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

This protocol describes sample preparation and data reduction required for successful neutron spin echo studies of collective membrane fluctuations of relevance to biological functions and practical applications of lipid membranes. NSE directly accesses membrane dynamics on the nanosecond timescales of key membrane functions with the unique advantage of isotope sensitivity for probing selective membrane features inaccessible with other techniques. Studies of membrane dynamics on the nanoscale are essential to understanding the molecular mechanism's underlying membrane properties and membrane-protein interactions implicated in various cell pathologies.

The method provides researchers new to NSE with detailed guidelines on designing, preparing, and characterizing lipid vesicles for successful experiments and subsequent data reduction analysis and interpretation. Demonstrating the procedure will be Teshani Kumarage and Julie Nguyen, a graduate student and an undergraduate student from the laboratory. Working inside a hood, prepare the lipid suspension by dissolving accurately-weighed lipids in one milliliter of solvent with manual mixing.

Dry the lipid solution by gently streaming an inert gas in the vial while slowly rotating it at an angle. To thoroughly remove the residual solvent, place the vials overnight in a vacuum oven at 35 degrees Celsius. On the next day, hydrate the lipid film with two milliliters of heavy water to obtain a lipid concentration of 50 milligrams per milliliter and vortex the hydrated lipid suspension until the lipid film is fully dissolved.

Next, perform five freeze-thaw cycles by storing the vial of hydrated lipid suspension at minus 80 degrees Celsius until frozen. And then transferring it to a 35 degrees Celsius water bath to thaw the lipid suspension. Vortex the thawed suspension until homogenous before proceeding to the next cycle.

Before starting the experiment, assemble the extruder setup using a poly-bicarbonate membrane between two membrane supports and add two paper filters on each side to provide additional support. Use airtight glass syringes to hydrate the polycarbonate membrane by passing 0.3 milliliters of heavy water through the membrane assembly several times. After hydrating the membrane, insert a one milliliter gas-tight syringe with a prepared milky white color lipid solution into one end and an empty syringe into the opposite end of the extruder apparatus.

Once the syringes are connected, place the assembly into the extruder block. Program the pump by holding down the Rate button to enter the extrusion rate and press the Diameter button to enter the syringe diameter. Then, press Withdraw until the light turns on.

Press start and wait for the sample to start dispensing into the empty syringe. Hit the Stop button just before the sample syringe is fully empty. Record the dispensed volume, then hold the Rate button until phase one appears on the screen.

Keeping the Withdraw light off, press the volume button to enter the dispensed volume recorded earlier. Press the Rate button again and use the right most up arrow to access phase two. Press volume to enter the same value of the dispensed volume recorded earlier.

In this phase, press the Withdraw button until the Withdraw light is on. Repeat the cycle for phase three by pressing the volume button until LP:SE appears on the screen and set it to 20. Finally, press the Rate button, access phase four, and hit the volume button to get to the Stop function to finish the pump setup.

After the pump is programmed, press Start to begin the extrusion cycle. Perform 15 to 20 extrusion cycles before collecting the transparent opal blue extruded lipid suspension in a clean vial for measurements. Open the DAVE software and select reduce NSE data from the Data Reduction menu.

Upload the data files over different Q-values using the Open echo Files from the file menu. The uploaded files will show up under the available data sets. Right click on the selected file and label it according to the measurement it corresponds to like Sample, Cell, or Resolution.

Using the Data Set tab, group the detector pixels in 2 X 2 to improve the signal to noise ratio. Apply the same binning to all files of Resolution, Cell, and Sample. Inspect the data over all pixel groups and mask those with poor signals by pressing the End key on the keyboard.

Press Enter to access a pop-up window to apply the same mask to all four of your times. Masked pixels will turn green. Ensure that the collected data is in the form of an echo signal.

Start fitting the resolution file from the uploaded file list by right clicking on the desired file and selecting Fit Operations, Fit Echoes Resolution from the pop-up menu. Ensure that the fits of the echo signals yield reasonable fitting parameters. To inspect the error associated with each fitting parameter over the entire detector, select Image Options, and then select the fitting parameter of interest.

Then, right click on the detector image to access a pop-up window showing an error bar map. If the fit over a specific pixel is unsatisfactory, refit the signal over that pixel by selecting it, pressing the Fitting tab, and then pressing Fit Pixel. Input new starting parameters for the phase and period in the Fitting tab.

Reduce the sample file by selecting the corresponding file from the uploaded and labeled file list. Inspect all pixels and mask the poor ones as described above. Then, right click on the file and select Fit Operations, Import Phases.

For the resolution file, fit echo signals as described before with the values of the period unchanged and echo phase point imported from the resolution fits. Input the beam center for all data files by accessing the General tab and entering X and Y beam center values recorded from the experiment. Once the fits are complete, calculate the normalized intermediate scattering function by clicking right on the desired sample file from the list of fitted files and selecting Calculate I of Q from the pop-up menu.

Enter the required information for the resolution and cell files and the number of Q-arcs in the pop-up window, then press OK to view results. Notice that the data in cyan shows a poor signal due to detector edge effects and should be eliminated when compiling different Q data sets. Finally, save the reduced data sets as ASCII files and save the entire session as a DAVE project in the desired folder.

In this study, the NSE measurements of liposomal samples prepared with different deuteration schemes were carried out. NSE measurements of membrane bending fluctuations are performed on fully contrasted liposomes. This deuteration scheme results in a large scattering length difference between the membrane core and deuterated fluid environment, significantly enhancing the scattering signal from the liposomal membranes and improving the measurement statistics of bending dynamics.

On the other hand, NSE measurements of membrane thickness fluctuations of liposomes show deviations relative to the Q to the third dependence of bending fluctuations. To isolate the thickness fluctuation signal, the obtained signal is divided by Q to the third and the excess dynamics are fitted to a Lorentzian function in Q.This method is contingent on good quality data, which is more achievable with samples of high concentrations and strong scattering signals. The dynamics probed by NSE can be synergistically explored by deuterium NMR relaxometry and molecular-dynamic simulations to illustrate how molecular lipid structures and packing motifs influence membrane functions.

NSE studies on lipid membranes shed new light on membrane biophysics, the intricate relations of membrane structure and dynamics, and how they impact membrane functions and membrane-protein interactions.