Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies

Summary

Biophysical and biochemical studies of interactions among membrane-embedded protein domains face many technical challenges, the first of which is obtaining appropriate study material. This article describes a protocol for producing and purifying disulfide-stabilized transmembrane peptide complexes that are suitable for structural analysis by solution nuclear magnetic resonance (NMR) and other analytical applications.

Abstract

Physical interactions among the lipid-embedded alpha-helical domains of membrane proteins play a crucial role in folding and assembly of membrane protein complexes and in dynamic processes such as transmembrane (TM) signaling and regulation of cell-surface protein levels. Understanding the structural features driving the association of particular sequences requires sophisticated biophysical and biochemical analyses of TM peptide complexes. However, the extreme hydrophobicity of TM domains makes them very difficult to manipulate using standard peptide chemistry techniques, and production of suitable study material often proves prohibitively challenging. Identifying conditions under which peptides can adopt stable helical conformations and form complexes spontaneously adds a further level of difficulty. Here we present a procedure for the production of homo- or hetero-dimeric TM peptide complexes from materials that are expressed in E. coli, thus allowing incorporation of stable isotope labels for nuclear magnetic resonance (NMR) or non-natural amino acids for other applications relatively inexpensively. The key innovation in this method is that TM complexes are produced and purified as covalently associated (disulfide-crosslinked) assemblies that can form stable, stoichiometric and homogeneous structures when reconstituted into detergent, lipid or other membrane-mimetic materials. We also present carefully optimized procedures for expression and purification that are equally applicable whether producing single TM domains or crosslinked complexes and provide advice for adapting these methods to new TM sequences.

Introduction

This protocol details a procedure we have developed to produce disulfide-stabilized complexes of transmembrane (TM) peptides for structural studies using solution NMR. The procedure takes advantage of the robust expression afforded by the ΔtrpLE1413 fusion system (see below) and allows TM peptide complexes of defined composition to be generated using sophisticated stable-isotope labeling techniques for modern multi-dimensional NMR experiments. We have employed these techniques to determine several NMR structures that revealed important new information about how lymphocyte-activating immunoreceptors are assembled from multiple membrane protein subunits t....

Protocol

1. Cloning and Construct Design

Clone the sequences of interest into the pMM-peptide vector (which can be provided on request) using HindIII and BamHI restriction sites (see Figure 1). The double-stranded DNA insert should incorporate, in the following order: a HindIII site; a single methionine codon for CNBr cleavage; the E. coli codon-optimized peptide coding sequence; a stop codon; a BamHI site. All other methionines in the peptide should be eliminated and the dipeptide sequence asp-pro should be avoided as it will also be cleaved in acidic conditions.

A unique cysteine residue should b....

Results

The level of expression achieved for trpLE fusions is variable and heavily dependent on the amino-acid sequence of the attached peptide. Figure 3 shows the SDS-PAGE analysis of pre-induction (lane 1) and time-of-harvest (lane 2) samples from a culture that yielded approximately 120 mg of pure, intact trpLE-DAP12TM fusion from 1 liter of culture and 4 ml nickel matrix. All of the trpLE-DAP12TM fusion was localized to the inclusion body pellet (lane 4) as opposed to supernatant (lane 3).

Discussion

Expression of trpLE-TM fusions. In our experience, trpLE-TM fusions are poorly expressed in rich culture medium at 37 °C, and the culture conditions described here have proven successful for many different sequences containing from one to four TM domains with yields ranging from 50 to 150 mg/L of pure, intact fusion. Fusions encoding three- or four-TM GPCR fragments (human CCR5 TM1-TM3 and TM4-TM7, respectively) or a core catalytic fragment of human signal peptide peptidase (four TM domains; see ref ^15<.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Cyanogen bromide	ALDRICH	P.No- C91492,CAS-506-68-3	HAZARDOUS SUBSTANCE. DANGEROUS GOODS. Very toxic by inhalation, in contact with skin and if swallowed. Contact with acids liberates very toxic gas. Very toxic to aquatic organisms, may cause long-term adverse effects in the aquatic environment.
Trifluoroacetic acid	SIGMA-ALDRICH	P.CODE-1000984387, CAS Number 76-05-1	HAZARDOUS SUBSTANCE. DANGEROUS GOODS., Causes severe burns. Harmful by inhalation. Harmful to aquatic organisms, may cause long-term adverse effects in the aquatic environment.
2-Mercapt–thanol	SIGMA-ALDRICH	P.No M7154, CAS Number 60-24-2	HAZARDOUS SUBSTANCE. DANGEROUS GOODS. Toxic by inhalation, in contact with skin and if swallowed. Irritating to skin. Risk of serious damage to eyes. May cause sensitization by skin contact. Very toxic to aquatic organisms, may cause long-term adverse effects in the aquatic environment.
1,1,1,3,3,3-Hexafluoro-2-propanol	SIGMA-ALDRICH	Product Number 52512, CAS-No. 920-66-1	HAZARDOUS SUBSTANCE. DANGEROUS GOODS. Harmful by inhalation and if swallowed. Causes burns.
Formic acid	Merck KGaA	K41186564	Flammable liquid and vapour. Causes severe skin burns and eye damage.
Urea	UNIVAR, AJAX FINECHEM	Product Number, 817, CAS-No 57-13-6	When heated, decomposes to carbon dioxide and ammonia; if burned, emits small amounts of nitrogen oxides. Can cause redness and irritation of skin and eyes.
GUANIDINE HYDROCHLORIDE	Amresco	P.No-M110, CAS Number: 50-01-1	Harmful if swallowed, Causes serious eye irritation,Causes skin irritation, Acute Toxicity Oral, Skin Irritant, Eye Irritant.
TRITON X-100	SIGMA	Product Number- T8532 CAS No: 9002-93-1	Triton X-100 is a nonionic detergent, 100% active ingredient, which is often used in biochemical applications to solubilize proteins. Triton X-100 has no antimicrobial properties and considered a comparatively mild non-denaturing detergent
His-Select Nickel-Affinity gel	SIGMA-ALDRICH	Catalog Num- P6611	IS-Select Nickel Affinity Gel is an immobilized metal- ion affinity chromatography (IMAC) product. The HIS-Select Nickel Affinity gel is a proprietary quadridentate chelate on beaded agarose charged with nickel that is designed to specifically bind histidine containing proteins.
(-)-Glutathione, oxidized	SIGMA-ALDRICH	Catalog num 150568
Misonix S-3000 sonicator	QSONICA	S-3000 (discontinued)	Max power output 600 watts. 1/2-inch replaceable-tip probe takes 1/2-inch high-intensity, high-volume tips and a range of high-intensity, low-volume tips. Closest models currently available from this company are Q500 and Q700.
RP-HPLC: BioLogic DuoFlow chromatography system, Software Version 5.3	Bio-Rad Laboratories	Catalog Num 760-0047, Config No: AU500571, Serial No: 484BR3705	Peptides binds to reverse phase HPLC columns in high aqueous mobile phase and are eluted from RP HPLC columns with high organic mobile phase. In RP HPLC peptides are separated based on their hydrophobic character. Peptides can be separated by running a linear gradient of the organic solvent.
Prep HT C3 ZORBAX 300SB-Analytical HPLC Column, 21.2 x 150 mm, 5 μm particle size	Agilent	Product No: 895150-909	Reversed-phase HPLC colum
NuPAGE 12% Bis-Tris Gels	Life Technologies	NP0341BOX	Pre cast gels for protein electrophoresis
Slide-A-Lyzer G2 Dialysis Cassettes, 3.5K MWCO	Thermo Scientific	Product No: 87724	Sample dialysis