本文介绍了中子自旋回波（NSE）研究脂质膜的样品制备、数据约化和数据分析的实验方案。通过对脂质进行合理的氘代标记，可探测介观尺度的膜动力学行为，这些尺度正是重要生命过程发生的时间和空间范围。
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勘误通知
Important: There has been an erratum issued for this article. View Erratum Notice
摘要
摘要
脂质双分子层构成了细胞膜的主要基质，是营养物质交换、蛋白质-膜 相互作用以及病毒出芽等其他重要细胞过程的主要平台。为了实现高效的生物学活性，细胞膜必须具有足够的刚性以维持细胞及其区室的完整性，同时又需具备足够的流动性，以允许膜组分（如蛋白质和功能结构域）扩散并相互作用。这种弹性与流动性的精细平衡及其对生物学功能的影响，要求我们更深入地理解在介观尺度长度和时间范围内的集体膜动力学，例如膜形变和蛋白质结合事件。在能够有效探测这一动态范围的技术中，中子自旋回波（NSE）光谱技术是其中之一。结合氘标记技术，NSE可用于直接探测弯曲和厚度涨落，以及特定膜特征的介观动力学。本文简要描述了NSE技术，并概述了在脂质体膜上进行NSE实验的步骤，包括样品制备和氘化方案的详细信息，以及数据采集与归约的指导说明。本文还介绍了用于提取关键膜参数的数据分析方法，例如弯曲刚度模量、面积可压缩模量和平面内黏度。为了说明NSE研究的生物学意义，本文讨论了NSE探测膜现象的若干典型实例，即添加剂对膜弯曲刚度的影响、结构域形成对膜涨落的影响，以及膜-蛋白质相互作用的动力学特征。
引言
细胞膜及其功能的理解在过去几十年中取得了显著进展。过去认为细胞膜是被动的脂质双分子层，仅用于界定细胞边界并承载膜蛋白1 逐渐转变为一种动态模型，其中脂质双分子层在调控重要的生物过程（包括细胞信号传导、分子交换和蛋白质功能）中发挥着关键作用 — 仅举几例2,3,4,5,6细胞膜具有高度动态性，持续发生重塑和分子重分布，这一认识推动了科学界对膜结构的研究超越平衡态结构的探索7,8,9因此，已开发出多种方法用于研究生物及仿生脂质膜中的各种动态模式。迄今为止，大多数研究主要集中在分子的扩散运动上10,11,12,13
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方案
1. 实验所需的氘代方案
- 进行弯曲涨落测量时，需在D2O（D 99.9%）或D2O缓冲液（例如用D2O代替H2O配制的磷酸盐缓冲液）中制备完全质子化的脂质体。使用完全质子化的DMPC（C36H72NO8P）和DSPC（C44H88NO8P），其质量按
计算为133.4 mg，其中XDMPC和XDSPC分别为DMPC和DSPC的摩尔分数，在此分别设为0.7和0.3，MwDMPC和MwDSPC分别为677.9 g/mol和790.1 g/mol。类似地，mDSPC = 66.6 mg。该氘化方案可增强膜（NSLD ~ 0 × 10-6 Å-2）与氘化缓冲液（NSLD ~ 6.4....
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结果
通过中子自旋回声（NSE）研究膜弯曲涨落时，通常在约 (0.04 - 0.2) Å-1 的 Q 范围内进行。该 Q 范围对应于介于膜厚度与脂质体半径之间的中间长度尺度，此处弯曲动力学占主导地位。在更宽的 Q 范围内测量可揭示其他动态模式，包括脂质体扩散和膜内动力学。有关 NSE 可探测的膜动力学转变的更多细节，请参阅以下相关文献25,71。需要特别强调的是，NSE 信号与以下表达式成正比：
，其中 Icoh 和 Iinc 分别代表样品的相干散射强度和非相干散射强度。因此，建议将 NSE 脂质体样品制备在氘代缓冲液中（即使用 D2<.......
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讨论
中子自旋回声（NSE）是一种在多种条件下测量脂质膜介观动力学的强大且独特的技术。NSE的有效应用取决于样品质量、中子对比度以及在给定样品中可探测的动力学范围。因此，要成功进行NSE实验并采集高质量数据，必须完成若干关键步骤。在NSE实验前，使用实验室常规方法对脂质体悬浮液进行表征，是确保中子束时间高效利用的关键步骤。例如，通过动态光散射（DLS）可测定挤出法制备的脂质体的粒径分布（或扩散系数），该方法在各实验室或共享平台中普遍可用84。冷冻电子显微镜是近年来在脂质体样品上得到验证的另一种表征方法，通过获取脂质体悬浮液冷冻超薄切片的高分辨率显微图像，可有效评估脂质体的单层结构65、结构域形成85,86，或添加剂（如纳米颗粒76和蛋白质87）的掺入情况。此外，小角X射线散射（SAXS）也可用于表征膜结构
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披露
作者声明无利益冲突，无可披露内容。
致谢
R. Ashkar 感谢 M. Nagao、L.-R. Stingaciu 和 P. Zolnierczuk 提供了许多有益的讨论，以及在他们各自束线上的中子自旋回波实验中给予的频繁协助。作者承认使用了美国国家标准与技术研究院（NIST）和美国橡树岭国家实验室（ORNL）的中子自旋回波谱仪。NIST 的中子自旋回波谱仪由高分辨率中子散射中心资助，该中心是美国国家标准与技术研究院与美国国家科学基金会根据协议号 DMR-1508249 建立的合作项目。ORNL 散裂中子源的中子自旋回波谱仪由美国能源部基础能源科学办公室下属的科学用户设施部门资助。橡树岭国家实验室由 UT-Battelle, LLC 根据美国能源部合同号 DE-AC05-00OR22725 进行管理。
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材料
| 姓名 | 公司 | 目录编号 | 评论 |
|---|---|---|---|
| 氯仿（生物技术级） | Sigma Aldrich | 496189 | 生物技术级，≥99.8%，含0.5-1.0%乙醇作为稳定剂 |
| 循环水浴锅 | Julabo | SE-12 | 带智能泵的加热循环器，可编程温度设置，并配有外部传感器接口用于测量与控制 |
| 氧化氘 | Cambridge Isotopes Laboratories | DLM-4 | 氘代水；重水（D2O）（D，99.9%） |
| 数字半微量天平 | Mettler Toledo | MS105 | 半微量天平，量程120 g，可读性0.01 mg，配备高分辨率称重传感器、符合人体工学的门设计以及移液器校验功能 |
| 乙醇（分子生物学级） | Sigma Aldrich | E7023 | 200 proof乙醇，适用于分子生物学应用 |
| 玻璃移液管 | VWR | 36360-536 | 一次性钠钙玻璃巴斯德移液管 |
| 玻璃小瓶 | Thermo Scientific | B7990-1 | 硼硅酸盐玻璃小瓶，配有PTFE/硅胶隔垫盖 |
| 实验室级冷冻柜 | Fisher Scientific | IU2886D | 超低温冷冻柜（-86 至 -50 °C），用于脂质和蛋白质的长期储存 |
| 脂质（部分氘代或全氘代） | Avanti Polar Lipids | 根据脂质种类而定 | 脂质可从Avanti公司购买粉末形式或氯仿溶液形式，可根据需要选择特定含量和氘代模式 |
| Millipore纯水器 | Millipore Sigma | ZRQSVP3US | Direct-Q® 3 UV纯水系统，内置紫外灯，可提供纯水和超纯水，降低有机物含量，适用于生物 应用 |
| 小型挤出器套装 | Avanti Polar Lipids | 610020 | 小型挤出器套装包括小型挤出器、加热模块、2支气密型注射器、2个O型圈、聚碳酸酯膜和滤膜支撑装置 |
| 快接接头 | Grainger | 2YDA1 和 2YDA7 | 按钮式管路接头，适用于QuickConnect水循环应用，例如高温囊泡挤出 |
| 注射泵 | SyringePump.com | New Era-1000 | 完全可编程注射泵，支持注入和回吸操作；可设置多达41个泵送阶段，调节泵送速率、分配体积和挤出循环次数 |
| 超声波清洗仪 | Fisher Scientific | CPX2800 | 温度可控的超声波清洗仪，具备可编程功能，适用于脱气和超声处理 |
| 真空烘箱 | Thermo Scientific | 3608 | 0.7立方英尺真空烘箱，内置高温限温器，防止过热 |
| 涡旋混合器 | Fisher Scientific | 02-215-414 | 可变转速模拟控制，支持低转速启动以实现温和振荡，或高速混合以实现样品的剧烈涡旋 |
参考文献
- Singer, S. J., Nicolson, G. L. The fluid mosaic model of the structure of cell membranes. Science. 175 (4023), 720-731 (1972).
- Andersen, O. S., Koeppe, R. E. Bilayer thickness and membrane protein function: an energetic perspective. Annual ....
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重印与许可
勘误
Formal Correction: Erratum: Neutron Spin Echo Spectroscopy as a Unique Probe for Lipid Membrane Dynamics and Membrane-Protein Interactions
Posted by JoVE Editors on 8/06/2021. Citeable Link.
An erratum was issued for: Neutron Spin Echo Spectroscopy as a Unique Probe for Lipid Membrane Dynamics and Membrane-Protein Interactions. The Introduction, Protocol, and Representative Results sections have been updated.
In the Introduction, the fith pargraph was updated from:
Besides direct access to the length and time scale of membrane dynamics, NSE has the inherent capabilities of neutron isotope sensitivity52. Specifically, the ability of neutrons to interact differently with the isotopes of hydrogen, the most abundant element in biological systems, results in a different neutron scattering length density,34 or NSLD (the equivalent of the optical index of refraction50), when protium is substituted by deuterium. This enables an approach known as contrast variation, which is commonly used to highlight specific membrane features or conceal others — the latter scenario is referred to as contrast matching. A frequent application of contrast variation/matching is the substitution of water (NSLD = -0.56 × 10-6 Å-2) by heavy water or D2O (NSLD = 6.4 × 10-6 Å-2) to amplify the neutron signal from protiated lipid membranes (NSLD ~ 2 × 10-6 Å-2). This approach is highly effective in studies of membrane structure because the penetration of D2O into the headgroup region of the membrane allows accurate determination of the membrane thicknesses (see Figure 2A, left panel) and of the location of different lipid subgroups when more sophisticated models are applied53,54. This paper highlights some examples on the use of contrast variation for studies of collective dynamics in biomimetic membranes and select membrane features.
to:
Besides direct access to the length and time scale of membrane dynamics, NSE has the inherent capabilities of neutron isotope sensitivity52. Specifically, the ability of neutrons to interact differently with the isotopes of hydrogen, the most abundant element in biological systems, results in a different neutron scattering length density,34 or NSLD (the equivalent of the optical index of refraction50), when protium is substituted by deuterium. This enables an approach known as contrast variation, which is commonly used to highlight specific membrane features or conceal others — the latter scenario is referred to as contrast matching. A frequent application of contrast variation/matching is the substitution of water (NSLD = -0.56 × 10-6 Å-2) by heavy water or D2O (NSLD = 6.4 × 10-6 Å-2) to amplify the neutron signal from protiated lipid membranes (NSLD ~ 0 × 10-6 Å-2). This approach is highly effective in studies of membrane structure because the penetration of D2O into the headgroup region of the membrane allows accurate determination of the membrane thicknesses (see Figure 2A, left panel) and of the location of different lipid subgroups when more sophisticated models are applied53,54. This paper highlights some examples on the use of contrast variation for studies of collective dynamics in biomimetic membranes and select membrane features.
In the Protocol, step 1.1 was updated from:
For bending fluctuation measurements, make fully protiated liposomes in D2O (D 99.9%) or D2O-buffer (e.g., phosphate buffer prepared with D2O instead of H2O). Use fully protiated DMPC (C36H72NO8P) and DSPC (C44H88NO8P) with 133.4 mg, where XDMPC and XDSPC are the mole fractions of DMPC and DSPC, here set to 0.7 and 0.3, respectively, and MwDMPC and MwDSPC are the molar weights given by 677.9 g/mol and 790.1 g/mol, respectively. Similarly, mDSPC = 66.6 mg. This deuteration scheme increases the scattering contrast between the membrane (NSLD ~ 2 × 10-6 Å-2) and the deuterated buffer (NSLD ~ 6.4 × 10-6 Å-2) and amplifies the signal from membrane undulations (see Figure 2A left panel).
to:
For bending fluctuation measurements, make fully protiated liposomes in D2O (D 99.9%) or D2O-buffer (e.g., phosphate buffer prepared with D2O instead of H2O). Use fully protiated DMPC (C36H72NO8P) and DSPC (C44H88NO8P) with 133.4 mg, where XDMPC and XDSPC are the mole fractions of DMPC and DSPC, here set to 0.7 and 0.3, respectively, and MwDMPC and MwDSPC are the molar weights given by 677.9 g/mol and 790.1 g/mol, respectively. Similarly, mDSPC = 66.6 mg. This deuteration scheme increases the scattering contrast between the membrane (NSLD ~ 0 × 10-6 Å-2) and the deuterated buffer (NSLD ~ 6.4 × 10-6 Å-2) and amplifies the signal from membrane undulations (see Figure 2A left panel).
In the Representative Results, the fist pagargaph was updted from:
NSE studies accessing bending fluctuations are typically performed over a Q-range of ~ (0.04 - 0.2) Å-1. This Q-range corresponds to intermediate length scales between the membrane thickness and the liposomal radius, where bending dynamics dominate. Measurement over an extended Q-range can give access to additional dynamic modes, including liposomal diffusion and intramembrane dynamics. For more details on the cross-over in membrane dynamics accessed by NSE, check these relevant publications25,71. It is important to emphasize that NSE signals are proportional to: , where Icoh and Iinc are, respectively, the coherent and incoherent scattering intensity from the sample. Therefore, it is advisable to prepare NSE liposomal samples in deuterated buffers (i.e., buffers prepared with D2O instead of H2O) to minimize the incoherent scattering signal, mainly contributed by the hydrogen content of the sample. However, in some cases intermediate deuteration schemes (i.e., using mixtures of D2O and H2O) might be necessary to obtain optimal contrast conditions. Typically, NSE measurements of membrane bending fluctuations are performed on fully protiated liposomes in deuterated buffer, referred to as fully contrasted liposomes in Figure 5. This deuteration scheme results in a large NSLD difference between the membrane core (~2 × 10-6 Å-2) and its deuterated fluid environment (~6.4 × 10-6 Å-2), which significantly enhances the scattering signal from the liposomal membranes and improves the measurement statistics of bending dynamics. This contrast scheme (Figure 2A left panel) is frequently utilized in studies of bending rigidity of lipid membranes with single38,72 and multiple39,66 lipid components and in studies of membrane softening/stiffening by biological inclusions (e.g., cholesterol, drug molecules, peptides/proteins)36,37,73,74,75, and synthetic additives (e.g., nanoparticles)76,77.
to:
NSE studies accessing bending fluctuations are typically performed over a Q-range of ~ (0.04 - 0.2) Å-1. This Q-range corresponds to intermediate length scales between the membrane thickness and the liposomal radius, where bending dynamics dominate. Measurement over an extended Q-range can give access to additional dynamic modes, including liposomal diffusion and intramembrane dynamics. For more details on the cross-over in membrane dynamics accessed by NSE, check these relevant publications25,71. It is important to emphasize that NSE signals are proportional to: , where Icoh and Iinc are, respectively, the coherent and incoherent scattering intensity from the sample. Therefore, it is advisable to prepare NSE liposomal samples in deuterated buffers (i.e., buffers prepared with D2O instead of H2O) to minimize the incoherent scattering signal, mainly contributed by the hydrogen content of the sample. However, in some cases intermediate deuteration schemes (i.e., using mixtures of D2O and H2O) might be necessary to obtain optimal contrast conditions. Typically, NSE measurements of membrane bending fluctuations are performed on fully protiated liposomes in deuterated buffer, referred to as fully contrasted liposomes in Figure 5. This deuteration scheme results in a large NSLD difference between the membrane core (~0 × 10-6 Å-2) and its deuterated fluid environment (~6.4 × 10-6 Å-2), which significantly enhances the scattering signal from the liposomal membranes and improves the measurement statistics of bending dynamics. This contrast scheme (Figure 2A left panel) is frequently utilized in studies of bending rigidity of lipid membranes with single38,72 and multiple39,66 lipid components and in studies of membrane softening/stiffening by biological inclusions (e.g., cholesterol, drug molecules, peptides/proteins)36,37,73,74,75, and synthetic additives (e.g., nanoparticles)76,77.
In the Representative Reults, Figure 2 was updated from:
Figure 2: Examples of possible deuteration schemes in NSE experiments on lipid membranes. (A) Left: Fully contrasted membranes, e.g., protiated membranes in deuterated buffer, showing the NSLD profile along the normal to the membrane surface. The difference in the NSLD between the headgroup (~2 × 10-2 Å-2) and tail region (~4.5 × 10-6 Å-2) of the membrane is due to the headgroup hydration with deuterated buffer. Right: Tail-contrast matched membranes such that the hydrocarbon tail region of the membrane has the same NSLD as the buffer, as shown in the corresponding NSLD profile along the membrane normal. (B) Domain-forming membranes with two neutron contrast schemes where the domains (center) or the matrix (left) are contrast-matched to the buffer, enabling selective studies of matrix or domain dynamics, respectively. This figure has been modified from Nickels et al., JACS 201541. (C) Asymmetric membranes prepared by cyclodextrin exchange between protiated and deuterated lipid vesicles, resulting in the deuteration of one membrane leaflet while keeping the other leaflet protiated. This allows studies of the bending dynamics of the protiated leaflet and provides insights into the mechanical coupling between opposing leaflets in asymmetric membranes. This figure has been modified from Rickeard et al., Nanoscale 202040. Please click here to view a larger version of this figure.
to:
Figure 2: Examples of possible deuteration schemes in NSE experiments on lipid membranes. (A) Left: Fully contrasted membranes, e.g., protiated membranes in deuterated buffer, showing the NSLD profile along the normal to the membrane surface. The difference in the NSLD between the tail region (~0 × 10-2 Å-2) and headgroup region (~4.5 × 10-6 Å-2) of the membrane is due to the headgroup hydration with deuterated buffer. Right: Tail-contrast matched membranes such that the hydrocarbon tail region of the membrane has the same NSLD as the buffer, as shown in the corresponding NSLD profile along the membrane normal. (B) Domain-forming membranes with two neutron contrast schemes where the domains (center) or the matrix (left) are contrast-matched to the buffer, enabling selective studies of matrix or domain dynamics, respectively. This figure has been modified from Nickels et al., JACS 201541. (C) Asymmetric membranes prepared by cyclodextrin exchange between protiated and deuterated lipid vesicles, resulting in the deuteration of one membrane leaflet while keeping the other leaflet protiated. This allows studies of the bending dynamics of the protiated leaflet and provides insights into the mechanical coupling between opposing leaflets in asymmetric membranes. This figure has been modified from Rickeard et al., Nanoscale 202040. Please click here to view a larger version of this figure.
