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 dipeptide sequence asp-pro should be avoided as it will also be cleaved in acidic conditions.
A unique cysteine residue should be introduced into each peptide sequence according to the desired positioning of the disulfide crosslink in the final complex, and all other cysteines should be mutated to serine. The plasmid carries a kanamycin resistance cassette for selection.
2. Expression of trpLE-peptide Fusion
<ol>
 <li>Make up and sterile filter M9/Kan minimal growth medium (0.1 mM CaCl2, 2 mM MgSO4, 3 g/L KH2PO4, 7.5 g/L Na2HPO4&middot;2H2O, 0.5 g/L NaCl, 1 g/L NH4Cl, 4 g/L glucose, 50 mg/L kanamycin sulfate). Supplement with Centrum Complete A to Zinc to provide B-vitamins and trace metals: dissolve 1 tablet in 40 ml H2O with mixing for 30 min, centrifuge to remove insoluble material and sterile filter the orange supernatant. Store stock solution at 4 &deg;C in the dark for up to 2 weeks and use 4 ml/L in M9. 
 NOTE: 15N-enriched NH4Cl, 13C-enriched glucose or other stable-isotope-labeled precursors may be included in M9 medium at this stage if desired.</li>
 <li>Inoculate a 100 ml starter culture from freshly transformed BL21(DE3) strain E. coli in M9/Kan medium. Grow overnight at 37 &deg;C with shaking at 200 rpm.</li>
 <li>The next morning, check the OD600 of the starter culture - this should be in the range of 2 or greater. Dilute the 100 ml starter culture into 1 L total volume of Centrum-supplemented M9 medium. Split into two 2 L baffled flasks (500 ml culture in each flask) and grow at 37 &deg;C shaking at 140 rpm until the OD600 reaches 0.6.</li>
 <li>Set the shaker temperature to 18 &deg;C and continue shaking for 1 hr, allowing cultures to cool completely before induction.</li>
 <li>Take a pre-induction sample for SDS-PAGE analysis (equivalent of 50 &mu;l from a culture at OD600 = 1), then add IPTG to 0.1 mM final concentration to induce expression of the trpLE-peptide fusion. Continue growing at 18 &deg;C/140 rpm overnight (16-20 hr) under IPTG induction.</li>
</ol>
3. Inclusion Body Preparation and Nickel Matrix Binding
<ol>
 <li>The final OD600 of overnight expression cultures in minimal medium is variable and should be between 2.5 and 5.0. Take a sample for SDS-PAGE analysis (equivalent of 50 &mu;l from a culture at OD600 = 1) and collect cells by centrifugation for 20 min at 5,000 x g.</li>
 <li>Resuspend cell pellet from 1 L culture in 50 ml lysis buffer (50 mM Tris-HCl pH 7.5, 20 mM &beta;-ME, 0.2 M NaCl). Cell suspensions can be stored frozen for later processing.</li>
 <li>Lyse cells by sonication on ice at maximum output for 1 min and rest on ice for 5 min. We use a Misonix Sonicator 3000 (max output 600 watts) with a &frac12;-inch high-intensity replaceable tip. Repeat sonication/cooling for three cycles.</li>
 <li>Collect insoluble inclusion body (IB) material by centrifugation for 15 min at 20,000 x g and discard the supernatant. A loose, thin layer of lighter-colored cell debris may be visible on top of the dense IB pellet; this can be washed away using water or buffer with gentle agitation. Steps 3.3 and 3.4 may be repeated to improve the purity of the IB fraction if desired. Samples of pellet and supernatant material should be taken for SDS-PAGE analysis to confirm trpLE-TM fusion localisation to inclusion bodies.</li>
 <li>Dissolve IB pellets in guanidine solution (6 M guanidine HCl, 50 mM Tris-HCl pH 8.0, 200 mM NaCl, 1% Triton X-100, 5 mM &beta;-ME), using 25-50 ml per liter of culture processed. This step will require several hours with agitation and occasional mild sonication.</li>
 <li>Clear the IB lysate by centrifugation for 1 hr at 75,000-100,000 x g and 20 &deg;C. Decant the supernatant and filter through a 0.2 &mu;m membrane.</li>
 <li>Combine the cleared IB lysate, in a 50 ml conical tube or a larger vessel that can be closed securely, with nickel affinity resin that has been washed with water and equilibrated to guanidine solution. Use 3-4 ml settled resin per liter of culture processed for fusions that express to a similar degree as trpLE-DAP12TM shown here (Figure 3, lane 2). Batch bind by incubating overnight at room temperature with gentle mixing.</li>
</ol>
4. On-column Oxidative Crosslinking
<ol>
 <li>Load the IB lysate/nickel resin suspension into an empty gravity flow column with porous bed support, adding continuously until the entire volume flows through. Collect and put aside the flowthrough, which may still contain unbound fusion protein.</li>
 <li>Wash the nickel resin by gravity flow with 5 bed volumes of urea solution (8 M urea, 50 mM Tris-HCl pH 8.0, 200 mM NaCl) containing 5 mM &beta;-ME.</li>
 <li>Wash the nickel resin again with 5 bed volumes of urea solution without &beta;-ME.</li>
 <li>Wash the nickel resin a third time with 5 bed volumes of urea solution that has been supplemented with 20 &mu;M CuSO4 and 2 mM oxidized glutathione. After this wash, close the column outlet and incubate 30 min at room temperature to allow maximal disulfide bond formation.</li>
</ol>
5. TFA Elution and Quantitation
CAUTION: Trifluoroacetic acid (TFA) causes severe burns on contact with skin and fumes are highly irritating. Concentrated TFA should be used only in an approved chemical fume hood with eye protection and non-latex gloves. Check the chemical compatibility of all materials used; polypropylene is compatible with all steps of this protocol.
<ol>
 <li>Wash out the oxidizing urea solution with 10 bed volumes of water and dry the column bed by applying a vacuum line to the column outlet.</li>
 <li>Close the column outlet and add 1.5 bed volumes of neat (99-100%) TFA. Stir the nickel resin with a small spatula to ensure even exposure to acid and incubate for 5 min. Open the column outlet and collect the acid eluate, pressurizing gently with a syringe or compressed air line if necessary to push out all of the liquid.</li>
 <li>Repeat the acid elution step with another 1.5 bed volumes of neat TFA. Work quickly at this step to avoid complete dissolution of the beaded agarose matrix. Protein yield can be determined at this point according to the Beer-Lambert equation and the theoretical molar extinction coefficient of the trpLE-peptide sequence. Dilute a sample of the TFA eluate (1:20 to 1:50) in trifluoroethanol (TFE) and measure the absorbance at 280 nm, using TFA/TFE at the same dilution as a blank.</li>
</ol>
6. CNBr Digestion
CAUTION: Cyanogen bromide (CNBr) is extremely toxic and should be handled only in an approved chemical fume hood. PPE including safety glasses, lab coat and non-latex gloves are absolutely required. CNBr is reactive to moisture and should be brought to room temperature before opening. Contact your institutional safety office for instructions on neutralization/disposal of CNBr-contaminated solutions and materials.
<ol>
 <li>Dilute the acid eluate to 80% final TFA concentration by dropwise addition of water while mixing gently. If a precipitate forms, add back a small volume (0.5 to 1 ml) of neat TFA until the solution clarifies, then re-correct to 80% with water. Take a 5 &mu;l pre-digest sample for SDS-PAGE analysis, freezing in liquid nitrogen and lyophilizing the sample immediately.</li>
 <li>Add CNBr crystals to approximately 0.2 g/ml, taking care to weigh the toxic chemical safely in closed, pre-weighed tubes or using a balance inside the chemical fume hood. Mix gently until completely dissolved. Flush and seal the reaction vessel under inert gas (nitrogen or argon stream) and incubate 3-4 hr at room temperature, protected from light.</li>
 <li>Take a 5 &mu;l post-digest sample for SDS-PAGE analysis and freeze and lyophilize immediately. Transfer the digest reaction to a regenerated cellulose dialysis cassette or tubing (cellulose acetate will dissolve in concentrated acid) with a molecular weight cutoff of 3.5 kDa or less, according to the size of the expected peptide complex. Dialyze overnight to 4 L of water in the chemical fume hood to reduce the TFA concentration and decompose any unreacted CNBr. Leave room in the dialysis cassette or tubing for a significant increase in volume (as much as two- to three-fold) during overnight dialysis.</li>
 <li>Remove the dialyzed reaction solution with suspended precipitate from the dialysis cassette or tubing (most of the protein will precipitate when the TFA concentration is reduced) and transfer the suspension to 50 ml conical tubes. Freeze and lyophilize to remove water and traces of CNBr/TFA. This step is likely to require several days. 
 CAUTION: Both the dialyzed digest reaction and the dialysis water should continue to be treated as toxic and handled/disposed with appropriate precautions.</li>
 <li>Samples of cultures from pre-induction and harvest stages, inclusion body supernatant and pellet, TFA eluate and CNBr-digested material may be analyzed by SDS-PAGE. Prepare samples by heating to 95 &deg;C in Invitrogen NuPAGE LDS sample buffer. TFA eluate and digest samples should be prepared in both reducing and non-reducing conditions to facilitate product identification and evaluation of oxidative crosslinking.</li>
 <li>Separate the samples on a 12% NuPAGE bis-tris pre-cast acrylamide gel using MES-SDS running buffer for optimal resolution of the smallest digest products.</li>
</ol>
7. Reversed-phase HPLC Purification of Disulfide-crosslinked Peptide Complexes
<ol>
 <li>Dissolve up to 100 mg of lyophilized digest products into 2-3 ml neat formic acid and load onto a preparative-scale (21.2 x 150 mm) Agilent ZORBAX SB-300 C3 PrepHT column in solvent A (typically 10-20% acetonitrile or 5-10% isopropanol in water with 0.1% TFA).</li>
 <li>Elute the digest products in a gradient of solvent B (acetonitrile or isopropanol with 0.1% TFA) over at least 5 column volumes. See DISCUSSION for suggestions on the selection of optimal gradient conditions for different peptide sequences.</li>
 <li>Take 50-100 &mu;l samples from each HPLC fraction for SDS-PAGE analysis and freeze and lyophilize immediately. Removal of HPLC solvents is rapid and should be complete in 1-2 hr.</li>
 <li>Prepare lyophilized HPLC samples for SDS-PAGE by heating to 95 &deg;C in Invitrogen NuPAGE LDS sample buffer with no reducing agent. Cool and split each sample evenly into 2 microcentrifuge tubes, adding 100 mM DTT to one of them and re-heating briefly.</li>
 <li>Separate the reduced and non-reduced HPLC samples on a 12% NuPAGE bis-tris pre-cast acrylamide gel with MES-SDS running buffer as above. Small, hydrophobic peptides often do not take up Coomassie stain well and may not run at their expected molecular weights. However, comparison of reduced and non-reduced samples should facilitate identification of the crosslinked peptide products and assessment of purity.</li>
 <li>Analyze the candidate peptide-containing fractions by matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (see DISCUSSION) to identify the species of interest and confirm its expected mass.</li>
 <li>Combine, freeze and lyophilize HPLC fractions containing the pure, crosslinked TM peptide complex and take a dry weight of the final product to determine yield. Fractions with high isopropanol content may not remain frozen. If peptide fractions dry down to a film rather than a fluffy lyophilized product, re-dissolve the film completely in a small volume of pure hexafluoroisopropanol (HFIP), freeze and lyophilize again. The HFIP-treated product should be a small, white cone that is easily tipped out and weighed.</li>
</ol>

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	<li>Call, M. E., Chou, J. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+view+into+the+blind+spot:+solution+NMR+provides+new+insights+into+signal+transduction+across+the+lipid+bilayer.">A view into the blind spot: solution NMR provides new insights into signal transduction across the lipid bilayer.</a> Structure. 18, 1559-1569 (2010).</li><li>Bocharov, E. V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+structure+of+the+dimeric+transmembrane+domain+of+the+growth+factor+receptor+ErbB2+presumably+corresponding+to+the+receptor+active+state.">Spatial structure of the dimeric transmembrane domain of the growth factor receptor ErbB2 presumably corresponding to the receptor active state.</a> The Journal of Biological Chemistry. 283, 6950-6956 (2008).</li><li>Mineev, K. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+structure+of+the+transmembrane+domain+heterodimer+of+ErbB1+and+ErbB2+receptor+tyrosine+kinases.">Spatial structure of the transmembrane domain heterodimer of ErbB1 and ErbB2 receptor tyrosine kinases.</a> J. Mol. Biol. 400, 231-243 (2010).</li><li>Mineev, K. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spatial+structure+and+dimer--monomer+equilibrium+of+the+ErbB3+transmembrane+domain+in+DPC+micelles.">Spatial structure and dimer--monomer equilibrium of the ErbB3 transmembrane domain in DPC micelles.</a> Biochim. Biophys Acta. 1808, 2081-2088 (2011).</li><li>Lau, T. L., Kim, C., Ginsberg, M. H., Ulmer, T. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+the+integrin+alphaIIbbeta3+transmembrane+complex+explains+integrin+transmembrane+signalling.">The structure of the integrin alphaIIbbeta3 transmembrane complex explains integrin transmembrane signalling.</a> The EMBO Journal. 28, 1351-1361 (2009).</li><li>Zhu, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+structure+of+a+receptor+with+two+associating+transmembrane+domains+on+the+cell+surface:+integrin+alphaIIbbeta3.">The structure of a receptor with two associating transmembrane domains on the cell surface: integrin alphaIIbbeta3.</a> Molecular Cell. 34, 234-249 (2009).</li><li>Call, M. 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E. x. p. r. e. s. s. i. o. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=purification,+and+isotope+labeling+of+cannabinoid+CB2+receptor+fragment,+CB2(180-233).">purification, and isotope labeling of cannabinoid CB2 receptor fragment, CB2(180-233).</a> Protein Expr. Purif. 38, 61-68 (2004).</li><li>Miozzari, G. F., Yanofsky, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Translation+of+the+leader+region+of+the+Escherichia+coli+tryptophan+operon.">Translation of the leader region of the Escherichia coli tryptophan operon.</a> J. Bacteriol. 133, 1457-1466 (1978).</li><li>North, C. L., Blacklow, S. 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The authors declare that they have no competing financial interests.

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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14.0K Views.  The Walter and Eliza Hall Institute of Medical Research. 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.

Video: Production of Disulfide-stabilized Transmembrane Peptide Complexes for Structural Studies

Steady-state, Pre-steady-state, and Single-turnover Kinetic Measurement for DNA Glycosylase Activity

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization of OGG1 is undertaken to measure the rates of 8-oxoG excision and product release. When the OGG1 concentration is lower than substrate DNA, time courses of product formation are biphasic; a rapid exponential phase (i.e. burst) of product formation is followed by a linear steady-state phase. The initial burst of product formation corresponds to the concentration of enzyme properly engaged on the substrate, and the burst amplitude depends on the concentration of enzyme. The first-order rate constant of the burst corresponds to the intrinsic rate of 8-oxoG excision and the slower steady-state rate measures the rate of product release (product DNA dissociation rate constant, koff). Here, we describe steady-state, pre-steady-state, and single-turnover approaches to isolate and measure specific steps during OGG1 catalytic cycling. A fluorescent labeled lesion-containing oligonucleotide and purified OGG1 are used to facilitate precise kinetic measurements. Since low enzyme concentrations are used to make steady-state measurements, manual mixing of reagents and quenching of the reaction can be performed to ascertain the steady-state rate (koff). Additionally, extrapolation of the steady-state rate to a point on the ordinate at zero time indicates that a burst of product formation occurred during the first turnover (i.e. y-intercept is positive). The first-order rate constant of the exponential burst phase can be measured using a rapid mixing and quenching technique that examines the amount of product formed at short time intervals (&lt;1 sec) before the steady-state phase and corresponds to the rate of 8-oxoG excision (i.e. chemistry). The chemical step can also be measured using a single-turnover approach where catalytic cycling is prevented by saturating substrate DNA with enzyme (E&gt;S). These approaches can measure elementary rate constants that influence the efficiency of removal of a DNA lesion.

Time courses for the glycosylase activity of 8-oxoguanine DNA glycosylase are biphasic exhibiting a burst of product formation and a linear steady-state phase. Utilizing quench-flow techniques, the burst and the steady-state rates can be measured, which correspond to excision of 8-oxoguanine and release of the glycosylase from the product DNA, respectively.

Human 8-oxoguanine DNA glycosylase (OGG1) excises the mutagenic oxidative DNA lesion 8-oxo-7,8-dihydroguanine (8-oxoG) from DNA. Kinetic characterization ...

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox reactions. The Cu (I) complex of the peptide model of a Cu (I) binding metallochaperone protein, which includes the sequence MTCSGCSRPG (underlined is conserved), was determined in solution under inert conditions by NMR spectroscopy.
NMR is a widely accepted technique for the determination of solution structures of proteins and peptides. Due to difficulty in crystallization to provide single crystals suitable for X-ray crystallography, the NMR technique is extremely valuable, especially as it provides information on the solution state rather than the solid state. Herein we describe all steps that are required for full three-dimensional structure determinations by NMR. The protocol includes sample preparation in an NMR tube, 1D and 2D data collection and processing, peak assignment and integration, molecular mechanics calculations, and structure analysis. Importantly, the analysis was first conducted without any preset metal-ligand bonds, to assure a reliable structure determination in an unbiased manner.

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

The NMR-solution structure of a metallochaperone model peptide with Cu (I) was determined, and a detailed protocol from sample preparation and 1D and 2D data collection to a three-dimensional structure is described.

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox ...

Thin-layer Chromatographic (TLC) Separations and Bioassays of Plant Extracts to Identify Antimicrobial Compounds

A common screen for plant antimicrobial compounds consists of separating plant extracts by paper or thin-layer chromatography (PC or TLC), exposing the chromatograms to microbial suspensions (e.g. fungi or bacteria in broth or agar), allowing time for the microbes to grow in a humid environment, and visualizing zones with no microbial growth.&#160;The effectiveness of this screening method, known as bioautography, depends on both the quality of the chromatographic separation and the care taken with microbial culture conditions.&#160;This paper describes standard protocols for TLC and contact bioautography&#160;with a novel application to amino acid-fermenting bacteria. The extract is separated on flexible (aluminum-backed) silica TLC plates, and bands are visualized under ultraviolet (UV) light.&#160;Zones are cut out and incubated face down onto agar inoculated with the test microorganism.&#160;Inhibitory bands are visualized by staining the agar plates with tetrazolium red.&#160;The method is applied to the separation of red clover (Trifolium pratense cv. Kenland) phenolic compounds and their screening for activity against Clostridium sticklandii, a hyper ammonia-producing bacterium (HAB) that is native to the bovine rumen.&#160;The TLC methods apply to many types of plant extracts&#160;and other bacterial species (aerobic or anaerobic), as well as fungi, can be used as test organisms if culture conditions are modified to fit the growth requirements of the species.

Thin-layer Chromatographic TLC Separations and Bioassays of Plant Extracts to Identify Antimicrobial Compounds

Methods are described for thin-layer chromatographic (TLC) separation of plant extracts and contact bioautography to identify antibacterial metabolites. The methods are applied to the screening of red clover phenolic compounds inhibiting hyper&#160;ammonia-producing bacteria (HAB) native to the bovine rumen.

A common screen for plant antimicrobial compounds consists of separating plant extracts by paper or thin-layer chromatography (PC or TLC), exposing the ...

Capillary Electrophoresis to Monitor Peptide Grafting onto Chitosan Films in Real Time

Free-solution capillary electrophoresis (CE) separates analytes, generally charged compounds in solution through the application of an electric field. Compared to other analytical separation techniques, such as chromatography, CE is cheap, robust and effectively requires no sample preparation (for a number of complex natural matrices or polymeric samples). CE is fast and can be used to follow the evolution of mixtures in real time (e.g., chemical reaction kinetics), as the signals observed for the separated compounds are directly proportional to their quantity in solution.
Here, the efficiency of CE is demonstrated for monitoring the covalent grafting of peptides onto chitosan films for subsequent biomedical applications. Chitosan&#39;s antimicrobial and biocompatible properties make it an attractive material for biomedical applications such as cell growth substrates. Covalently grafting the peptide RGDS (arginine - glycine - aspartic acid - serine) onto the surface of chitosan films aims at improving cell attachment. Historically, chromatography and amino acid analysis have been used to provide a direct measurement of the amount of grafted peptide. However, the fast separation and absence of sample preparation provided by CE enables equally accurate yet real-time monitoring of the peptide grafting process. CE is able to separate and quantify the different components of the reaction mixture: the (non-grafted) peptide and the chemical coupling agents. In this way the use of CE results in improved films for downstream applications.
The chitosan films were characterized through solid-state NMR (nuclear magnetic resonance) spectroscopy. This technique is more time-consuming and cannot be applied in real time, but yields a direct measurement of the peptide and thus validates the CE technique.

Free solution capillary electrophoresis is a fast, cheap and robust analytical method that enables the quantitative monitoring of chemical reactions in real time. Its utility for rapid, convenient and precise analysis is demonstrated here through analysis of covalent peptide grafting onto chitosan films for improved cell adhesion.

Free-solution capillary electrophoresis (CE) separates analytes, generally charged compounds in solution through the application of an electric field. ...

Controlled Photoredox Ring-Opening Polymerization of O-Carboxyanhydrides Mediated by Ni/Zn Complexes

Here, we describe an effective protocol that combines photoredox Ni/Ir catalysis with the use of a Zn-alkoxide for efficient ring-opening polymerization, allowing for the synthesis of isotactic poly(&#945;-hydroxy acids) with expected molecular weights (&#62;140 kDa) and narrow molecular weight distributions (Mw/Mn &#60; 1.1). This ring-opening polymerization is mediated by Ni and Zn complexes in the presence of an alcohol initiator and a photoredox Ir catalyst, irradiated by a blue LED (400 - 500 nm). The polymerization is performed at a low temperature (-15 &#176;C) to avoid undesired side reactions. The complete monomer consumption can be achieved within 4 - 8 hours, providing a polymer close to the expected molecular weight with narrow molecular weight distribution. The resulted number-average molecular weight shows a linear correlation with the degree of polymerization up to 1000. The homodecoupling 1H NMR study confirms that the obtained polymer is isotactic without epimerization. This polymerization reported herein offers a strategy for achieving rapid, controlled O-carboxyanhydrides polymerization to prepare stereoregular poly(&#945;-hydroxy acids) and its copolymers bearing various functional side-chain groups.

Controlled Photoredox Ring-Opening Polymerization of O-Carboxyanhydrides Mediated by Ni/Zn Complexes

A protocol for the controlled photoredox ring-opening polymerization of O-carboxyanhydrides mediated by Ni/Zn complexes is presented.

Here, we describe an effective protocol that combines photoredox Ni/Ir catalysis with the use of a Zn-alkoxide for efficient ring-opening polymerization, ...

Preparation of Stable Bicyclic Aziridinium Ions and Their Ring-Opening for the Synthesis of Azaheterocycles

Bicyclic aziridinium ions were generated by the removal of an appropriate leaving group through internal nucleophilic attack by nitrogen atom in the aziridine ring. The utility of bicyclic aziridinium ions, specifically 1-azoniabicyclo[3.1.0]hexane and 1-azoniabicyclo[4.1.0]heptane tosylate highlighted in the aziridine ring openings by the nucleophile with the release of the ring strain to yield the corresponding ring-expanded azaheterocycles such as pyrrolidine, piperidine and azepane with diverse substituents on the ring in regio- and stereospecific manner. Herein, we report a simple and convenient method for the preparation of the stable 1-azabicyclo[4.1.0]heptane tosylate followed by selective ring opening via a nucleophilic attack either at the bridge or at the bridgehead carbon to yield piperidine and azepane rings, respectively. This synthetic strategy allowed us to prepare biologically active natural products containing piperidine and azepane motif including sedamine, allosedamine, fagomine and balanol in highly efficient manner.

Bicyclic aziridinium ions such as 1-azoniabicyclo[4.1.0]heptane tosylate were generated from 2-[4-tolenesulfonyloxybutyl]aziridine, which was utilized for the preparation of substituted piperidines and azepanes via regio- and stereospecific ring-expansion with various nucleophiles. This highly efficient protocol allowed us to prepare diverse azaheterocycles including natural products such as fagomine, febrifugine analogue and balanol.

Bicyclic aziridinium ions were generated by the removal of an appropriate leaving group through internal nucleophilic attack by nitrogen atom in the ...

Novel Techniques for Observing Structural Dynamics of Photoresponsive Liquid Crystals

We discuss in this article the experimental measurements of the molecules in liquid crystal (LC) phase using the time-resolved infrared (IR) vibrational spectroscopy and time-resolved electron diffraction. Liquid crystal phase is an important state of matter that exists between the solid and liquid phases and it is common in natural systems as well as in organic electronics. Liquid crystals are orientationally ordered but loosely packed, and therefore, the internal conformations and alignments of the molecular components of LCs can be modified by external stimuli. Although advanced time-resolved diffraction techniques have revealed picosecond-scale molecular dynamics of single crystals and polycrystals, direct observations of packing structures and ultrafast dynamics of soft materials have been hampered by blurry diffraction patterns. Here, we report time-resolved IR vibrational spectroscopy and electron diffractometry to acquire ultrafast snapshots of a columnar LC material bearing a photoactive core moiety. Differential-detection analyses of the combination of time-resolved IR vibrational spectroscopy and electron diffraction are powerful tools for characterizing structures and photoinduced dynamics of soft materials.

Here, we present the protocols of differential-detection analyses of time-resolved infrared vibrational spectroscopy and electron diffraction which enable observations of the deformations of local structures around photoexcited molecules in a columnar liquid crystal, giving an atomic perspective on the relationship between the structure and the dynamics of this photoactive material.

We discuss in this article the experimental measurements of the molecules in liquid crystal (LC) phase using the time-resolved infrared (IR) vibrational ...

Analyzing Protein Architectures and Protein-Ligand Complexes by Integrative Structural Mass Spectrometry

Proteins are an important class of biological macromolecules that play many key roles in cellular functions including gene expression, catalyzing metabolic reactions, DNA repair and replication. Therefore, a detailed understanding of these processes provides critical information on how cells function. Integrative structural MS methods offer structural and dynamical information on protein complex assembly, complex connectivity, subunit stoichiometry, protein oligomerization and ligand binding. Recent advances in integrative structural MS have allowed for the characterization of challenging biological systems including large DNA binding proteins and membrane proteins. This protocol describes how to integrate diverse MS data such as native MS and ion mobility-mass spectrometry (IM-MS) with molecular dynamics simulations to gain insights into a helicase-nuclease DNA repair protein complex. The resulting approach provides a framework for detailed studies of ligand binding to other protein complexes involved in important biological processes.

Mass spectrometry (MS) has emerged as an important tool for the investigation of structure and dynamics of macromolecular assemblies. Here, we integrate MS-based approaches to interrogate protein complex formation and ligand binding.

Proteins are an important class of biological macromolecules that play many key roles in cellular functions including gene expression, catalyzing ...

Preparation of SNS Cobalt(II) Pincer Model Complexes of Liver Alcohol Dehydrogenase

Chemical model complexes are prepared to represent the active site of an enzyme. In this protocol, a family of tridentate pincer ligand precursors (each possessing two sulfur and one nitrogen donor atom functionalities (SNS) and based on bis-imidazole or bis-triazole compounds) are metallated with CoCl2&#183;6H2O to afford tridentate SNS pincer cobalt(II) complexes. Preparation of the cobalt(II) model complexes for liver alcohol dehydrogenase is facile. Based on a quick color change upon adding the CoCl2&#183;6H2O to acetonitrile solution that contains the ligand precursor, the complex forms rapidly. Formation of the metal complex is complete after allowing the solution to reflux overnight. These cobalt(II) complexes serve as models for the zinc active site in liver alcohol dehydrogenase (LADH). The complexes are characterized using single crystal X-ray diffraction, electrospray mass spectrometry, ultra-violet visible spectroscopy, and elemental analysis. To accurately determine the structure of the complex, its single crystal structure must be determined. Single crystals of the complexes that are suitable for X-ray diffraction are then grown via slow vapor diffusion of diethyl ether into an acetonitrile solution that contains the cobalt(II) complex. For high quality crystals, recrystallization typically takes place over a 1 week period, or longer. The method can be applied to the preparation of other model coordination complexes and can be used in undergraduate teaching laboratories. Finally, it is believed that others may find this recrystallization method to obtain single crystals beneficial to their research.

Preparation of SNS CobaltII Pincer Model Complexes of Liver Alcohol Dehydrogenase

The preparation of SNS pincer cobalt(II) model complexes of liver alcohol dehydrogenase is presented here. The complexes can be prepared by reacting the ligand precursor with CoCl2&#183;6H2O and can then be recrystallized by allowing diethyl ether to slowly diffuse into an acetonitrile solution that contains the cobalt complex.

Chemical model complexes are prepared to represent the active site of an enzyme. In this protocol, a family of tridentate pincer ligand precursors (each ...

Directed Assembly of Elastin-like Proteins into defined Supramolecular Structures and Cargo Encapsulation In Vitro

Tailored proteinaceous building blocks are versatile candidates for the assembly of supramolecular structures such as minimal cells, drug delivery vehicles and enzyme scaffolds. Due to their biocompatibility and tunability on the genetic level, Elastin-like proteins (ELP) are ideal building blocks for biotechnological and biomedical applications. Nevertheless, the assembly of protein based supramolecular structures with distinct physiochemical properties and good encapsulation potential remains challenging.
Here we provide two efficient protocols for guided self-assembly of amphiphilic ELPs into supramolecular protein architectures such as spherical coacervates, fibers and stable vesicles. The presented assembly protocols generate Protein Membrane-Based Compartments (PMBCs) based on ELPs with adaptable physicochemical properties. PMBCs demonstrate phase separation behavior and reveal method dependent membrane fusion and are able to encapsulate chemically diverse fluorescent cargo molecules. The resulting PMBCs have a high application potential as a drug formulation and delivery platform, artificial cell, and compartmentalized reaction space.

At the interface of organic and aqueous solvents, tailored amphiphilic elastin-like proteins assemble into complex supramolecular structures such as vesicles, fibers and coacervates triggered by environmental parameters. The described assembly protocols yield Protein Membrane-Based Compartments (PMBCs) with tunable properties, enabling the encapsulation of various cargo.