Bio-conjugation of anti-human CD3 monoclonal antibodies to magnetic nanoparticles by using cyanogen bromide: A potential for cell sorting and noninvasive diagnosis.

The conjugation of monoclonal antibodies with superparamagnetic iron oxide nanoparticles (SPIONs) has appeared as a potential multifunctional clinical tool, which can effectively diagnose cancers and monitor their treatment, specifically. Despite the presence of different methods for conjugating antibodies to iron oxide nanoparticles, novel cost-effective and simpler conjugation techniques should be performed in this regard. In current study, an anti-CD3 monoclonal antibody was conjugated to the Fe3O4 coated by carboxymethyl dextran (CMD) using cyanogen bromide (CNBr). Moreover, EDC/NHS techniques were applied as a positive control. The experimental results showed that the Conjugation was performed and the presence of the antibody conjugated to the MNPs in human xenograft tumors was confirmed using Prussian blue (PB) staining, following magnetic resonance imaging (MRI), 30 min after injection. This conjugation method was shown to be able to separate CD3+ T lymphocytes efficiently from whole blood with high purity. Accordingly, this type of bio-conjugation method can be utilized in the future for cell sorting, and can be applied for adopted cell therapies such as CAR-T cell (Chimeric antigen receptor T cell) therapy, as well as targeted MRI imaging.

Preparation of Caveolin-1 for NMR Spectroscopy Experiments.

Caveolin-1 is a 20.5 kDa integral membrane protein that is involved in a myriad of cellular processes including signal transduction, relieving mechano-stresses on the cell, endocytosis, and most importantly caveolae formation. As a consequence, there is intense interest in characterizing caveolin-1 structurally. Out of the many available structural techniques, nuclear magnetic resonance (NMR) spectroscopy is particularly well suited to investigations on integral membrane proteins like caveolin-1 that have significant unstructured regions and unusual topologies. However, the technique requires relatively large amounts of protein (i.e. concentrations in the 0.5-5 mM range), and obtaining these amounts can be difficult especially for highly hydrophobic membrane proteins such as caveolin-1. Herein, we describe a robust protocol for the preparation of caveolin-1 for structural studies using NMR.

Free and Immobilized Lecitase™ Ultra as the Biocatalyst in the Kinetic Resolution of (E)-4-Arylbut-3-en-2-yl Esters.

The influence of buffer type, co-solvent type, and acyl chain length was investigated for the enantioselective hydrolysis of racemic 4-arylbut-3-en-2-yl esters using Lecitase™ Ultra (LU). Immobilized preparations of the Lecitase™ Ultra enzyme had significantly higher activity and enantioselectivity than the free enzyme, particularly for 4-phenylbut-3-en-2-yl butyrate as the substrate. Moreover, the kinetic resolution with the immobilized enzyme was achieved in a much shorter time (24-48 h). Lecitase™ Ultra, immobilized on cyanogen bromide-activated agarose, was particularly effective, producing, after 24 h of reaction time in phosphate buffer (pH 7.2) with acetone as co-solvent, both (R)-alcohols and unreacted (S)-esters with good to excellent enantiomeric excesses (ee 90-99%). These conditions and enzyme were also suitable for the kinetic separation of racemic (E)-4-phenylbut-3-en-2-yl butyrate analogs containing methyl substituents on the benzene ring (4b,4c), but they did not show any enantioselectivity toward (E)-4-(4'-methoxyphenyl)but-3-en-2-yl butyrate (4d).

Quantitative Mass Spectrometric Assay of Whole and CNBr-Cleaved Amyloid-ß Peptides in Human Brain.

Various amyloid-ß (Aß) peptides accumulate in brain in Alzheimer's disease, and the amounts of specific peptide variants may have pathological significance. The quantitative determination of these variants is challenging because losses inevitably occur during tissue processing and analysis. This report describes the use of stable-isotope-labeled Aß peptides as internal standards for quantitative mass spectrometric assays, and the use of cyanogen bromide (CNBr) to remove C-terminal residues beyond Met35. The removal of residues beyond Met35 reduces losses due to aggregation, and facilitates the detection of post-translationally modified Aß peptides. Results from 8 human brain samples suggest that the tissue concentrations of the 42-residue Aß peptide tend to be similar in different patients. Concentrations of the 40-residue Aß peptide are more variable, and may be greater or lesser than the 42-residue peptide. The concentration of the CNBr cleavage product closely matches the sum of the 40-residue and 42-residue peptide concentrations, indicating that these two Aß peptides account for most of the C-terminal variants in these patients. CNBr treatment facilitated the detection of post-translational modifications such as pyroglutamyl and hexose-modified Aß peptides.

Heterologous production of a new lasso peptide brevunsin in Sphingomonas subterranea.

A shuttle vector pHSG396Sp was constructed to perform gene expression using Sphingomonas subterranea as a host. A new lasso peptide biosynthetic gene cluster, derived from Brevundimonas diminuta, was amplified by PCR and integrated to afford a expression vector pHSG396Sp-12697L. The new lasso peptide brevunsin was successfully produced by S. subterranea, harboring the expression vector, with a high production yield (10.2 mg from 1 L culture). The chemical structure of brevunsin was established by NMR and MS/MS experiments. Based on the information obtained from the NOE experiment, the three-dimensional structure of brevunsin was determined, which indicated that brevunsin possessed a typical lasso structure. This expression vector system provides a new heterologous production method for unexplored lasso peptides that are encoded by bacterial genomes.

Electron transfer dissociation of synthetic and natural peptides containing lanthionine/methyllanthionine bridges.

RATIONALE: The modes of cleavage of lanthionine/methyllanthionine bridges under electron transfer dissociation (ETD) were investigated using synthetic and natural lantipeptides. Knowledge of the mass spectrometric fragmentation of lanthionine/methyllanthionine bridges may assist in the development of analytical methods for the rapid discovery of new lantibiotics. The present study strengthens the advantage of ETD in the characterization of posttranslational modifications of peptides and proteins. METHODS: Synthetic and natural lantipeptides were obtained by desulfurization of peptide disulfides and cyanogen bromide digestion of the lantibiotic nisin, respectively. These peptides were subjected to electrospray ionization collision-induced dissociation tandem mass spectrometry (CID-MS/MS) and ETD-MS/MS using an HCT ultra ETDII ion trap mass spectrometer. MS3 CID was performed on the desired product ions to prove cleavage of the lanthionine/methyllanthionine bridge during ETD-MS/MS. RESULTS: ETD has advantages over CID in the cleavage of the side chain of lanthionine/methyllanthionine bridges. The cleavage of the N-Cα backbone peptide bond followed by C-terminal side chain of the lanthionine bridge results in formation of c•+ and z+ ions. Cleavage at the preceding peptide bond to the C-terminal side chain of lanthionine/methyllanthionine bridges yields specific fragments with the cysteine/methylcysteine thiyl radical and dehydroalanine. CONCLUSIONS: ETD successfully cleaves the lanthionine/methyllanthionine bridges of synthetic and natural lantipeptides. Diagnostic fragment ions of ETD cleavage of lanthionine/methyllanthionine bridges are the N-terminal cysteine/methylcysteine thiyl radical and C-terminal dehydroalanine. Detection of the cysteine/methylcysteine thiyl radical and dehydroalanine in combined ETD-CID-MS may be used for the rapid identification of lantipeptide natural products.

Stabilization of multimeric sucrose synthase from Acidithiobacillus caldus via immobilization and post-immobilization techniques for synthesis of UDP-glucose.

Sucrose synthases (SuSys) have been attracting great interest in recent years in industrial biocatalysis. They can be used for the cost-effective production of uridine 5'-diphosphate glucose (UDP-glucose) or its in situ recycling if coupled to glycosyltransferases on the production of glycosides in the food, pharmaceutical, nutraceutical, and cosmetic industry. In this study, the homotetrameric SuSy from Acidithiobacillus caldus (SuSyAc) was immobilized-stabilized on agarose beads activated with either (i) glyoxyl groups, (ii) cyanogen bromide groups, or (iii) heterogeneously activated with both glyoxyl and positively charged amino groups. The multipoint covalent immobilization of SuSyAc on glyoxyl agarose at pH 10.0 under optimized conditions provided a significant stabilization factor at reaction conditions (pH 5.0 and 45 °C). However, this strategy did not stabilize the enzyme quaternary structure. Thus, a post-immobilization technique using functionalized polymers, such as polyethyleneimine (PEI) and dextran-aldehyde (dexCHO), was applied to cross-link all enzyme subunits. The coating of the optimal SuSyAc immobilized glyoxyl agarose with a bilayer of 25 kDa PEI and 25 kDa dexCHO completely stabilized the quaternary structure of the enzyme. Accordingly, the combination of immobilization and post-immobilization techniques led to a biocatalyst 340-fold more stable than the non-cross-linked biocatalyst, preserving 60% of its initial activity. This biocatalyst produced 256 mM of UDP-glucose in a single batch, accumulating 1 M after five reaction cycles. Therefore, this immobilized enzyme can be of great interest as a biocatalyst to synthesize UDP-glucose.

Understanding the viscoelastic behavior of arterial elastin in glucose via relaxation time distribution spectrum.

Elastin is a critical extracellular matrix protein that provides many biological tissues with resilience. In elastic arteries such as aorta, elasticity is crucial for energy storage and transmission of the pulsatile blood flow. As one of the main mechanisms of aging, non-enzymatic glycation can greatly compromise the mechanical properties of the long-lived elastin. In this study, effect of glucose on the viscoelastic behavior of purified porcine aortic elastin was investigated through stress relaxation tests and the corresponding relaxation time distribution spectra. Elastin was incubated in 2M glucose solution at 37°C for 4, 7, 14, 21 or 28 days. Biaxial stress relaxation tests were performed to study the viscoelastic property of elastin. Elastin samples with glucose treatment show increased stress relaxation with incubation time. Continuous relaxation time distribution spectra were obtained from the stress relaxation data using Tikhonov regularization method. Generally the spectra of both untreated and treated elastin have a broad range of relaxation time constants and multiple peaks located between 0.1-10,000s. The intensity of the short-term peak (0.1-10s) increases after glucose exposure whereas the intensity of the long-term peak (> 100s) decreases. The dominant peaks, i.e., the long-term peak of untreated tissue and the short-term peak of glucose treated tissue, suggest different relaxation mechanisms. The initial stress level dependency of stress relaxation was studied and the results suggested that the intensity of all the peaks increases with higher initial stresses. A multi-exponential model was developed to describe the stress relaxation behavior with material parameters obtained directly from the continuous relaxation spectrum. To fully characterize the relaxation processes, a multi-exponential model with four exponential terms, located between 0.001-1s, 1-10s, 10-100s, and 100-10,000s and obtained directly from the corresponding relaxation spectrum, appears to best capture the stress relaxation behavior of elastin before and after glucose exposure.

Different Covalent Immobilizations Modulate Lipase Activities of Hypocrea pseudokoningii.

Enzyme immobilization can promote several advantages for their industrial application. In this work, a lipase from Hypocrea pseudokoningii was efficiently linked to four chemical supports: agarose activated with cyanogen bromide (CNBr), glyoxyl-agarose (GX), MANAE-agarose activated with glutaraldehyde (GA) and GA-crosslinked with glutaraldehyde. Results showed a more stable lipase with both the GA-crosslinked and GA derivatives, compared to the control (CNBr), at 50 °C, 60 °C and 70 °C. Moreover, all derivatives were stabilized when incubated with organic solvents at 50%, such as ethanol, methanol, n-propanol and cyclohexane. Furthermore, lipase was highly activated (4-fold) in the presence of cyclohexane. GA-crosslinked and GA derivatives were more stable than the CNBr one in the presence of organic solvents. All derivatives were able to hydrolyze sardine, açaí (Euterpe oleracea), cotton seed and grape seed oils. However, during the hydrolysis of sardine oil, GX derivative showed to be 2.3-fold more selectivity (eicosapentaenoic acid (EPA)/docosahexaenoic acid (DHA) ratio) than the control. Additionally, the types of immobilization interfered with the lipase enantiomeric preference. Unlike the control, the other three derivatives preferably hydrolyzed the R-isomer of 2-hydroxy-4-phenylbutanoic acid ethyl ester and the S-isomer of 1-phenylethanol acetate racemic mixtures. On the other hand, GX and CNBr derivatives preferably hydrolyzed the S-isomer of butyryl-2-phenylacetic acid racemic mixture while the GA and GA-crosslink derivatives preferably hydrolyzed the R-isomer. However, all derivatives, including the control, preferably hydrolyzed the methyl mandelate S-isomer. Moreover, the derivatives could be used for eight consecutive cycles retaining more than 50% of their residual activity. This work shows the importance of immobilization as a tool to increase the lipase stability to temperature and organic solvents, thus enabling the possibility of their application at large scale processes.

Expression of Mastoparan B, a Venom Peptide, Via Escherichia coli C43 (DE3) Coupled with an Artificial Oil Body-Cyanogen Bromide Technology Platform.

BACKGROUND: Mastoparan B (MPB) is a venom peptide isolated from Vespa basalis (black-bellied hornet), one of the dangerous vespine wasps found in Taiwan. MPB is a tetradecapeptide (LKLKSIVSWAKKVL), amphiphilic venom peptide, with a molecular mass of 1.6 kDa. MPB belongs to an evolutionarily conserved component of the innate immune response against microbes. In this study, we attempted to modify a reliable oleosin-based fusion expression strategy coupled with the artificial oil body (AOB)-cyanogen bromide (CNBr) platform to produce bioactive MPB. OBJECTIVES: The aim of this study was to develop an artificial oil body (AOB)-cyanogen bromide (CNBr) platform to produce the bioactive form of mastoparan B (MPB), which in a manner identical to that of its native counterpart. METHODS: The plasmid pET30-His6-rOle(127M→L)-MPB was constructed, and then four different E. coli strains- BL21(DE3), BL21(DE3)pLysS, C41(DE3), and C43(DE3) were tested to identify the most suitable host for the pET30-His6-rOle(127M→L)-MPB fusion protein expression. We optimized the expression conditions by testing different growth temperatures, isopropyl-ß-D-thiogalactoside (IPTG) concentrations, and post-induction collection times. Afterwards, the His6-rOle(127M→L)-MPB protein was purified by one-step nickel-chelated affinity chromatography (Ni2+-NTA) under denaturing conditions. The purified His6-rOle(127M→L)-MPB was selectively cleaved by thrombin protease to remove the His6-tag and the leader peptide from the N-terminus. Subsequently, rOle(127M→L)-MPB protein was constituted into AOB and incubated with CNBr for a cleavage reaction, which resulted in the release of the MPB from rOle(127M→L)-MPB protein via AOB. The purified MPB was identified by MALDI-MS and HPLC analysis, and its bioactivity was examined by antimicrobial testing. RESULTS: After a 2-h induction period, the E. coli C43(DE3) was found to be superior to BL21(DE3) and the other protease-deficient strains as an expression host. And, the optimal His6-rOle(127M→L)-MPB expression at 37°C for 2 h after induction with 5 µM IPTG. The purified MPB showed that a single major peak was detected by HPLC/UV detection with a retention time of 22.5 minutes, which was approximately 90% pure. The putative MPB, and over two-third of the peptide sequence was verified by the MALDI-MS analysis. Finally, the purified MPB was examined by a broth dilution-antimicrobial susceptibility test. These results indicated that the purified MPB was bioactive and very effective in anti-bacterial (E. coli J96) activity. Here, we successfully used the oleosin-based fusion expression strategy coupled with the artificial oil body (AOB)-cyanogen bromide (CNBr) platform to produce bioactive MPB peptide which, in a manner identical to that of its native counterpart. CONCLUSION: In this study, the recombinant oleosin based fusion strategy coupled with AOB-CNBr purification platform open a new avenue for the production of active MPB and facilitate the studies and applications of the peptide in the future for medicinal applications such as hypotension and antibacterial effect.

Selective detection of cyanogen halides by BN nanocluster: a DFT study.

The electronic sensitivity and adsorption behavior toward cyanogen halides (X-CN; X = F, Cl, and Br) of a B12N12 nanocluster were investigated by means of density functional theory calculations. The X-head of these molecules was predicted to interact weakly with the BN cluster because of the positive σ-hole on the electronic potential surface of halogens. The X-CN molecules interact somewhat strongly with the boron atoms of the cluster via the N-head, which is accompanied by a large charge transfer from the X-CN to the cluster. The change in enthalpy upon the adsorption process (at room temperature and 1 atm) is about -19.2, -23.4, and -30.5 kJ mol-1 for X = F, Cl, and Br, respectively. The LUMO level of the BN cluster is largely stabilized after the adsorption process, and the HOMO-LUMO gap is significantly decreased. Thus, the electrical conductivity of the cluster is increased, and an electrical signal is generated that can help to detect these molecules. By increasing the atomic number of X, the signal will increase, which makes the sensor selective for cyanogen halides. Also, it was indicated that the B12N12 nanocluster benefits from a short recovery time as a sensor.

Green fluorescent protein as a scaffold for high efficiency production of functional bacteriotoxic proteins in Escherichia coli.

The availability of simple, robust, and cost-effective methods for the large-scale production of bacteriotoxic peptides such as antimicrobial peptides (AMPs) is essential for basic and pharmaceutical research. However, the production of bacteriotoxic proteins has been difficult due to a high degree of toxicity in bacteria and proteolytic degradation. In this study, we inserted AMPs into the Green fluorescent protein (GFP) in a loop region and expressed them as insoluble proteins in high yield, circumventing the inherent toxicity of AMP production in Escherichia coli. The AMPs inserted were released by cyanogen bromide and purified by chromatography. We showed that highly potent AMPs such as Protegrin-1, PMAP-36, Buforin-2, and Bactridin-1 are produced in high yields and produced AMPs showed similar activities compared to chemically synthesized AMPs. We increased the yield more than two-fold by inserting three copies of Protegrin-1 in the GFP scaffold. The immunogold electron micrographs showed that the expressed Protegrin-1 in the GFP scaffold forms large and small size aggregates in the core region of the inclusion body and become entirely nonfunctional, therefore not influencing the proliferation of E. coli. Our novel method will be applicable for diverse bacteriotoxic peptides which can be exploited in biomedical and pharmaceutical researches.

Hemocompatibility evaluation for peptide fragments of human serum albumin cleaved by cyanogens bromide.

We have previously demonstrated that peptide fragments of human serum albumin can be developed into potential renal targeting drug carriers. However, the interactions of these peptide fragments with red blood cells and plasma components are not evaluated well and there is yet no report on the evaluation of the hemocompatibility of peptide fragments. In this study, three kinds of peptide fragments were prepared and identified by amino acid analysis, and the blood compatibility of the peptide fragments was investigated by measuring blood coagulation, platelet and complement activation and hemolysis activity. Results indicated that all the peptide fragments prepared were highly hemocompatible without causing any clot formation, red blood cell aggregation or immune response. In addition, data from the cytotoxicity assay using HeLa cells and Madin-Darby canine kidney cells suggested that these peptide fragments do not induce toxicity towards either cell lines at concentrations up to 5 mg/ml. Therefore, it can be concluded that peptide fragments exhibit good hemocompatibility with no unwanted effect on the viability of renal cells, preliminarily demonstrating that it is safe to use peptide fragments as renal targeting drug carriers.

Facile method to sequence cyclic peptides/peptoids via one-pot ring-opening/cleavage reaction.

A facile method for sequence determination of cyclic peptides/peptoids is described. Macrocyclic peptides/peptoids of 3-10 residues were efficiently synthesized through thioether formation. One-pot reaction of thioether-embedded cyclic peptides/peptoids involving cyanogen bromide-mediated ring-opening and cleavage provides linearized molecules, which can be efficiently sequenced by tandem mass spectrometry.

C-terminal protein characterization by mass spectrometry: isolation of C-terminal fragments from cyanogen bromide-cleaved protein.

A sample preparation method for protein C-terminal peptide isolation from cyanogen bromide (CNBr) digests has been developed. In this strategy, the analyte was reduced and carboxyamidomethylated, followed by CNBr cleavage in a one-pot reaction scheme. The digest was then adsorbed on ZipTipC18 pipette tips for conjugation of the homoserine lactone-terminated peptides with 2,2'-dithiobis (ethylamine) dihydrochloride, followed by reductive release of 2-aminoethanethiol from the derivatives. The thiol-functionalized internal and N-terminal peptides were scavenged on activated thiol sepharose, leaving the C-terminal peptide in the flow-through fraction. The use of reversed-phase supports as a venue for peptide derivatization enabled facile optimization of the individual reaction steps for throughput and completeness of reaction. Reagents were replaced directly on the support, allowing the reactions to proceed at minimal sample loss. By this sequence of solid-phase reactions, the C-terminal peptide could be recognized uniquely in mass spectra of unfractionated digests by its unaltered mass signature. The use of the sample preparation method was demonstrated with low-level amounts of a whole, intact model protein. The C-terminal fragments were retrieved selectively and efficiently from the affinity support. The use of covalent chromatography for C-terminal peptide purification enabled recovery of the depleted material for further chemical and/or enzymatic manipulation. The sample preparation method provides for robustness and simplicity of operation and is anticipated to be expanded to gel-separated proteins and in a scaled-up format to high-throughput protein profiling in complex biological mixtures.

N-terminal protein characterization by mass spectrometry after cyanogen bromide cleavage using combined microscale liquid- and solid-phase derivatization.

A sample preparation method for protein N-terminal peptide isolation from cyanogen bromide (CNBr) protein digests has been developed. In this strategy, the CNBr cleavage was preceded by protein α- and ε-amine acetylation and carboxyamidomethylation in a one-pot reaction scheme. The peptide mixture was adsorbed on ZipTipC18 pipette tips for reaction of the newly generated N-termini with sulfosuccinimidyl-2-(biotinamido) ethyl-1, 3-dithiopropionate. In the subsequent steps, the peptides were exposed in situ to hydroxylamine for reversal of potential hydroxyl group acylation, followed by reductive release of the disulfide-linked biotinamido moiety from the derivatives. The selectively thiol group-functionalized internal and C-terminal peptides were reversibly captured by covalent chromatography on activated thiol-sepharose, leaving the N-terminal fragment in the flow-through fraction. The use of the reversed-phase support as a venue for postcleavage serial modification proved instrumental to ensure throughput and completeness of derivatization. By this sequence of solid-phase reactions, the N-terminal peptide could be recognized uniquely in the MALDI-mass spectra of unfractionated digests by its unaltered mass signature. The use of the sample preparation method was demonstrated with low-picomole amounts of model protein. The N-terminal CNBr fragments were retrieved selectively from the affinity support. The sample preparation method provides for robustness and simplicity of operation using standard equipment available in most biological laboratories and is anticipated to be readily expanded to gel-separated proteins.

Preparation of uniformly (13)C,(15)N-labeled recombinant human amylin for solid-state NMR investigation.

A number of diseases are caused by the formation of amyloid fibrils. Detailed understanding of structural features of amyloid fibers is of great importance for our understanding of disease progression and design of agents for diagnostics or potential prevention of protein aggregation. In lack of 3D crystal ordering, solid-state NMR forms the most suited method to determine the structures of the fibrils with atomic resolution. To exploit this potential, large amounts of isotopic-labeled protein need to be obtained through recombinant protein expression. However, expression and purification of amyloidogenic proteins in large amounts remains challenging due to their aggregation potential, toxicity for cells and difficult purification. In this work, we report a method for the production of large amounts of uniformly labeled (13)C,(15)N-human amylin, being one of the most amyloidogenic peptides known. This method utilizes inclusion bodies-directed expression and cheap chemical cleavage with cyanogen bromide in order to minimize the cost of the procedure compared to the use of less efficient proteolytic enzymes. We demonstrate the formation of amylin fibrils in vitro characterized using biophysical methods and electron microscopy, show toxicity towards human cells, and demonstrate that produced material may form the basis for structure determination using solid-state NMR.

Coupling antibody to cyanogen bromide-activated sepharose.

Antibodies will be immobilized on a cyanogen bromide-activated Sepharose for subsequent use in pull-down assays or immunoaffinity purification.

Cloning, expression and mutational studies of a trypsin inhibitor that retains activity even after cyanogen bromide digestion.

A winged bean trypsin inhibitor (WbTI-2) of molecular mass ∼20kDa, has been cloned and expressed in Escherichiacoli with full activity like the one from seed protein. It completely inhibits trypsin at an enzyme:inhibitor molar ratio of 1:2. PCR with cDNA and genomic DNA using same primers produced about 550 base pair product, which indicated it to be an intronless gene. Through site-directed mutagenesis, the Arg64 has been confirmed as the P1 residue. For the presence of five methionine residues in WbTI-2, cyanogen bromide (CNBr) digestion was carried out. Out of three fragments the one (about 65% of original size) containing the reactive site loop retained 50% activity.

Guided reconstitution of membrane protein fragments.

Structural analysis by NMR of G protein-coupled receptors (GPCRs) has proven to be extremely challenging. To reduce the number of peaks in the NMR spectra by segmentally labeling a GPCR, we have developed a Guided Reconstitution method that includes the use of charged residues and Cys activation to drive heterodimeric disulfide bond formation. Three different cysteine-activating reagents: 5-5'-dithiobis(2-nitrobenzoic acid) [DTNB], 2,2'-dithiobis(5-nitropyridine) [DTNP], and 4,4'-dipyridyl disulfide [4-PDS] were analyzed to determine their efficiency in heterodimer formation at different pHs. Short peptides representing the N-terminal (NT) and C-terminal (CT) regions of the first extracellular loop (EL1) of Ste2p, the Saccharomyces cerevisiae alpha-factor mating receptor, were activated using these reagents and the efficiencies of activation and rates of heterodimerization were analyzed. Activation of NT peptides with DTNP and 4-PDS resulted in about 60% yield, but heterodimerization was rapid and nearly quantitative. Double transmembrane domain protein fragments were biosynthesized and used in Guided Reconstitution reactions. A 102-residue fragment, 2TM-tail [Ste2p(G31-I120C)], was heterodimerized with CT-EL1-tail(DTNP) at pH 4.6 with a yield of ∼75%. A 132-residue fragment, 2TMlong-tail [Ste2p(M1-I120C)], was expressed in both unlabeled and (15)N-labeled forms and used with a peptide comprising the third transmembrane domain, to generate a 180-residue segmentally labeled 3TM protein that was found to be segmentally labeled using [(15)N,(1)H]-HSQC analysis. Our data indicate that the Guided Reconstitution method would be applicable to the segmental labeling of a membrane protein with 3 transmembrane domains and may prove useful in the preparation of an intact reconstituted GPCR for use in biophysical analysis and structure determination.