Name: high-resolution neutron spectroscopy to study picosecond-nanosecond dynamics of proteins and hydration water
Uploaded: 2022-04-28T13:10:00
Description: Watch this Scientific Journal Video about High-Resolution Neutron Spectroscopy to Study Picosecond-Nanosecond Dynamics of Proteins and Hydration Water at JoVE.com

The authors are grateful to Michaela Zamponi at the J&#252;lich Centre for Neutron Science at the Heinz Maier-Leibnitz Zentrum, Garching, Germany, for part of the neutron scattering experiments conducted on the instrument SPHERES. This work has benefited from the activities of the Deuteration Laboratory (DLAB) consortium funded by the European Union under Contracts HPRI-2001-50065 and RII3-CT-2003-505925, and from UK Engineering and Physical Sciences Research Council (EPSRC)-funded activity within the Institut Laue Langevin EMBL DLAB under Grants GR/R99393/01 and EP/C015452/1. Support by the European Commission under the 7th Framework Programme through the Key Action: Strengthening the European Research Area, Research Infrastructures is acknowledged [Contract 226507 (NMI3)]. Kevin Pounot and Christian Beck thank the Federal Ministry of Education and Research (BMBF, grant number 05K19VTB) for funding of their postdoctoral fellowships.

1. Prepare the deuterated buffer for proteins in the liquid state
<ol>
	<li>Dissolve all components of the buffer in pure D2O.</li>
	<li>If the pH electrode was calibrated in H2O, adjust the pD according to the formula pD = pH + 0.4 using NaOD or DCl34. 
	NOTE: The use of D2O instead of H2O might affect protein solubility and the buffer conditions might need to be adapted, (e.g., by a slight change in salt concentration).</li>
</o...

Neutron spectroscopy is the only method that allows probing the ensemble-averaged ps-ns dynamics of protein samples regardless of the size of the protein or the complexity of the solution when deuteration is used6. Specifically, by probing self-diffusion of protein assemblies in solution, the hydrodynamic size of such assemblies can be unambiguously determined. Nonetheless, the method is commonly limited by the low neutron flux, which implies long acquisition times and the requirement of high amou...

The neutron is a charge-less and massive particle that has been successfully used over the years to probe samples in various fields from fundamental physics to biology1. For biological applications, small-angle neutron scattering, inelastic neutron scattering, and neutron crystallography and reflectometry are extensively used2,3,4. Inelastic neutron scattering provides an ensemble-averaged measurement of the dynamics without requiring specific labeling per se, and a signal quality that does not depend on the size or the protein5.&#160;The measurement can be done using a highly complex environment for the protein under study that mimics the intracellular medium, such as a deuterated bacterial lysate or even in vivo3,6,7. Different experimental setups can be used to study the dynamics, namely i) time-of-flight-giving access to sub-ps-ps dynamics, ii) backscattering-giving access to ps-ns dynamics, and iii) spin-echo-giving access to dynamics from ns to hundreds of ns. Neutron backscattering makes use of the Bragg&#39;s law 2d sin&#952; = n&#955;, where d is the distance between planes in a crystal,&#160;&#952; the scattering angle, n the scattering order, and&#160;&#955; the wavelength. The use of crystals for backscattering toward the detectors allows for achieving a high resolution in energy, typically ~0.8 &#181;eV. To measure the energy exchange, either a Doppler drive carrying a crystal in backscattering is used to define and tune the incoming neutron wavelength8,9,10 (Figure 1), or a time-of-flight setup can be used at the cost of a decrease in energy resolution11.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/63664/63664fig01.jpg" /> 
Figure 1: Sketch of a neutron backscattering spectrometer with a Doppler drive. The incoming beam hits the phase space transformation (PST) chopper42, which increases the flux at the sample position. It is then backscattered towards the sample by the Doppler drive, which selects an energy E1 (cyan arrow). The neutrons are then scattered by the sample (with different energies represented by the color of the arrows) and the analyzers, made of Si 111 crystals, will only backscatter neutrons with a specific energy E0 (red colored arrows here). Hence, the momentum transfer q is obtained from the detected position of the neutron on the detector array, and the energy transfer is obtained from the difference E1- E0. The time-of-flight expected for the neutron pulse produced by the PST is used to discard the signal from the neutrons scattered directly toward the detector tubes. Abbreviation: PST = phase space transformation. <a href="https://www.jove.com/files/ftp_upload/63664/63664fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
For backscattering spectroscopy, the main contribution to the signal from hydrogen proton-rich samples, such as proteins, comes from incoherent scattering, for which the scattering intensity Sinc(q, &#969;) is shown by Eq (1)12
<img alt="Equation 1" src="/files/ftp_upload/63664/63664eq01.jpg" style="margin: auto;" /> (1)
Where &#963;inc is the incoherent cross-section of the element considered, k&#39; is the norm of the scattered wavevector, k the norm of the incoming wavevector, q (= k - k&#39;) the momentum transfer, rj(t) the position vector of atom j at time t, and&#160;&#969; the frequency corresponding to the energy transfer between the incoming neutron and the system. The angular brackets denote the ensemble average. Hence, incoherent scattering probes the ensemble-averaged single-particle self-correlation of atom positions with time and gives the self-dynamics averaged over all atoms in the system and different time origins (ensemble average). The scattering function is the Fourier transform in time of the intermediate scattering function I(q, t), which can be viewed as the Fourier transform in space of the van Hove correlation function shown by Eq (2):
<img alt="Equation 2" src="/files/ftp_upload/63664/63664eq02.jpg" style="margin: auto;" /> (2)
Where &#961;(r,t) is the probability density of finding an atom at position r and time t13.
For a Fickian diffusion process, the self-diffusion function results (see Eq (3)) after a double Fourier transform in a scattering function consisting in a Lorentzian of line width given by &#947; = Dq2.
<img alt="Equation 10" src="/files/ftp_upload/63664/63664eq10.jpg" style="margin: auto;" /> (3)
More sophisticated models were developed and found useful such as the jump diffusion model by Singwi and Sj&#246;lander for ps-ns internal protein dynamics14 or the rotation model by Sears for hydration water15,16,17.
On the neutron backscattering (NBS) instrument IN16B8,9 at the ILL, Grenoble, France (Supplemental Figure S1), a setup commonly used with proteins consists of Si 111 crystals for the analyzers with a Doppler drive for tuning the incoming wavelength (Supplemental Figure S2A), thereby giving access to the momentum transfer range ~0.2 &#197;-1 &#60; q &#60; ~2 &#197;-1 and energy transfer range of -30 &#181;eV &#60; <img alt="Equation 3" src="/files/ftp_upload/63664/63664eq03.jpg" style="margin: auto;" /> &#60; 30 &#181;eV-corresponding to timescales ranging from a few ps to a few ns and distances of a few &#197;. In addition, IN16B offers the possibility to perform elastic and inelastic fixed-window scans (E/IFWS)10, which include data acquisition at a fixed energy transfer. As the flux is limited when working with neutrons, E/IFWS allows maximization of the flux for one energy transfer, thus reducing the acquisition time needed to obtain a satisfying signal-to-noise ratio. A more recent option is the backscattering and time-of-flight spectrometer (BATS) mode11, which allows measurement of a wide range of energy transfers, (e.g., -150 &#181;eV &#60; <img alt="Equation 3" src="/files/ftp_upload/63664/63664eq03.jpg" style="margin: auto;" /> &#60; 150 &#181;eV), with a higher flux than with the Doppler drive, yet at the cost of a lower energy resolution (Supplemental Figure S2B).
An important property of neutron scattering is that the incoherent cross section&#160;&#963;inc has a 40 times higher value for hydrogen than for deuterium and is negligible for other elements commonly found in biological samples. Therefore, the dynamics of proteins in a liquid environment can be studied by using a deuterated buffer, and the powder state allows for the study of either protein internal dynamics with hydrogenated protein powder hydrated with D2O, or the study of hydration water for perdeuterated protein powder hydrated with H2O. In the liquid state, neutron backscattering typically allows simultaneously accessing of the center-of-mass self-diffusion of proteins (Fickian-type diffusion) and their internal dynamics. The latter are backbone and side-chain motions usually described by the so-called jump diffusion model or others3,18. In hydrogenated protein powders, the protein diffusion is absent and only internal dynamics needs to be modeled. For hydration water, the contributions of translational and rotational motions of water molecules present a different dependence on the momentum transfer q, which allows for their distinction in the data analysis process17.
This paper illustrates the neutron backscattering method with the study of proteins that were found to be able to unfold, aggregate into a canonical form consisting of stacks of &#946;-strands-the so-called cross-&#946; pattern19,20-and form elongated fibers. This is the so-called amyloid aggregation, which is extensively studied due to its central role in neurodegenerative disorders such as Alzheimer&#39;s or Parkinson&#39;s diseases21,22. The study of the amyloid proteins is also motivated by the functional role they can play23,24 or their high potential for the development of novel biomaterials25. The physicochemical determinants of the amyloid aggregation remain unclear, and no general theory of amyloid aggregation is available, despite tremendous progress during the past years21,26.
Amyloid aggregation implies changes in protein structure and stability with time, the study of which naturally implies dynamics, linked to protein conformation stability, protein function, and protein energy landscape27. Dynamics is directly linked to the stability of a specific state through the entropic contribution for the fastest motions28, and protein function can be sustained by motions on various timescales from sub-ps for light-sensitive proteins29 to ms for domain motions, which can be facilitated by picosecond-nanosecond dynamics30.
Two examples of using neutron backscattering spectroscopy to study amyloid proteins will be presented, one in the liquid state to study protein dynamics and one in the hydrated powder state to study hydration water dynamics. The first example concerns the aggregation of lysozyme into &#181;m sized spheres (called particulates) followed in real time5, and the second a comparison of water dynamics in native and aggregated states of the human protein tau31.
Lysozyme is an enzyme involved in immune defense and is composed of 129 amino acid residues. Lysozyme can form particulates in deuterated buffer at pD of 10.5 and at a temperature of 90 &#176;C. With neutron scattering, we showed that the time evolution of the center-of-mass diffusion coefficient of lysozyme follows the single exponential kinetics of thioflavin T fluorescence (a fluorescent probe used to monitor the formation of amyloid cross-&#946; patterns32), indicating that the formation particulate superstructures and cross-&#946; patterns occur in a single step with the same rate. Moreover, the internal dynamics remained constant throughout the aggregation process, which can be explained either by a fast conformational change that cannot be observed on NBS instruments, or by the absence of significant change in protein internal energy upon aggregation.
The human protein tau is an intrinsically disordered protein (IDP) consisting of 441 amino acids for the so-called 2N4R isoform, which is notably involved in Alzheimer&#39;s disease33. Using neutron backscattering on powders of perdeuterated protein tau, we showed that hydration water dynamics is increased in the fiber state, with a higher population of water molecules undergoing translational motions. The result suggests that an increase in hydration water entropy might drive the amyloid fibrillation of tau.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Aluminum sample holder</td>
			<td></td>
			<td></td>
			<td>Not commercially available. Either the local contact on the instrument can provide them or they can be manufactured based on a technical drawing that can be provided by the local contact.</td>
		</tr>
		<tr>
			<td>Deuterium chloride, 35 wt. % in D2O, &#8805;99 atom % D</td>
			<td>Sigma-Aldrich</td>
			<td> 
			543047</td>
			<td></td>
		</tr>
		<tr>
			<td>Deuterium oxide (D, 99.9%)</td>
			<td>Eurisotop</td>
			<td>DLM-4DR-PK</td>
			<td></td>
		</tr>
		<tr>
			<td>Dow Corning high-vacuum silicone grease</td>
			<td>Sigma-Aldrich</td>
			<td>Z273554-1EA</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol 96%, EMSURE Reag. Ph Eur</td>
			<td>Sigma-Aldrich</td>
			<td>1.5901</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass dessicator</td>
			<td>VWR</td>
			<td>&#160; 75871-660</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass dessicator plate, 140 mm</td>
			<td>VWR</td>
			<td>89038-068</td>
			<td></td>
		</tr>
		<tr>
			<td>Indium wire, 1.0 mm (0.04 in) dia, Puratronic, 99.999%</td>
			<td>Alfa Aesar</td>
			<td>00470.G1</td>
			<td></td>
		</tr>
		<tr>
			<td>Lysozyme from chicken egg white dialyzed, lyophilized, powder, ~100,000 U/mg</td>
			<td>Sigma-Aldrich</td>
			<td>62970</td>
			<td></td>
		</tr>
		<tr>
			<td>nPDyn</td>
			<td></td>
			<td>v3.x</td>
			<td>see github.com/kpounot/nPDyn, model functions fot fitting also included in the software</td>
		</tr>
		<tr>
			<td>OHAUS AX324 Adventurer balance, internal calibration</td>
			<td>Dutscher</td>
			<td>92641</td>
			<td></td>
		</tr>
		<tr>
			<td>Phosphorus pentoxide, ReagentPlus, 99%</td>
			<td>Sigma-Aldrich</td>
			<td>214701</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette ErgoOne 0.5-10 &#956;L</td>
			<td>Starlab</td>
			<td>S7100-0510</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette ErgoOne 100-1,000 &#956;L</td>
			<td>Starlab</td>
			<td>S7100-1000</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette ErgoOne 20-200 &#956;L</td>
			<td>Starlab</td>
			<td>S7100-2200</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tip TipOne 1,000 &#956;L</td>
			<td>Starlab</td>
			<td>S1111-6001</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tip TipOne 10 &#956;L</td>
			<td>Starlab</td>
			<td>S1111-3200</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipette tip TipOne 200 &#956;L</td>
			<td>Starlab</td>
			<td>S1111-0206</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium deuteroxide solution, 40 wt. % in D2O, 99.5 atom % D</td>
			<td>Sigma-Aldrich</td>
			<td>372072</td>
			<td></td>
		</tr>
	</tbody>
</table>

high-resolution neutron spectroscopy to study picosecond-nanosecond dynamics of proteins and hydration water

Neutron scattering offers the possibility to probe the dynamics within samples for a wide range of energies in a nondestructive manner and without labeling other than deuterium. In particular, neutron backscattering spectroscopy records the scattering signals at multiple scattering angles simultaneously and is well suited to study the dynamics of biological systems on the ps-ns timescale. By employing D2O-and possibly deuterated buffer components-the method allows monitoring of both center-of-mass diffusion and backbone and side-chain motions (internal dynamics) of proteins in liquid state.
Additionally, hydration water dynamics can be studied by employing powders of perdeuterated proteins hydrated with H2O. This paper presents the workflow employed on the instrument IN16B at the Institut Laue-Langevin (ILL) to investigate protein and hydration water dynamics. The preparation of solution samples and hydrated protein powder samples using vapor exchange is explained. The data analysis procedure for both protein and hydration water dynamics is described for different types of datasets (quasielastic spectra or fixed-window scans) that can be obtained on a neutron backscattering spectrometer.
The method is illustrated with two studies involving amyloid proteins. The aggregation of lysozyme into &#181;m sized spherical aggregates-denoted particulates-is shown to occur in a one-step process on the space and time range probed on IN16B, while the internal dynamics remains unchanged. Further, the dynamics of hydration water of tau was studied on hydrated powders of perdeuterated protein. It is shown that translational motions of water are activated upon the formation of amyloid fibers. Finally, critical steps in the protocol are discussed as to how neutron scattering is positioned regarding the study of dynamics with respect to other experimental biophysical methods.

The aggregation of lysozyme into particulates was performed at 90 &#176;C with a protein concentration of 50 mg/mL in a deuterated buffer (0.1 M NaCl at pD 10.5). The formation of particulates is triggered by the temperature increase to 90 &#176;C and occurs within 6 h (Supplemental Figure S8). The data acquisition was performed on IN16B, as described in the protocol above (data are permanently curated by the ILL and accessible at&#160;http://dx.doi.org/10.5291/ILL-DATA.8-04-811).
<p class="jove_cont...

Watch this Scientific Journal Video about High-Resolution Neutron Spectroscopy to Study Picosecond-Nanosecond Dynamics of Proteins and Hydration Water at JoVE.com

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

Neutron backscattering spectroscopy offers a nondestructive and label-free access to the ps-ns dynamics of proteins and their hydration water. The workflow is presented with two studies on amyloid proteins: on the time-resolved dynamics of lysozyme during aggregation and on the hydration water dynamics of tau upon fiber formation.

Neutron scattering offers the possibility to probe the dynamics within samples for a wide range of energies in a nondestructive manner and without ...

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.

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