Name: production of disulfide-stabilized transmembrane peptide complexes for structural studies
Uploaded: 2013-03-06T13:00:00
Description: Watch this Scientific Journal Video about Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies at JoVE.com

Funding for this work is provided by the National Health and Medical Research Council of Australia (NHMRC project grant 1011352 to M.E.C and M.J.C.; Independent Research Institutes Infrastructure Support Scheme [IRIISS] grant to WEHI) and the Victorian Government (VESKI Innovation Fellowship to M.E.C.; Victorian State Government Operational Infrastructure Support to WEHI). M.E.C. is a Queen Elizabeth II Fellow of the Australian Research Council. E.F.X.B acknowledges support from the Norma Hilda Schuster Scholarship Program at the University of Melbourne.

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 dipept...

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Expression of trpLE-TM fusions. In our experience, trpLE-TM fusions are poorly expressed in rich culture medium at 37 &deg;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<...

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 &Delta;trpLE1413 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 through interactions among their TM domains (recently reviewed in 1). These techniques are applicable to many other membrane protein systems as well as a wide range of downstream analytical methods in addition to solution NMR. While the example given here utilizes native cysteine residues to create a complex that mimics the naturally disulfide-bonded protein, the design is equally well suited for creating engineered disulfide bonds to stabilize complexes that are normally held together by weaker, non-covalent interactions such as homo- and hetero-dimeric TM complexes of epidermal growth factor receptor (EGFR)-family proteins2-4 or heterodimeric &alpha;&beta; integrin complexes5,6.
Extremely hydrophobic peptide sequences such as those derived from the lipid bilayer-spanning portions of TM proteins are exceedingly difficult subjects for biochemical and biophysical studies. In addition to being very challenging to manipulate using standard protein and peptide chemistry techniques, they are often quite toxic to cells and are therefore difficult to produce recombinantly. We7,8 and others9-11 have had significant success expressing such difficult peptide sequences in bacteria as in-frame carboxy-terminal fusions to a modified version of the &Delta;trpLE1413 sequence derived from the E. coli trp operon12. The ~13 kDa trpLE polypeptide encoded by this sequence can be produced at high levels under a T7 promoter and is entirely localized to inclusion bodies where problems relating to toxicity and/or instability are circumvented. Modification of the sequence by addition of an amino-terminal 9-histidine tag13 and elimination of internal methionine and cysteine residues from the trpLE sequence14 allowed trpLE-peptide fusions to be purified by metal-ion affinity chromatography and digested using cyanogen bromide (CNBr) to release the desired peptide sequence. We have successfully used this system to express more than a dozen different sequences as trpLE fusions representing membrane protein fragments that contain as many as four TM domains (7,8 and unpublished results; see also DISCUSSION section).
The key innovation in this protocol is the identification of conditions under which the unstructured and very poorly soluble trpLE-TM fusions can be efficiently disulfide-crosslinked in the context of a streamlined workflow. Several aspects of high-yield expression, handling and purification of peptide products have also been thoroughly optimized, and the recommendations presented here are equally relevant for production of non-disulfide-crosslinked (monomeric) TM peptide products.

<table border="1">
	<tbody>
		<tr>
			<td>Name of Reagent/Material</td>
			<td>Company</td>
			<td>Catalogue Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Cyanogen bromide</td>
			<td>ALDRICH</td>
			<td>P.No- C91492,CAS-506-68-3</td>
			<td>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.</td>
		</tr>
		<tr>
			<td>Trifluoroacetic acid</td>
			<td>SIGMA-ALDRICH</td>
			<td>P.CODE-1000984387, CAS Number 76-05-1</td>
			<td>HAZARDOUS SUBSTANCE. DANGEROUS GOODS., Causes severe burns. Harmful by inhalation. Harmful to aquatic organisms, may cause long-term adverse effects in the aquatic environment.</td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>SIGMA-ALDRICH</td>
			<td>P.No M7154, CAS Number 60-24-2</td>
			<td>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.</td>
		</tr>
		<tr>
			<td>1,1,1,3,3,3-Hexafluoro-2-propanol</td>
			<td>SIGMA-ALDRICH</td>
			<td>Product Number 52512, CAS-No. 920-66-1</td>
			<td>HAZARDOUS SUBSTANCE. DANGEROUS GOODS. Harmful by inhalation and if swallowed. Causes burns.</td>
		</tr>
		<tr>
			<td>Formic acid</td>
			<td>Merck KGaA</td>
			<td>K41186564</td>
			<td>Flammable liquid and vapour. Causes severe skin burns and eye damage.</td>
		</tr>
		<tr>
			<td>Urea</td>
			<td>UNIVAR, AJAX FINECHEM</td>
			<td>Product Number, 817, CAS-No 57-13-6</td>
			<td>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.</td>
		</tr>
		<tr>
			<td>GUANIDINE HYDROCHLORIDE</td>
			<td>Amresco</td>
			<td>P.No-M110, CAS Number: 50-01-1</td>
			<td>Harmful if swallowed, Causes serious eye irritation,Causes skin irritation, Acute Toxicity Oral, Skin Irritant, Eye Irritant.</td>
		</tr>
		<tr>
			<td>TRITON X-100</td>
			<td>SIGMA</td>
			<td>Product Number- T8532 CAS No: 9002-93-1</td>
			<td>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</td>
		</tr>
		<tr>
			<td>His-Select Nickel-Affinity gel</td>
			<td>SIGMA-ALDRICH</td>
			<td>Catalog Num- P6611</td>
			<td>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.</td>
		</tr>
		<tr>
			<td>(-)-Glutathione, oxidized</td>
			<td>SIGMA-ALDRICH</td>
			<td>Catalog num 150568</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Misonix S-3000 sonicator</td>
			<td>QSONICA</td>
			<td>S-3000 (discontinued)</td>
			<td>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.</td>
		</tr>
		<tr>
			<td>RP-HPLC: BioLogic DuoFlow chromatography system, Software Version 5.3</td>
			<td>Bio-Rad Laboratories</td>
			<td>Catalog Num 760-0047, Config No: AU500571, Serial No: 484BR3705</td>
			<td>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.</td>
		</tr>
		<tr>
			<td>Prep HT C3 ZORBAX 300SB-Analytical HPLC Column, 21.2 x 150 mm, 5 &mu;m particle size</td>
			<td>Agilent</td>
			<td>Product No: 895150-909</td>
			<td>Reversed-phase HPLC colum</td>
		</tr>
		<tr>
			<td>NuPAGE 12% Bis-Tris Gels</td>
			<td>Life Technologies</td>
			<td>NP0341BOX</td>
			<td>Pre cast gels for protein electrophoresis</td>
		</tr>
		<tr>
			<td>Slide-A-Lyzer G2 Dialysis Cassettes, 3.5K MWCO</td>
			<td>Thermo Scientific</td>
			<td>Product No: 87724</td>
			<td>Sample dialysis</td>
		</tr>
	</tbody>
</table>

production of disulfide-stabilized transmembrane peptide complexes for structural studies

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.

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).
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Watch this Scientific Journal Video about Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies at JoVE.com

Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies

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.

Physical  interactions among the lipid-embedded alpha-helical domains of membrane  proteins play a crucial role in folding and assembly of membrane ...

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