
Department of Chemistry, Binghamton University, the State University of New York, Binghamton, NY, 13902, USA.
Chem Phys Lipids. 2021 May;236:105071. doi: 10.1016/j.chemphyslip.2021.105071. Epub 2021 Mar 11.
The cellular membrane disruption induced by the aggregation of Aβ peptide has been proposed as a plausible cause of neuronal cell death during Alzheimer's disease. The molecular-level details of the Aβ interaction with cellular membranes were previously probed using solid state NMR (ssNMR), however, due to the limited sensitivity of the latter, studies were limited to samples with high Aβ-to-lipid ratio. The dynamic nuclear polarization (DNP) is a technique for increasing the sensitivity of NMR. In this work we demonstrate the feasibility of DNP-enhanced ssNMR studies of Aβ peptide interacting with various model liposomes: (1) a mixture of zwitterionic 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) and negatively charged 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG); (2) a mixture of POPC, POPG, cholesterol, sphingomyelin and ganglioside GM1; (3) the synaptic plasma membrane vesicles (SPMVs) extracted from rat brain tissues. In addition, DNP-ssNMR was applied to capturing changes in Aβ conformation taking place upon the peptide insertion into POPG liposomes. The signal enhancements under conditions of DNP allow carrying out informative 2D ssNMR experiments with about 0.25 mg of Aβ peptides (i.e. reaching Aβ-to-lipid ratio of 1:200). In the studied liposome models, the C NMR chemical shifts at many C-labelled sites of Aβ are characteristic of β-sheets. In addition, in POPG liposomes the peptide forms hydrophobic contacts F19-L34 and F19-I32. Both the chemical shifts and hydrophobic contacts of Aβ in POPG remain the same before and after 8 h of incubation. This suggests that conformation at the C-labelled sites of the peptide is similar before and after the insertion process. Overall, our results demonstrate that DNP helps to overcome the sensitivity limitation of ssNMR, and thereby expand the applicability of ssNMR for charactering the Aβ peptide interacting with lipids.
细胞外膜的破坏是由 Aβ 肽的聚集引起的，这被认为是阿尔茨海默病期间神经元细胞死亡的一个合理原因。之前使用固态 NMR（ssNMR）研究了 Aβ 与细胞膜的相互作用的分子水平细节，但是，由于后者的灵敏度有限，因此研究仅限于 Aβ 与脂质的高比例的样品。动态核极化（DNP）是一种提高 NMR 灵敏度的技术。在这项工作中，我们证明了 DNP 增强的 ssNMR 研究 Aβ 肽与各种模型脂质体相互作用的可行性：（1） 一种两性离子 1-棕榈酰-2-油酰基-甘油-3-磷酸胆碱（POPC）和带负电荷的 1-棕榈酰基-2-油酰基-sn-甘油-3-磷酸-（1'-rac-甘油）（POPG）的混合物;（2）POPC、POPG、胆固醇、神经鞘磷脂和神经节苷脂 GM1 的混合物;（3）从大鼠脑组织中提取的突触质膜小泡（SPMVs）。此外，应用 DNP-ssNMR 捕获肽插入 POPG 脂质体时 Aβ 构象的变化。在 DNP 条件下的信号增强允许使用约 0.25 mg 的 Aβ 肽进行信息丰富的 2D ssNMR 实验（即达到 Aβ 与脂质的比例为 1：200）。在所研究的脂质体模型中，许多 C 标记的 Aβ 位点的 C NMR 化学位移具有β-折叠的特征。此外，在 POPG 脂质体中，肽形成疏水性接触 F19-L34 和 F19-I32。在孵育 8 小时前后，POPG 脂质体中的 Aβ 的 C NMR 化学位移和疏水性接触保持不变。这表明肽的 C 标记位点的构象在插入过程前后相似。总的来说，我们的结果表明，DNP 有助于克服 ssNMR 的灵敏度限制，从而扩大 ssNMR 用于表征与脂质相互作用的 Aβ 肽的适用性。
