Neutron Spin Echo Spectroscopy as a Unique Probe for Lipid Membrane Dynamics and Membrane-Protein Interactions

Published: May 27th, 2021

DOI:

10.3791/62396

Teshani Kumarage^1,2, Julie Nguyen¹, Rana Ashkar^1,2

¹Department of Physics, Virginia Tech, ²Center for Soft Matter and Biological Physics, Virginia Tech

ERRATUM NOTICE

Important: There has been an erratum issued for this article. Read more …

Lipid bilayers form the main matrix of cell membranes and are the primary platform for nutrient exchange, protein-membrane interactions, and viral budding, among other vital cellular processes. For efficient biological activity, cell membranes should be rigid enough to maintain the integrity of the cell and its compartments yet fluid enough to allow membrane components, such as proteins and functional domains, to diffuse and interact. This delicate balance of elastic and fluid membrane properties, and their impact on biological function, necessitate a better understanding of collective membrane dynamics over mesoscopic length and time scales of key biological processes, e.g., membrane deformations and protein binding events. Among the techniques that can effectively probe this dynamic range is neutron spin echo (NSE) spectroscopy. Combined with deuterium labeling, NSE can be used to directly access bending and thickness fluctuations as well as mesoscopic dynamics of select membrane features. This paper provides a brief description of the NSE technique and outlines the procedures for performing NSE experiments on liposomal membranes, including details of sample preparation and deuteration schemes, along with instructions for data collection and reduction. The paper also introduces data analysis methods used to extract key membrane parameters, such as the bending rigidity modulus, area compressibility modulus, and in-plane viscosity. To illustrate the biological importance of NSE studies, select examples of membrane phenomena probed by NSE are discussed, namely, the effect of additives on membrane bending rigidity, the impact of domain formation on membrane fluctuations, and the dynamic signature of membrane-protein interactions.

Erratum

Erratum: Neutron Spin Echo Spectroscopy as a Unique Probe for Lipid Membrane Dynamics and Membrane-Protein Interactions

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 Equation 1 133.4 mg, where X_DMPC and X_DSPC are the mole fractions of DMPC and DSPC, here set to 0.7 and 0.3, respectively, and Mw_DMPC and Mw_DSPC are the molar weights given by 677.9 g/mol and 790.1 g/mol, respectively. Similarly, m_DSPC = 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 Equation 1 133.4 mg, where X_DMPC and X_DSPC are the mole fractions of DMPC and DSPC, here set to 0.7 and 0.3, respectively, and Mw_DMPC and Mw_DSPC are the molar weights given by 677.9 g/mol and 790.1 g/mol, respectively. Similarly, m_DSPC = 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: Equation 5 , where I_coh and I_inc 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: Equation 5 , where I_coh and I_inc 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.