Photocrosslinking/label transfer: A key step in mapping short alpha-neurotoxin binding site on nicotinic acetylcholine receptor

We developed a novel radioactive short bifunctional photoprobe, which could be coupled through a cleavable bond to an engineered cysteinyl residue on an analogue of a nicotinic acetylcholine receptor-specific alpha-neurotoxin. This cysteine was put on the tip of loop II in place of Arg33, a major residue for the interaction with the receptor. To facilitate the purification of the nAChR labeled subunits, we tagged the ligand with a desthiobiotin moiety. After irradiation of the photosensitive toxin-nAChR complex, gel electrophoresis showed that most of the radioactivity was attached to the alpha subunit (59%), followed by the gamma subunit (28%), with the delta subunit (13%) being less labeled. On a preparative scale, the labeled subunits were purified on streptavidin beads before separation on SDS-PAGE. "In-gel" CNBr cleavage of the labeled alpha subunit followed by Edman degradation of the purified peptides showed that alphaTyr190 and alphaTyr198 were the most labeled residues, with a less important labeling on alphaCys192. We believe that the novel photoactivatable probe will be of great use to identify key residues of ligands interacting with macromolecules.     

Site-directed disulfide reduction using an affinity reagent: Application on the nicotinic acetylcholine receptor

The aim of this study was to present a new concept of site-directed reduction of disulfide bonds based upon the use of an affinity ligand harbouring a readily oxidizable dithiol. The cysteine bond involved in the acetylcholine binding site of the AChoR was specifically reduced by a carbamylcholine analogue. The ligand, in its oxidized form, was characterized by an affinity constant of 20 μM for the agonist binding site. In its dithiol form, it specifically reduced the disulfide between Cys-192 and Cys-193 on the -subunits of the nicotinic acetylcholine receptor. This reduction needed 10 times lower concentration when carried out with site-directed reducing agent (ARA) than with DTT, and was highly specific for the -subunits. The contribution of the carbamylcholine moiety of the site-directed reducing agent was clearly demonstrated in kinetic studies where reduction abilities of ARA, DTT and the methylated analogue of ARA (MeRA) were compared. At the same concentration (20 μM), DTT and MeRA had a 25 times lower initial rate of reduction than ARA. With 200 μM of DTT this initial reduction was still 4 times lower. Furthermore, the use of a maleimido undecagold cluster which specifically labeled the reduced nicotinic receptor opens the way to structural analysis of the agonist binding site by electron microscopy. These results demonstrate the potency of this kind of site-directed reducing agent for structural study of receptors or enzymes involving a disulfide bond in their active site.     

The covalent tagging of the cell surface insulin receptor in intact cells with the generation of an insulin-free, functional receptor. A new approach to the study of receptor dynamics

A new method is described in which the cell surface insulin receptor can be radioactively tagged in a specific manner with a small insulin-free probe. After protecting the amino groups of insulin essential for binding and bio-activity, insulin is coupled to the heterobifunctional, cleavable cross-linking reagent SASD (sulfosuccinimidyl 2-(p-azidosalicylamido)-1,3'-dithiopropionate), via displacement of the N-hydroxysuccinimide moiety of SASD. Removal of the protecting groups results in the formation of 2-(p-azidosalicylamido)-1,3'-dithiopropionate (ASD)-insulin with insulin receptor binding activity equivalent to unmodified insulin. Iodination of ASD-insulin results in the incorporation of 125I into both the azidohydroxybenzoyl moiety of SASD and a tyrosine residue of insulin. Following binding of 125I-ASD-insulin to intact monolayers of 3T3-C2 cells, radiolabel is incorporated exclusively into a 135-kDa protein in a manner dependent upon the length of exposure of the cells to short wavelength ultraviolet light. This protein corresponds in molecular weight to the alpha subunit of the insulin receptor. Labeling of this protein can be inhibited by excess unlabeled insulin. Reduction of the disulfide bond of ASD with 10 mM glutathione causes the release of the 125I-insulin portion of the reagent from the receptor complex, with the iodinated photoactivated end of ASD covalently attached to the receptor. Insulin receptor labeled in this manner retains its ability to bind insulin. General metabolic processes of the intact cells do not appear to be perturbed by this labeling procedure, and the cellular processing of the insulin receptor does not appear to be modified by the covalent labeling of the receptor protein. This procedure therefore provides a way to specifically label the cell surface insulin receptor in a manner which does not perturb the normal functioning of the labeled cell and equally importantly, does not perturb the normal cellular processing of the insulin receptor itself.     

The sidedness of the COOH terminus of the acetylcholine receptor delta subunit

The nicotinic acetylcholine receptor from Torpedo sp. occurs as a dimer, disulfide-cross-linked between delta subunits. We determined the sidedness of the COOH terminus of the acetylcholine receptor delta subunit by locating the delta-delta disulfide relative to the membrane and by identifying the Cys residue forming the disulfide. We used receptor-rich native membrane vesicles isolated from Torpedo californica electric tissue and characterized as to orientation and intactness. These vesicles had not been extracted and retained v ("43-kDa protein") as a marker of the cytoplasmic surface. Using the reduction of v as an assay of permeability, we showed that two reductants, 2-mercaptoethanesulfonate and reduced glutathione, were relatively impermeant. Both of these reductants reduced the delta-delta disulfide in sealed right-side-out vesicles equally in the presence and absence of saponin, and 2-mercaptoethanesulfonate reduced this disulfide equally in the presence and absence of Triton X-100. By contrast, surfactants enhanced the reduction of dimer in inside-out and sequestered vesicles. We conclude that the disulfide is extracellular. To identify the Cys residue forming the disulfide, we labeled the sulfhydryls both in receptor dimer and in monomer generated by mild reduction of dimer. By high performance liquid chromatography and NH2-terminal sequencing of cyanogen bromide fragments of labeled delta-delta dimer and delta monomer, we found that the penultimate residue, delta-Cys-500, uniquely formed an intersubunit disulfide and that this disulfide was uniquely reduced when receptor dimer was reduced to monomer. Therefore, the delta COOH terminus is extracellular.     

Three possible disulfides in the acetylcholine receptor alpha-subunit

The cysteinyl residues of the acetylcholine receptor alpha-subunit of Torpedo californica were analyzed. All seven cysteines could be accounted for. Three possible disulfide bridges and one unpaired cysteine were indicated. The disulfide linkages were as follows: Cys128 to Cys142; Cys192 to Cys193; Cys412 to Cys418 (Cys222 is unpaired). The identification of cysteinyl residues was accomplished by a modified protein blot procedure. Cysteinyl residues of intact nicotinic acetylcholine receptor were selectively biotinylated with 3-(N-maleimidopropionyl)biocytin and subsequently detected by the 125I-labeled avidin overlay of blotted Staphylococcus aureus V8 proteolyzed alpha-subunits. Two pairs of cysteines (Cys128/Cys142 and Cys412/Cys418) could be demonstrated only after Na(BH4) reduction of the acetylcholine receptor. Cysteine residues 192 and 193 are particularly sensitive to reduction; 0.1 mM dithiothreitol is sufficient.     

Delineation of bacterial luciferase aldehyde site by bifunctional labeling reagents

Previously we have established that a highly reactive cysteinyl group on the alpha subunit is at the aldehyde site of the (alpha beta) dimeric Vibrio harveyi luciferase. Three isomeric bifunctional reagents have been synthesized and used to further delineate the luciferase aldehyde site. These probes differ in their relative positions of and distances between the two functional groups active in chemical and photochemical labelings, respectively. Each of the probes can effectively and reversibly inactivate luciferase by forming a disulfide linkage primarily to the reactive cysteinyl residue. Upon subsequent photolysis, a diazoacetate arm in each probe was activated for photochemical labeling of amino acid residues within reach. After reductive regeneration of the reactive cysteinyl residue, 0.35-0.40 probe per dimeric luciferase was found to have been photochemically incorporated, correlating well with the degree of irreversible enzyme inactivation. Low but significant amounts of the three isomeric probes initially attached to the alpha reactive cysteine through a disulfide have been found to photochemically tag certain residues on beta. The latter residues are estimated to be no more than 8-11 A away from the alpha reactive cysteine. Thus the reactive cysteinyl residue, and hence the aldehyde site, must be at or near the alpha beta subunit interface. Furthermore, the structural integrity of the microenvironment surrounding this reactive cysteinyl residue is crucial to luciferase activity. An HPLC method for the isolation of luciferase alpha and beta subunits has also been developed.     

Insulin receptors are bivalent as demonstrated by photoaffinity labeling.

Insulin receptors in human placental membranes were photoaffinity-labeled with a radioactive human insulin-like growth factor I (hIGF-I) photoprobe N epsilon B28-monoazidobenzoyl 125I-hIGF-I either alone or together with a non-radioactive insulin photoprobe N epsilon B29-monoazidobenzoyl insulin. Precipitation of the solubilized receptors with anti-insulin antibody showed that receptors labeled with the radioactive hIGF-I photoprobe were detected in the immunoprecipitate only when photolabeling was carried out in the presence of the non-radioactive insulin photoprobe. Comparable results were obtained in converse experiments using a radioactive insulin photoprobe N epsilon B29-monoazidobenzoyl 125I-insulin, a non-radioactive hIGF-I photoprobe N epsilon B28-monoazidobenzoyl hIGF-I, and an antibody to hIGF-I. The amount of radioactive receptors precipitated by either the anti-insulin antibody or the anti-hIGHF-I antibody was close to the expected amount. These observations demonstrate that the insulin receptor is bivalent being capable of binding two molecules of ligand.     

Proposed mechanisms for binding of apo[a] kringle type 9 to apo B-100 in human lipoprotein[a].

The protein component of human lipoprotein[a] consists primarily of two apolipoproteins, apo[a] and apo B-100, linked through a cystine disulfide(s). In the amino acid sequence of apo bd, Cys4057 located within a plasminogen kringle 4-like repeat sequence (3991-4068) is believed to form a disulfide bond with a specific cysteine residue in apo B-100. Our fluorescence-labeling experiments and molecular modeling studies have provided evidence for possible interactions between this apo[a] kringle type and apo B-100. The fluorescent probe, fluorescein-5-maleimide, was used in parallel experiments to label free sulfhydryl moieties in lipoprotein[a] and low-density lipoprotein (LDL). In apo B-100 of LDL, Cys3734 was labeled with the probe, but this site was not labeled in autologous lipoprotein[a]. The result strongly implicates Cys3734 of apo B-100 as the residue forming the disulfide linkage with Cys4057 of apo[a]. To explore possible noncovalent interactions between apo B-100 and apo[a], the crystallographic coordinates for plasminogen kringle 4 were used to generate molecular models of the apo[a] kringle-repeat sequence (3991-4068, LPaK9), the only plasminogen kringle 4 type repeat in apo[a] having an extra cysteine residue not involved in an intramolecular disulfide bond. The Cys4057 residue (henceforth designated as Cys67 in the LPaK9 sequence) is believed to form an intermolecular disulfide bond with a cysteine of apo B-100. In computer graphics molecular models of LPaK9, Cys67 is located on the surface of the kringle near the lysine ligand binding site. Selected segments of the LDL apo B-100 sequence that contain free sulfhydryl cysteines were subjected to energy minimization and docking with the ligand binding site and adjacent regions of the LPaK9 model. In the docking experiments, apo B-100 segment 3732-3745 (PSCKLDFREIQIYK) displayed the best fit and the largest number of van der Waals contacts with models of LPaK9. Other apo B-100 peptides with sulfhydryl cysteine were found to be less compatible when minimized with this kringle. These results support and extend previously suggested mechanisms for a complex interaction between apo[a] and apo B-100 that involve more than a simple covalent disulfide bond.     

Denmotoxin, a three-finger toxin from the colubrid snake Boiga dendrophila (Mangrove Catsnake) with bird-specific activity

Boiga dendrophila (mangrove catsnake) is a colubrid snake that lives in Southeast Asian lowland rainforests and mangrove swamps and that preys primarily on birds. We have isolated, purified, and sequenced a novel toxin from its venom, which we named denmotoxin. It is a monomeric polypeptide of 77 amino acid residues with five disulfide bridges. In organ bath experiments, it displayed potent postsynaptic neuromuscular activity and irreversibly inhibited indirectly stimulated twitches in chick biventer cervicis nerve-muscle preparations. In contrast, it induced much smaller and readily reversible inhibition of electrically induced twitches in mouse hemidiaphragm nerve-muscle preparations. More precisely, the chick muscle alpha(1)betagammadelta-nicotinic acetylcholine receptor was 100-fold more susceptible compared with the mouse receptor. These data indicate that denmotoxin has a bird-specific postsynaptic activity. We chemically synthesized denmotoxin, crystallized it, and solved its crystal structure at 1.9 A by the molecular replacement method. The toxin structure adopts a non-conventional three-finger fold with an additional (fifth) disulfide bond in the first loop and seven additional residues at its N terminus, which is blocked by a pyroglutamic acid residue. This is the first crystal structure of a three-finger toxin from colubrid snake venom and the first fully characterized bird-specific toxin. Denmotoxin illustrates the relationship between toxin specificity and the primary prey type that constitutes the snake's diet.     

Conformational analysis of a toxic peptide from Trimeresurus wagleri which blocks the nicotinic acetylcholine receptor.

The 22-residue toxic peptide (WTX1) from the venom of the Southeast Asian snake Trimeresurus wagleri has multiple sites of action, but its lethal effect has been attributed to blocking the postsynaptic acetylcholine receptor at the neuromuscular junction. The 3-dimensional structure of WTX1 was studied using 2-dimensional nuclear magnetic resonance spectroscopy, circular dichroism, and computer simulations. In aqueous solution, WTX1 was shown to have extended and flexible "tails" defined by a short, rigid disulfide-bonded loop. The flexible regions can undergo structural rearrangement when moved from an aqueous to a less polar environment and may contribute to its effectiveness at different receptor sites. By substituting Gly or Phe for His at position 10, significant effects on the disulfide bond formation and, thereby, the activity of the peptide were observed. These results suggest that even subtle differences in single residues can have profound effects on the dynamics of folding, disulfide bond formation, and activity of this toxic peptide.     

Muscarinic acetylcholine receptors. Peptide sequencing identifies residues involved in antagonist binding and disulfide bond formation

Muscarinic acetylcholine receptors (mAChR) were purified from rat brain and labeled either with the site-directed affinity label [3H]propylbenzilylcholine mustard (PrBCM) or with the sulfhydryl-specific label [3H]N-ethylmaleimide (NEM), using a protocol designed to give selective incorporation of the label into disulfide-bonded cysteines. m1 mAChRs were purified from CHO-K1 cells stably expressing the cloned receptor sequence and labeled with [3H]PrBCM. The labeled receptors were cleaved with the lysine-specific protease Lys-C and, after fractionation of the products, subcleaved with cyanogen bromide. Two major CNBr cleavage products were found with a molecular mass of approximately 3.9 and approximately 2.4 kDa, labeled either by [3H]PrBCM or [3H]NEM. The results obtained from CNBr cleavage of purified forebrain receptors were consistent with those obtained from the purified cloned m1 mAChR. Edman degradation was applied to the CNBr peptides. The results were compatible with the attachment of the [3H]PrBCM label to a conserved aspartic acid residue in transmembrane helix 3 of the mAChR (corresponding to Asp-105, m1 sequence) and of [3H]NEM to a conserved cysteine residue (corresponding to Cys-98, m1 sequence). These results support the hypothesis that the cysteine residue participates in a disulfide bond on the extracellular surface of the mAChRs and related G-protein-coupled receptors, while the aspartic acid residue is involved in binding the positively charged headgroup of muscarinic antagonists.     

Acetylcholine receptor binding site contains a disulfide cross-link between adjacent half-cystinyl residues

A conserved feature of all nicotinic receptors is the presence of a readily reducible disulfide bond adjacent to the acetylcholine binding site. Previously we showed that in intact receptor from Torpedo californica electric tissue reduction of this disulfide followed by affinity alkylation with 4-(N-maleimido)benzyltri[3H] methylammonium iodide specifically and uniquely labels the alpha subunit residues Cys-192 and Cys-193. To identify all of the half-cystinyl residues contributing to the binding site disulfide(s), we have now reduced receptor under mild conditions and alkylated with a mixture of 4-(N-maleimido)benzyltri[3H]methylammonium iodide and N-[1-14C]ethylmaleimide and find that Cys-192 and Cys-193 are labeled exclusively. Furthermore, from unreduced receptor we have isolated two cyanogen bromide peptides of alpha, one containing Cys-192 and Cys-193, and the other containing Cys-128 and Cys-142 (which are the other potential contributors to the binding site disulfide(s]. These isolated peptides incorporate iodo[1-14C]acetamide only following reduction by dithiothreitol. Our results demonstrate that: 1) the binding site disulfide is between Cys-192 and Cys-193; 2) Cys-128 is disulfide-cross-linked to Cys-142; and 3) under conditions that reduce Cys-192 and Cys-193 completely, Cys-128 and Cys-142 remain cross-linked. At the acetylcholine binding site, agonists induce a local conformational change that stabilizes the binding site disulfide against reduction. We suggest that a transition between two stable conformations of the vicinal disulfide, both involving a nonplanar cis peptide bond between Cys-192 and Cys-193, is associated with receptor activation by agonists.     

Efficient approach for profiling photoaffinity labeled peptides with a cleavable biotinyl photoprobe

Based on the application of our recent biotinyl photoprobe with a cleavable N-acylsulfonamide, an efficient process has been developed for profiling photoaffinity labeled peptides among a large excess of unlabeled concomitants. N-acylsulfonamide group was found to be stable under the usual S-pyridylethylation condition of cysteine residues whereas the group was easily cleaved by N-alkylation with iodoacetic acid in acidic condition. The selective nature between two common protein alkylation reactions was evaluated with l-glutamate dehydrogenase (GDH) using an acidic amino acid photoprobe with biotinylated acylsulfonamide function. The labeled GDH was successfully subjected to S-pyridylethylation keeping the biotin tag intact, and then was easily released from streptavidin matrix with high purity via iodoacetic acid-mediated alkylation under mild condition at pH 5.0.     

Identification of the fifth axial heme ligand of chloroperoxidase

Chloroperoxidase from Caldariomyces fumago catalyzes the peroxidative chlorination of organic acceptor molecules. From a variety of spectroscopic data, it had long been thought that chloroperoxidase possessed an active site structure similar to that of cytochrome P-450cam. Resonance Raman studies conducted with isotopically substituted enzyme proved conclusively that the fifth axial ligand to the ferriprotoporphyrin IX moiety of chloroperoxidase is indeed a cysteine thiolate (Bangcharoenpaurpong, O., Champion, P. M., Hall, K. S., and Hager, L. P. (1986) Biochemistry 25, 2374-2378). In this study, Ellman's reagent, 5,5'-dithiobis(2-nitrobenzoic acid), was used to ascertain which of the 3 cysteine residues in the primary structure of chloroperoxidase serves as the fifth axial heme ligand; two of the cysteine residues were earlier shown to be involved in a disulfide linkage. Apoprotein was labeled under denaturing conditions with 5,5'-dithiobis(2-nitrobenzoic acid). A unique peptide, containing the thionitrobenzoate adduct, was isolated via reverse phase HPLC following digestion with endoproteinase Glu-C. Amino acid and Edman sequence analysis revealed the fifth axial ligand in chloroperoxidase to be cysteine 29. Under reducing and denaturing conditions, incubation of apochloroperoxidase with Ellman's reagent resulted in 3 labeled residues. Proteolysis and isolation of the labeled peptides using reverse phase HPLC and subsequent Edman sequence analysis detected and identified the thionitrobenzoate adducts of each of the three cysteinyl peptides of chloroperoxidase.     

The Intermolecular Disulfide Bridge of Human Glial Cell Line-Derived Neurotrophic Factor: Its Selective Reduction and Biological Activity of the Modified Protein

Recombinant human glial cell line-derived neurotrophic factor has been implicated to have therapeutic potential in the treatment of neurodegenerative diseases. The mature protein is a single polypeptide of 134 amino acid residues and functions as a disulfide-linked dimer. Reduction of the protein with dithiothreitol at pH 7.0 and in the absence of denaturant showed that the single intermolecular cystine bridge was reduced preferentially. Direct alkylation of the generated free sulfhydryl group using iodoacetamide or iodoacetate without denaturant was incomplete. Unfolding the protein in 6 M guanidine hydrochloride prior to the modification showed rapid disulfide scrambling. However, the sulfhydryl-modifying reagent N-ethylmaleimide was able to label quantitatively the free cysteinyl residue in the absence of any added chaotropic agent. By a combination of peptide mapping, Edman degradation, and mass spectrometric analysis, the labeled residue was identified to be Cys¹⁰¹, hence verifying the location of the intermolecular disulfide bond. The modified protein behaved as a noncovalent dimer when chromatographed through a Superdex 75 column under nondenaturing conditions and was comparable in biological activity to an unmodified control sample. The results therefore indicate that the intermolecular disulfide bridge of the protein is not essential for its biological function.     

Expression and functional characterization of mutant human CXCR4 in insect cells: role of cysteinyl and negatively charged residues in ligand binding

Human CXCR4 was expressed in Sf9 insect cells using the Bac-to-Bac baculovirus expression system. The recombinant receptor exhibited ligand binding activities with a K(d) value (3.3 nM) comparable to that of the native receptor. The role of four conserved cysteinyl residues was explored by site-directed mutagenesis. Each cysteine was individually changed to an alanine residue. All of the four mutants showed decreased ligand binding activity with increased K(d) values although comparable levels of receptor expression were observed. These results suggest that each of these four cysteinyl residues may be important for the ligand binding of the receptor. Evidence suggests that the ionic interaction may be involved in ligand binding. Point mutation of several relatively conserved acidic residues (Asp-10, Asp-262, Glu-275, and Glu-277) to an alanine residue greatly decreased the ligand binding activity and affinity. Since SDF-1alpha is a highly basic protein, these acidic residues may interact with the basic residues of SDF-1alpha by ionic pairing in addition to other molecular interactions and play an important role in ligand binding.     

The acetylcholine receptor as part of a protein complex in receptor-enriched membrane fragments from Torpedo californica electric tissue

The acetylcholine receptor from Torpedo californica electric tissue consisting of polypeptide chains of molecular weight 42000 (+/- 2000) is part of a protein complex. Cross-linking experiments with bifunctional reagents have shown that this complex has possibly a pentameric structure with a molecular weight of 270000 (+/- 30000). Besides the receptor subunit (alpha-chain), at least three further classes of polypeptide chains are part of the complex: beta (Mr 48000), gamma (Mr 62000) and delta (Mr 68000). This can be shown by cross-linking the proteins extracted from receptor-enriched membrane fractions with a cleavable reagent: From the 270000 molecular weight particle the four predominant polypeptide chains of the membrane, alpha, beta, gamma, and delta, can be obtained. The gamma-polypeptide chains appear to form a dimer connected by an inter-chain disulphide bridge.     

Efficient approach to synthesis of two-chain asymmetric cysteine analogs of receptor-binding region of transforming growth factor-alpha.

The putative receptor-binding region of human transforming growth factor-alpha (TGF alpha) has been shown to be contributed by two fragments: an A-chain (residue 12-18) and a 17-residue carboxyl fragment (residue 34-50) that includes a disulfide-containing C-loop (residue 34-43). An approach to the synthesis of two-chain analogs containing an intermolecular disulfide linked A-chain and the 17-residue carboxyl fragment (C-fragment) possessing receptor-binding activity is described. The synthesis was achieved by the solid-phase method using the Boc-benzyl protecting group strategy. The single Cys of the A-chain was activated as a mixed disulfide with 2-thiopyridine to form the intermolecular disulfide bond with Cys41 or Cys46 of the C-fragment on the resin support. Prior to this reaction, the acetamido (Acm) protecting group of Cys41 or Cys46 was removed by Hg(OAc)2 on the resin support. The peptide and side chain protecting groups including the S-methylbenzyl moiety of the Cys34 and Cys43 were concomitantly cleaved by high HF. The intramolecular disulfide with two unprotected Cys was formed in the presence of an intermolecular disulfide. This intramolecular disulfide bond formation was usually not feasible under the traditionally-held scheme at basic pH since disulfide interchange would occur faster than intramolecular oxidation. To prevent the disulfide interchange, a new method was devised. The intramolecular disulfide bond oxidation was mediated by dimethylsulfoxide at an acidic pH, at which the disulfide interchange reaction was suppressed. The desired product was obtained with a 60-70% yield.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structure of unfolded and refolded recombinant derived [Ala125]interleukin 2

Naturally occurring interleukin 2 (IL-2) contains an odd number (three) of cysteinyl residues and thus is susceptible to the formation of a variety of intramolecular and intermolecular disulfide bonds. The cysteine at residue 125 has been replaced with an alanine residue by site-directed mutagenesis, and hence, this analogue can form only one intrachain disulfide bond. When expressed at high levels in Escherichia coli, this recombinant DNA derived IL-2 analogue is insoluble, reduced, and inactive. The protein was solubilized by denaturants and, after purification, was oxidized to form an intramolecular disulfide bond. Circular dichroism (CD) has been used to investigate the effects of various denaturants on the unfolding-refolding process of the purified, oxidized protein. A similar conformation is obtained when [Ala125]interleukin 2 [IL-2(Ala-125)] is refolded from 6 M guanidine hydrochloride, 8 M urea, or 5% acetic acid. The resultant protein, refolded from these denaturants, is monomeric and has activity comparable to or greater than that reported for naturally derived IL-2. In addition to this form, aggregates, as evidenced from gel filtration, are obtained. The specific activities of these are greatly reduced, and CD spectra indicated that they have much less helical content than the monomeric form of the protein. CD spectra also showed that the tertiary structure of IL-2(Ala-125) is entirely different in the presence of sodium dodecyl sulfate (SDS) from that of the monomeric form in the absence of SDS.     

Peptide mapping of the insulin-binding site of the 130-kDa subunit of the insulin receptor by means of a novel cleavable radioactive photoprobe

A radioactive photoaffinity probe for the insulin receptor was prepared by derivatizing insulin at its B29 lysine with a novel crosslinking reagent having a cleavable azo linkage. Insulin receptors purified from human placental membranes were photoaffinity labeled with this probe. The photolabeled receptor was treated with dithionite to cleave the azo linkage, thereby removing the insulin ligand and transferring the radioactivity to the receptor protein. The radioactive labeled subunit was isolated and digested with elastase for peptide mapping and separation by high performance liquid chromatography. Results obtained indicated that it will be feasible to use this new photoaffinity probe to obtain radioactive peptides representing the insulin-binding site(s) on the receptor subunit.