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

Summary

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.

Abstract

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 µm 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.

Introduction

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 used^2,3,4. 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 protein

Protocol

1. Prepare the deuterated buffer for proteins in the liquid state

1. Dissolve all components of the buffer in pure D₂O.
2. 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).

2. Prepare the H₂O-hydrated powders of perdeuterated protein

1. Prepare the sample hol....

Results

The aggregation of lysozyme into particulates was performed at 90 °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 °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 http://dx.doi.org/10.5291/ILL-DATA.8-04-811).

Discussion

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.......

Materials

Name	Company	Catalog Number	Comments
Aluminum sample holder			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.
Deuterium chloride, 35 wt. % in D2O, ≥99 atom % D	Sigma-Aldrich	543047
Deuterium oxide (D, 99.9%)	Eurisotop	DLM-4DR-PK
Dow Corning high-vacuum silicone grease	Sigma-Aldrich	Z273554-1EA
Ethanol 96%, EMSURE Reag. Ph Eur	Sigma-Aldrich	1.5901
Glass dessicator	VWR	75871-660
Glass dessicator plate, 140 mm	VWR	89038-068
Indium wire, 1.0 mm (0.04 in) dia, Puratronic, 99.999%	Alfa Aesar	00470.G1
Lysozyme from chicken egg white dialyzed, lyophilized, powder, ~100,000 U/mg	Sigma-Aldrich	62970
nPDyn		v3.x	see github.com/kpounot/nPDyn, model functions fot fitting also included in the software
OHAUS AX324 Adventurer balance, internal calibration	Dutscher	92641
Phosphorus pentoxide, ReagentPlus, 99%	Sigma-Aldrich	214701
Pipette ErgoOne 0.5-10 μL	Starlab	S7100-0510
Pipette ErgoOne 100-1,000 μL	Starlab	S7100-1000
Pipette ErgoOne 20-200 μL	Starlab	S7100-2200
Pipette tip TipOne 1,000 μL	Starlab	S1111-6001
Pipette tip TipOne 10 μL	Starlab	S1111-3200
Pipette tip TipOne 200 μL	Starlab	S1111-0206
Sodium deuteroxide solution, 40 wt. % in D2O, 99.5 atom % D	Sigma-Aldrich	372072