High-Resolution Neutron Spectroscopy to Study Picosecond-Nanosecond Dynamics of Proteins and Hydration Water

Neutron backscatterings measures global diffusion and internal dynamics of proteins and hydration water without radiation damage. Neutron spectroscopy simultaneously accesses spatial and time correlations on a molecular level. In soft matter and biological materials, it's particularly interesting to obtain indirect information on local order on these long range disorder systems.

So neutron backscattering can be used to study dynamics in biology, chemistry, or material sense. Another strong field is energy, with studies, for instance, on batteries and fuel cells. Neutron backscattering has been used successfully to study the molecular dynamics of proteins involved in neurodegenerative diseases such as Alzheimer's and Parkinson's.

The results obtained help us understand better the molecular hallmarks of these pathologies and might pave the way to develop better diagnostics. To prepare the sample holder, start by thoroughly cleaning a flat aluminum sample holder and its indium wire seal and screws with water, followed by ethanol, and then let it dry. Weigh the different parts of the sample holder, including the bottom, lid, and indium wire, separately on a precision balance.

Place the one millimeter indium wire seal into the groove of the bottom part of the sample holder, leaving a small overlap where the two ends join. Add around 100 milligrams of lyophilized protein such that it fills the inner surface of the bottom part of the sample holder. Next, place the sample holder in a desiccator with a Petri dish containing phosphorus pentoxide powder for 24 hours to completely dry the protein powder.

Weigh the dried bottom part of the sample holder containing the indium seal and the dried powder to obtain the mass of the dried sample. Remove the phosphorus pentoxide from the desiccator and put a Petri dish with deuterium oxide as well as the sample inside for hydrating the powder. Repeat the drying and hydrating three times for complete hydrogen to deuterium exchange.

Measure the powder mass regularly to check the hydration level and when the hydration is slightly above the desired level, wait for the mass to decrease slowly. Once the desired hydration is obtained, quickly put the lid on the bottom part and close the sample holder first with four screws to stop the vapor exchange. Place and tighten all the remaining screws until no gap is visible between the bottom part and the lid.

Weigh the sealed sample holder to check for any potential hydration loss via leaks after the neutron experiment. To prepare the liquid state sample, dissolve the protein in the deuterated buffer. Using water, determine the appropriate volume of liquid to be put in the sample holder.

Before handling any material, ensure the ionizing radiation dose is less than 100 microsieverts per hour. Then, thoroughly dry the sample stick and remove any previous sample. Place the sample on the sample stick, check for proper centering relative to the beam center, and insert the sample stick into the cryo furnace.

To acquire data in Nomad, go to the execution tab and drag and drop a furnace cryostat controller into the launchpad. Set the temperature to 200 Kelvin. Use the fast mode and a timeout of 30 minutes so that the temperature has time to stabilize.

Click on the refresher icon to run the temperature ramp in the background for data acquisition during the temperature decrease. Drag and drop the IN16 Doppler settings controller into the launchpad. Set the speed profile to accurate velocity, the set by to max delta E, the energy offset to 0.00 micro electronvolt, and the number of channels to 128 to obtain an elastic fixed window scans configuration.

Drag and drop a count controller into the launchpad, fill the subtitle field with a name that allows for easy identification of the data, and set scans of 30 seconds with 60 repetitions. Next, drag and drop the IN16 Doppler settings controller into the launchpad. Set the speed profile to sign, the set by to speed with a value of 4.5 meters per second, and the number of channels to 2048 to obtain a quasi elastic neutron scattering configuration.

Drag and drop a count controller and fill the subtitle field with a name that allows for easy identification of the data. Set scans of 30 minutes with four repetitions. For the temperature ramp, drag and drop a furnace cryostat controller, set the temperature to 310 Kelvin, and set the ramp to set point with a delta of 0.05 Kelvin for six seconds.

Use a timeout of 200 minutes. Use a for loop with 65 repetitions. Inside, insert an IN16 doppler settings controller, followed by the count controller and set a single scan of 30 seconds.

Subsequently, insert IN16 Doppler settings as described previously, but using an energy offset of 1.5 micro electronvolt and 1024 channels. Then, insert the count controller and set a single scan of three minutes. To acquire the last quasi elastic neutron scattering of 312 Kelvin, drag and drop the IN16 Doppler settings and count controllers configured as demonstrated previously.

Then, press the start button to run the script. The lysosome elastic and inelastic fixed window scans showed an increase in signal at low momentum transfer and low energy transfer, while the signal at high energy transfer and low momentum transfer decreased. The initial center of mass diffusion coefficient was 15 angstrom squared per nanosecond, which then exponentially decreased over time.

At temperatures higher than 220 Kelvin, the hydration water around the tau fibers was significantly more mobile than around the tau monomers. The fraction of water molecules undergoing translational motion and both the translational and rotation diffusion coefficients of the water molecules were increased around fibers. The protein concentration should be 2000 milligram per ml or at least 20 milligram per ml for proteinated proteins due to neutron back scattering limitations from neutron flux or sample holder buffer background.

Neutron backscattering has been recently applied, for example, to understand how water, which is the natural environmental proteins, can be fully replaced by a synthetic polymer for bio technological application like, for example, drug delivery.