Study of Protein Dynamics via Neutron Spin Echo Spectroscopy

Summary

The present protocol describes methods for investigating the structure and dynamics of two model proteins that have an important role in human health. The technique combines bench-top biophysical characterization with neutron spin echo spectroscopy to access the dynamics at time and length scales relevant for protein interdomain motions.

Abstract

Most human body proteins' activity and functionality are related to configurational changes of entire subdomains within the protein crystal structure. The crystal structures build the basis for any calculation that describes the structure or dynamics of a protein, most of the time with strong geometrical restrictions. However, these restrictions from the crystal structure are not present in the solution. The structure of the proteins in the solution may differ from the crystal due to rearrangements of loops or subdomains on the pico to nanosecond time scale (i.e., the internal protein dynamics time regime). The present work describes how slow motions on timescales of several tens of nanoseconds can be accessed using neutron scattering. In particular, the dynamical characterization of two major human proteins, an intrinsically disordered protein that lacks a well-defined secondary structure and a classical antibody protein, is addressed by neutron spin echo spectroscopy (NSE) combined with a wide range of laboratory characterization methods. Further insights into protein domain dynamics were achieved using mathematical modeling to describe the experimental neutron data and determine the crossover between combined diffusive and internal protein motions. The extraction of the internal dynamic contribution to the intermediate scattering function obtained from NSE, including the timescale of the various movements, allows further vision into the mechanical properties of single proteins and the softness of proteins in their nearly natural environment in the crowded protein solution.

Introduction

Probing dynamics of soft matter with neutrons
Investigating the dynamical properties of proteins and peptides is a major part of biophysical research, and many well-developed methods exist today to access a wide range of energy landscapes¹. Relating the experimentally revealed dynamics of the proteins to their biological function is a far more difficult task, requiring complex mathematical models and computer-aided dynamics simulations. The importance of neutron spectroscopy for the analysis of protein motions has been emphasized in several well-received and widely recognized studies^1,

Protocol

The present work characterizes two proteins: a regular human antibody protein IgG, and the intrinsically disordered MBP. The lyophilized form of the proteins was obtained from commercial sources (see Table of Materials).

1. Protein sample preparation

1. Prepare a 50 mM sodium phosphate + 0.1 M NaCl buffer by weighing and dissolving the respective solid reagents in heavy water (D₂O) (see Table of Materials). This is the deut.......

Discussion

NSE spectroscopy delivers a unique and detailed view of the dynamics of proteins, which other spectroscopic techniques cannot produce. Measurements over an extended time scale provide observations of both the proteins' translational and rotational diffusion, as presented here. The segmental dynamics and other internal oscillations reveal themselves as a strong decay of the coherent scattering function S(Q, t) at a short time scale and are well separated from the overall diffusional relaxation processes. The .......

Materials

Name	Company	Catalog Number	Comments
Bovine MBP protein solution	Sigma-Aldrich	M1891	lyophilized powder reconstituted in  D2O
D2O - heavy water	Sigma-Aldrich	Product No. 151882	liquid
Dionized water	in house	-	for washing / cleanning cells
DLS instrument	Zetasizer Nano ZS, FZ-Jülich	-	dynamic light scattering instrument
Elastic scattering standards	SNS-NSE, ORNL	-	Al2O3  and Graphite powders
Ethanol	Sigma-Aldrich	65350-M	70% ethanol for cleaning cells
IgG protein solution	Sigma-Aldrich	I4506	lyophilized powder reconstituted in  D2O
KWS-2 instrument	JCNS outstation at the MLZ,  Garching, Germany	-	small angle neutron instrument
Liquinox dish detergent	Alconox	-	Phosphate-free liquid lab glassware cleaner
Na2HPO4·7H2O	Sigma-Aldrich	Product No.S9390	disodium phosphate heptahydrate salt
NaCl	Sigma-Aldrich	Product No.S9888	sodium chloride salt
NaH2PO4·H2O	Sigma-Aldrich	Product No. S9638	monosodium phosphate monohydrate salt
Nanodrop spectrophotometer	Thermo Scientific	Catalog number: ND-2000	NanoDrop 2000/2000c Spectrophotometer
Neutron alignment camera	NeutronOptics, Grenoble	NOG210222	100 x 100 mm camera with Sony IMX249 CMOS sensor
Parafilm M - wax parafilm	Bemis	Parafilm M - 5259-04LC PM996	all-purpose laboratory film in cardboard dispenser
Phoenix-J-NSE Spectrometer	JCNS outstation at the MLZ,  Garching, Germany	-	neutron spectrometer
SasView	https://www.sasview.org/
SAXSpace, Anton Paar instrument	FZ-Jülich	-	small angle x-ray instrument
Slide-A-Lyzer dialysis membranes	Thermo Scientific	88400-88405	Slide-A-Lyzer mini dialysis devices tubes of 3.5 K MWCO
SNS Remote Analysis Cluster	Neutron Science Remote Analysis (sns.gov)	https://analysis.sns.gov
SNS-NSE spectrometer	ORNL, Oak Ridge, TN, USA	-	neutron spectrometer
Sterile syringe filters	VWR	N.A. PN:28145-501	0.2 µm pore size filters
Temperature Forcing System (TFS)	SP Scientific	Part Number 100004055	sample environment equipment
Urea -d4	Sigma-Aldrich	Product No. 176087	deuterated Urea salt
Viscometer	FZ-Jülich	-	falling ball viscometer