Abstract
Biology
ERRATUM NOTICE
Important: There has been an erratum issued for this article. Read more …脂质双层体是细胞膜的主要基质,是营养交换、蛋白膜相互作用和病毒萌芽等重要细胞过程的主要平台。对于有效的生物活性,细胞膜应足够刚性,以保持细胞及其隔间的完整性,但流动性足以使膜成分(如蛋白质和功能领域)扩散和相互作用。这种弹性和流体膜特性的微妙平衡及其对生物功能的影响,需要更好地了解关键生物过程(例如膜变形和蛋白质结合事件)的中观长度和时间尺度上的集体膜动力学。能够有效探测这一动态范围的技术包括中子自旋回波(NSE)光谱学。结合二元标签,NSE可用于直接访问弯曲和厚度波动以及选择膜特征的中等动力学。本文简要介绍了NSE技术,概述了在脂膜上进行NSE实验的程序,包括样品制备和去子宫化方案的细节,以及数据收集和减少说明。本文还介绍了用于提取关键膜参数的数据分析方法,如弯曲刚性模态、区域可压缩模态和平面粘度。为了说明NSE研究的生物学重要性,探讨了NSE所探讨的膜现象的选例,即添加剂对膜弯曲刚性的影响、域形成对膜波动的影响以及膜蛋白相互作用的动态特征。
Erratum
Erratum: Neutron Spin Echo Spectroscopy as a Unique Probe for Lipid Membrane Dynamics and Membrane-Protein InteractionsAn 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.
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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.
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