Paramagnetic Relaxation Enhancement for Detecting and Characterizing Self-Associations of Intrinsically Disordered Proteins

Summary

A protocol for the application of paramagnetic relaxation enhancement NMR spectroscopy to detect weak and transient inter- and intra-molecular interactions in intrinsically disordered proteins is presented.

Abstract

Intrinsically disordered proteins and intrinsically disordered regions within proteins make up a large and functionally significant part of the human proteome. The highly flexible nature of these sequences allows them to form weak, long-range, and transient interactions with diverse biomolecular partners. Specific yet low-affinity interactions promote promiscuous binding and enable a single intrinsically disordered segment to interact with a multitude of target sites. Because of the transient nature of these interactions, they can be difficult to characterize by structural biology methods that rely on proteins to form a single, predominant conformation. Paramagnetic relaxation enhancement NMR is a useful tool for identifying and defining the structural underpinning of weak and transient interactions. A detailed protocol for using paramagnetic relaxation enhancement to characterize the lowly-populated encounter complexes that form between intrinsically disordered proteins and their protein, nucleic acid, or other biomolecular partners is described.

Introduction

Intrinsic disorder (ID) describes proteins (IDPs) or regions within proteins (IDRs) that do not spontaneously fold into stable secondary or tertiary structures but are biologically active. Generally, the function of IDP/IDRs is to facilitate specific yet reversible interactions with biomolecules at physiological conditions¹. Thus, IDPs and IDRs are involved in a range of cellular functions, including recruitment, organization, and stabilization of multi-protein complexes, for example, the assembly and activity of the spliceosome², recruitment and organization of components at sites of DNA damage³,....

Protocol

General requirements for the protocol: protein purification facilities, UV-Vis spectrometer, high-field NMR spectrometer and operating software, post-processing analysis software including; NMRPipe³², Sparky³³, (or CCPN Analysis³⁴, or NMRViewJ³⁵).

1. Recombinant expression and purification of a protein for PRE measurements

1. Design an expression construct for the protein of interest so that there is a single cysteine residue present. Multiple mutations will be required to introduce a free cysteine at different positions in the protein o....

Results

Intramolecular ¹H_N-Γ₂ PREs were recorded on a self-associating, intrinsically disordered fragment (residues 171-264) derived from the low-complexity domain of the RNA-binding protein EWSR1⁴² (Figure 3). Residues in close sequential proximity to the spin-label attachment point (e.g., residue 178 or 260 in Figure 3) are expected to be significantly broadened and are not detectable in the spectrum. R.......

Discussion

A method for characterizing transient interactions that exist at low populations between intrinsically disordered proteins and various binding partners using PRE has been presented. In the example shown, the protein is self-associating, and thus the PRE may arise from a combination of inter and intramolecular interactions. This method is readily extended to heterogeneous samples where the interactions between two different proteins may be characterized. Complementary information about how different regions of the protein.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
0.45 µm and 0.22 µm syringe filters	Millipore Sigma	SLHVM33RS SLGVR33RS	Filter lysate before first purification step and before size exclusion chromatography.
100 mm Petri Dish	Fisher	FB0875713	Agar plates for bacterial transformation.
14N Ammonium chloride	Sigma Aldrich	576794	Use of 15N in M9 medium will produce an NMR visible protein, 14N will produces an NMR invisible protein
15N Ammonium chloride	Sigma Aldrich	299251	Use of 15N in M9 medium will produce an NMR visible protein, 14N will produces an NMR invisible protein
3 L Fernbach baffled flask	Corning	431523	Bacterial expression culture
3-Maleimido-Proxyl	Sigma Aldrich	253375	Nitroxide spin label
50 mL conical centrifuge tubes	Thermo Fisher	14-432-22	Solution/protein storage
Amicon centrifugal filter	Millipore Sigma	UFC900308	Protein concentration
Ampicillan	Sigma Aldrich	A5354	Antibiotic for a selective marker, exact choice depends on the expression construct plasmid
Analytical balance	Oahus	30061978	Explorer Pro, for weighing reagents
Ascorbic acid	Sigma Aldrich	AX1775	Reduces nitroxide spin label
Autoclave			Sterilize glassware and culture media
Calcium chloride	Sigma Aldrich	C4901	M9 media component
Centrifuge bottles	Thermo Fisher	010-1459	Harvest E. coli cells after recombinant protein expression
Centrifuge, hand-crank	Thomas Scientific	0241C68	Boekel hand-driven, low-speed centrifuge with 15 mL buckets that can accommodate NMR tubes
Chelex 100	Sigma Aldrich	C7901	Remove contaminating paramagnetic compounds from buffer solutions
Computer workstation			Linux or Mac OS compatable with NMR data processing and analysis software packages such as NMRPipe and Sparky
Deuterium oxide	Sigma Aldrich	151882	Needed for NMR lock signal
Dextrose	Sigma Aldrich	D9434	M9 media component
Dibasic Sodium Phosphate	Sigma Aldrich	S5136	M9 media component
Ellman's reagent (5,5-dithio-bis-(2-nitrobenzoic acid)	Thermo Fisher	22582	Quantification of free cystiene residues
High speed centrifuge tubes	Thermo Fisher	3114-0050	Used to clear bacterial lysate.
High-field NMR instrument (600 - 800 MHz)	Bruker		Equiped with a multichannel cryogenic probe and temperature control
IMAC column, HisTrap FF	Cytvia	17528601	Initial fractionation of crude bacterial lysate
Isopropyl B-D-thiogalactoside (IPTG)	Sigma Aldrich	I6758	Induces protein expression for genes under control of lac operator
LB agar	Thermo Fisher	22700025	Items are used for transforming E. coli to express protein of interest, substitions for any of these items with like products is acceptable.
LB broth	Thermo Fisher	12780052
Low-pressure chromatography system	Bio-Rad	7318300	BioRad BioLogic is used for low-pressure chomatograph such as running IMAC columns
Magnesium sulfate	Sigma Aldrich	M7506	M9 media component
Medium pressure chromatography system	Bio-Rad	7880007	BioRad NGC equipped with a multi-wavelength detector, pH and conductivity monitors, and automatic fraction collector
MEM vitamin solution	Sigma Aldrich	M6895	M9 media component
Microfluidizer	Avestin	EmulsiFlex-C3	Provides rapid and efficient bacterial cell lysis
Micropipettes	Thermo Fisher		Calibrated set of micropippetters with properly fitting disposable tips (available from multiple manufacturers e.g. Eppendorf)
Monobasic potassium phosphate	Sigma Aldrich	1551139	M9 media component
NMR pipettes	Sigma Aldrich	255688	To remove sample from NMR tube
NMR sample tube	NewEra	NE-SL5	Suitable for high-field NMR spectrometers
Preparative Centrifuge	Beckman Coulter	Avanti J-HC	Harvest E. coli cells after recombinant protein expression
Round bottom polystyrene centrifuge tubes	Corning	352057	Clear bacterial lysate
Shaking incubator	Eppendorf	S44I200005	Temperature controlled growth of E. coli starter and expression cultures
Sodium chloride	Sigma Aldrich	S5886	M9 media component
Sonicating water bath and vacuum source	Thomas Scientific		Used to degas buffer solutions
Sonicator	Thermo Fisher	FB505110	Used for bacterial cell lysis or shearing bacterial DNA
Spectrophotometer	Implen	OD600 Diluphotometer	Monitor growth of E.coli protein expression cultures
Superdex 200 16/600 size exculsion colum	Cytvia	28989333	Final protein purification step
Topspin software, version 3.2 or later	Bruker		Operating software for the NMR instrument
Transformation competent E. coli cells	Thermo Fisher	C600003	One Shot BL21 Star (DE3) chemically competent E. coli, other strains may be compatable
Tris(2-carboxyethyl)phosphine (TCEP)	ThermoFisher	20490	Reducing agent compatable with some sulfhydryl-reactive conjugations
UV-Vis spectrophotometer	Implen	NP80	Measure protein concentration.
Water bath, temperature controlled	ThermoFisher	FSGPD25	For heat shock step of bacterial transformation
Yeast extract	Sigma Aldrich	Y1625	For supplementing M9 media if required