A comparison of folding techniques in the chemical synthesis of the epidermal growth factor-like domain in neu differentiation factor alpha/beta.

The 52-residue alpha/beta chimera of the epidermal growth factor-like domain in neu differentiation factor (NDFealpha/beta) has been synthesized and folded to form a three disulfide bridge (Cys182-Cys196, Cys190-Cys210, Cys212-Cys221) containing peptide. We investigated two general strategies for the formation of the intramolecular disulfide bridges including, the single-step approach, which used fully deprotected and reduced peptide, and a sequential approach that relied on orthogonal cysteine protection in which specific pairs are excluded from the first oxidation step. Because there are 15 possible disulfide bridge arrangements in a peptide with six cysteines, the one-step approach may not always provide the desired disulfide pairing. Here, we compare the single-step approach with a systematic evaluation of the sequential approach. We employed the acetamidomethyl group to protect each pair of cysteines involved in disulfide bridges, i.e. Cys182 to Cys196, Cys190 to Cys210 and Cys212 to Cys221. This reduced the number of possible disulfide patterns from 15 to three in the first folding step. We compared the efficiencies of folding for each protected pair using RP-HPLC, mapped the disulfide connectivity of the predominant product and then formed the final disulfide from the partially folded intermediate via 12 oxidation. Only the peptide having the Cys182-Cys196 pair blocked with acetamidomethyl forms the desired disulfide isomer (Cys190-Cys210/Cys212-Cys221) as a single homogeneous product. By optimizing both approaches, as well as other steps in the synthesis, we can now rapidly provide large-scale syntheses of NDFealpha/beta and other novel EGF-like peptides.     

Controlled syntheses of natural and disulfide-mispaired regioisomers of alpha-conotoxin SI.

Methods are reported for the unambiguous syntheses of all three possible disulfide regioisomers with the sequence of alpha-conotoxin SI, a tridecapeptide amide from marine cone snail venom that binds selectively to the muscle subtype of nicotinic acetylcholine receptors. The naturally occurring peptide has two 'interlocking' disulfide bridges connecting Cys2-Cys7 and Cys3-Cys13 (2/7&3/13), while in the two mispaired isomers the disulfide bridges connect Cys2-Cys13 and Cys3-Cys7 (2/13 & 3/7, 'nested') and Cys2-Cys3 and Cys7-Cys13 (2/3 & 7/13, 'discrete'), respectively. Alignment of disulfide bridges was controlled at the level of orthogonal protection schemes for the linear precursors, assembled by Fmoc solid-phase peptide synthesis on acidolyzable tris(alkoxy)benzylamide (PAL) supports. Side-chain protection of cysteine was provided by suitable pairwise combination of the S-9H-xanthen-9-yl (Xan) and S-acetamidomethyl (Acm) protecting groups. The first disulfide bridge was formed from the corresponding bis(thiol) precursor obtained by selective deprotection of S-Xan, and the second disulfide bridge was formed by orthogonal co-oxidation of S-Acm groups on the remaining two Cys residues. It was possible to achieve the desired alignments with either order of loop formation (smaller loop before larger, or vice versa). The highest overall yields were obtained when both disulfides were formed in solution, while experiments where either the first or both bridges were formed while the peptide was on the solid support revealed lower overall yields and poorer selectivities towards the desired isomers.     

Selectivity of tryptophan residues in mediating photolysis of disulfide bridges in goat alpha-lactalbumin

Goat alpha-lactalbumin (GLA) contains four tryptophan (Trp) residues and four disulfide bonds. Illumination with near-UV light results in the cleavage of disulfide bridges and in the formation of free thiols. To obtain information about the reaction products, the illuminated protein was carbamidomethylated and digested with trypsin and the peptides were analyzed by mass spectrometry. Peptides containing Cys120Cam, Cys61Cam, or Cys91Cam were detected, as well as two peptides containing a new Cys-Lys cross-link. In one, Cys6 was cross-linked to Lys122, while the cross-link in the second was either a Cys91-Lys79 or Cys73-Lys93 cross-link; however, the exact linkage could not be defined. The results demonstrate photolytic cleavage of the Cys6-Cys120, Cys61-Cys77, and Cys73-Cys91 disulfide bonds. While photolysis of Cys6-Cys120 and Cys73-Cys91 disulfide bonds in GLA has been reported, cleavage of the Cys61-Cys77 disulfide bonds has not been previously detected. To examine the contribution of the individual Trp residues, we constructed the GLA mutants, W26F, W60F, W104F, and W118F, by replacing single Trp residues with phenylalanine (Phe). The substitution of each Trp residue led to less thiol production compared to that for wild-type GLA, showing that each Trp residue in GLA contributed to the photolytic cleavage of disulfide bridges. The specificity was expressed by the nature of the reaction products. No cleavage of the Cys6-Cys120 disulfide bridge was detected when the W26F mutant was illuminated, and no cleavage of the Cys73-Cys91 disulfide bridge was seen following illumination of W26F or W104F. In contrast, Cys61Cam, resulting from the cleavage of the Cys61-Cys77 disulfide bridge, was found following illumination of any of the mutants.     

Assignment of the three disulfide bonds of Selenocosmia huwena lectin-I from the venom of spider Selenocosmia huwena.

The positions of the disulfide bonds of Selenocosmia huwena lectin-I (SHL-I) from the venom of the Chinese bird spider S. huwena have been determined. The existence of three disulfide bonds in the native SHL-I was proved by matrix-assisted laser desorption ionization time-of-flight mass spectroscopic analysis. To map the disulfide bonds, native SHL-I was proteolytically digested. The resulting peptides were separated by reverse phase high-performance liquid chromatography. Matrix-assisted laser desorption ionization time-of-flight mass spectroscopic analysis indicated the presence of one disulfide bond Cys7-Cys19. The partially reduced peptides by using Tris-(2-carboxyethyl)-phosphine at pH 3.0 were purified by reverse phase high-performance liquid chromatography. Four M Guanidine-HCl was found to increase the yields of partially reduced peptides prominently. The free thiols were carboxamidomethlate by iodoacetamide. The specific location of another disulfide bond Cys2-Cys14 was proved by comparing N-terminal sequencing analysis of the partially reduced and alkylated SHL-I with that of the intact peptide. Finally, the three disulfide linkage of SHL-I could be assigned as Cys2-Cys14, Cys7-Cys19, Cys13-Cys26.     

Disulfide isomerization and thiol-disulfide exchange of long neurotoxins from the venom of Ophiophagus hannah

Selective reduction on the Cys28-Cys32 disulfide of Ophiophagus hannah neurotoxins, Oh-4 and Oh-5, revealed that isomerization of this disulfide linkage caused the two toxins to have distinct conformation and different retention time on a reversed-phase column. The Cys28-Cys32 disulfide of Oh-4 and Oh-5 was prone to form mixed disulfides with glutathione following pseudo-first-order kinetics. In addition to glutathionylated proteins, Oh-4 could be promoted to convert into Oh-5 by thiol compounds. Isomerization of Oh-5 into Oh-4 was not observed in the presence of thiol compounds. Dethiolation of glutathionylated proteins produced Oh-4 and Oh-5. Oxidation of the partially reduced toxin with reduced Cys28 and Cys32 was exclusively converted into Oh-5 regardless of the absence or presence of GSH/GSSG. Acrylamide quenching studies revealed difference in degree of exposure of the single Trp27 between Oh-4 and Oh-5. Synthesized peptides with substitution of Trp27 or Phe31 with Gly abolished entirely the formation of disulfide-linked dimeric product noted with the peptide of wild-type sequence. These results suggest that disulfide formation and isomerization of Cys28-Cys32 could be regulated by thiolation, and that the bulky aromatic residues Trp27 and Phe31 facilitate favorably the occurrence of disulfide isomerization of Cys28-Cys32.     

Stability and structure-forming properties of the two disulfide bonds of alpha-conotoxin GI

alpha-Conotoxin GI is a 13 residue snail toxin peptide cross-linked by Cys2-Cys7 and Cys3-Cys13 disulfide bridges. The formation of the two disulfide bonds by thiol/disulfide exchange with oxidized glutathione (GSSG) has been characterized. To characterize formation of the first disulfide bond in each of the two pathways by which the two disulfide bonds can form, two model peptides were synthesized in which Cys3 and Cys13 (Cono-1) or Cys2 and Cys7 (Cono-2) were replaced by alanines. Equilibrium constants were determined for formation of the single disulfide bonds of Cono-1 and Cono-2, and an overall equilibrium constant was measured for formation of the two disulfide bonds of alpha-conotoxin GI in pH 7.00 buffer and in pH 7. 00 buffer plus 8 M urea using concentrations obtained by HPLC analysis of equilibrium thiol/disulfide exchange reaction mixtures. The results indicate a modest amount of cooperativity in the formation of the second disulfide bond in both of the two-step pathways by which alpha-conotoxin GI folds into its native structure at pH 7.00. However, when considered in terms of the reactive thiolate species, the results indicate substantial cooperativity in formation of the second disulfide bond. The solution conformational and structural properties of Cono-1, Cono-2, and alpha-conotoxin GI were studied by 1H NMR to identify structural features which might facilitate formation of the disulfide bonds or are induced by formation of the disulfide bonds. The NMR data indicate that both Cono-1 and Cono-2 have some secondary structure in solution, including some of the same secondary structure as alpha-conotoxin GI, which facilitates formation of the second disulfide bond by thiol/disulfide exchange. However, both Cono-1 and Cono-2 are considerably less structured than alpha-conotoxin GI, which indicates that formation of the second disulfide bond to give the Cys2-Cys7, Cys3-Cys13 pairing induces considerable structure into the backbone of the peptide.     

Echistatin disulfide bridges: selective reduction and linkage assignment.

Echistatin is the smallest member of the disintegrin family of snake venom proteins, containing four disulfides in a peptide chain of 49 residues. Partial assignment of disulfides has been made previously by NMR and chemical approaches. A full assignment was made by a newly developed chemical approach, using partial reduction with tris-(2-carboxyethyl)-phosphine at acid pH. Reduction proceeded in a stepwise manner at pH 3, and the intermediates were isolated by high performance liquid chromatography. Alkylation of free thiols, followed by sequencer analysis, enabled all four bridges to be identified: (1) at 20 degrees C a single bridge linking Cys 2-Cys 11 was broken, giving a relatively stable intermediate; (2) with further treatment at 41 degrees C the bridges Cys 7-Cys 32 and Cys 8-Cys 37 became accessible to the reagent and were reduced at approx. equal rates; (3) the two bicyclic peptides produced in this manner were less stable and could be reduced at 20 degrees C to a peptide that retains a single bridge linking Cys 20-Cys 39; and (4) the monocyclic peptide can be reduced to the linear molecule at 20 degrees C. Some disulfide exchange occurred during alkylation of the bicyclic intermediates, but results unambiguously show the pattern to be [2-11; 7-32; 8-37; 20-39]. A comparison is made with kistrin, a longer disintegrin whose disulfide structure has been proposed from NMR analysis.     

Lasiocepsin, a novel cyclic antimicrobial peptide from the venom of eusocial bee Lasioglossum laticeps (Hymenoptera: Halictidae)

In the venom of eusocial bee Lasioglossum laticeps, we identified a novel unique antimicrobial peptide named lasiocepsin consisting of 27 amino acid residues and two disulfide bridges. After identifying its primary structure, we synthesized lasiocepsin by solid-phase peptide synthesis using two different approaches for oxidative folding. The oxidative folding of fully deprotected linear peptide resulted in a mixture of three products differing in the pattern of disulfide bridges. Regioselective disulfide bond formation significantly improved the yield of desired product. The synthetic lasiocepsin possessed antimicrobial activity against both Gram-positive and -negative bacteria, antifungal activity against Candida albicans, and no hemolytic activity against human erythrocytes. We synthesized two lasiocepsin analogs cyclized through one native disulfide bridge in different positions and having the remaining two cysteines substituted by alanines. The analog cyclized through a Cys8-Cys25 disulfide bridge showed reduced antimicrobial activity compared to the native peptide while the second one (Cys17-Cys27) was almost inactive. Linear lasiocepsin having all four cysteine residues substituted by alanines or alkylated was also inactive. That was in contrast to the linear lasiocepsin with all four cysteine residues non-paired, which exhibited remarkable antimicrobial activity. The shortening of lasiocepsin by several amino acid residues either from the N- or C-terminal resulted in significant loss of antimicrobial activity. Study of Bacillus subtilis cells treated by lasiocepsin using transmission electron microscopy showed leakage of bacterial content mainly from the holes localized at the ends of the bacterial cells.     

Assignment of the disulfide bonds of Ole e 1, a major allergen of olive tree pollen involved in fertilization.

The most prevalent allergen from olive tree pollen, Ole e 1, consists of a single polymorphic polypeptide chain of 145 amino acids which includes six cysteine residues at positions 19, 22, 43, 78, 90 and 131. By using an homogeneous form of the allergen expressed in Pichia pastoris, the array of the disulfide bridges has been elucidated. Specific proteolysis with thermolysin and reverse-phase HPLC separation of the peptides allowed the determination of the disulfide bond between Cys43 and Cys78. Another thermolytic product, which contained three peptides linked by the remaining four cysteines, was digested with Glu-specific staphylococcal V8 protease and the products isolated by reverse-phase HPLC. Amino acid compositions and Edman degradation of the peptide products indicated the presence of the disulfide bonds at Cys19-Cys90 and Cys22-Cys131. These data can help in the analysis of the three-dimensional structure of the protein as well as in studies of its allergenic determinants.     

Assignment of the disulfide bonds of huwentoxin-II by Edman degradation sequencing and stepwise thiol modification.

A novel strategy combining Edman degradation and thiol modification was developed to assign the three disulfides of huwentoxin-II (HWTX-II), an insecticidal peptide purified from the venom of the spider Selenocosmia huwena. Phenylthiohydantoin (Pth) derivatives of Cys and the elimination product, dehydroalanine (DeltaSer), can be observed in the Cys cycles during Edman degradation of native HWTX-II. The appearance of two products indicates that the disulfides of HWTX-II were split and that the free thiol group of the second half cystine has been generated. Information about the nature of the disulfide bridges of HWTX-II could be obtained from the sequencing signal if the nascent thiols were modified stepwise by 4-vinylpyridine. Using this method the disulfide bridges of HWTX-II were assigned as Cys4-Cys18, Cys8-Cys29 and Cys23-Cys34, which is different from that seen in HWTX-I, a neurotoxic peptide from the same spider. Using this strategy, one can assign the disulfide bonds of small proteins by sequencing and modification n - 1 times, where n is the number of disulfide bonds in the protein. The above assignment of the disulfide bonds of HWTX-II was confirmed by MALDI-TOF MS of tryptic fragments of HWTX-II. Some disulfide interchanging during proteolysis was observed by monitoring the kinetics of proteolysis of HWTX-II by MALDI-TOF MS.     

The N-terminal disulfide linkages of human insulin-like growth factor-binding protein-6 (hIGFBP-6) and hIGFBP-1 are different as determined by mass spectrometry.

The actions of insulin-like growth factors (IGFs) are modulated by a family of six high affinity binding proteins (IGFBPs 1-6). IGFBP-6 differs from other IGFBPs in having the highest affinity for IGF-II and in binding IGF-I with 20-100-fold lower affinity. IGFBPs 1-5 contain 18 conserved cysteines, but human IGFBP-6 lacks 2 of the 12 N-terminal cysteines. The complete disulfide linkages of IGFBP-6 were determined using electrospray ionization mass spectrometry of purified tryptic peptide complexes digested with combinations of chymotrypsin, thermolysin, and endoproteinase Glu-C. Numbering IGFBP-6 cysteines sequentially from the N terminus, the first three disulfide linkages are Cys1-Cys2, Cys3-Cys4, and Cys5-Cys6. The next two linkages are Cys7-Cys9 and Cys8-Cys10, which are analogous to those previously determined for IGFBP-3 and IGFBP-5. The C-terminal linkages are Cys11-Cys12, Cys13-Cys14, and Cys15-Cys16, analogous to those previously determined for IGFBP-2. Disulfide linkages of IGFBP-1 were partially determined and show that Cys1 is not linked to Cys2 and Cys3 is not linked to Cys4. Analogous with IGFBP-3, IGFBP-5, and IGFBP-6, Cys9-Cys11 and Cys10-Cys12 of IGFBP-1 are also disulfide-linked. The N-terminal linkages of IGFBP-6 differ significantly from those of IGFBP-1 (and, by implication, the other IGFBPs), which could contribute to the distinctive IGF binding properties of IGFBP-6.     

Determination of disulfide bond assignments and N-glycosylation sites of the human gastrointestinal carcinoma antigen GA733-2 (CO17-1A, EGP, KS1-4, KSA, and Ep-CAM)

The GA733-2 antigen is a cell surface glycoprotein highly expressed on most human gastrointestinal carcinoma and at a lower level on most normal epithelia. It is an unusual cell-cell adhesion protein that does not exhibit any obvious relationship to the four known classes of adhesion molecules. In this study, the disulfide-bonding pattern of the GA733-2 antigen was determined using matrix-assisted laser desorption/ionization mass spectrometry and N-terminal sequencing of purified tryptic peptides treated with 2-[2'-nitrophenylsulfonyl]-3-methyl-3-bromoindolenine or partially reduced and alkylated. Numbering GA733-2 cysteines sequentially from the N terminus, the first three disulfide linkages are Cys1-Cys4, Cys2-Cys6, and Cys3-Cys5, which is a novel pattern for a cysteine-rich domain instead of the expected epidermal growth factor-like disulfide structure. The next three disulfide linkages are Cys7-Cys8, Cys9-Cys10, and Cys11-Cys12, consistent with the recently determined disulfide pattern of the thyroglobulin type 1A domain of insulin-like growth factor-binding proteins 1 and 6. Analysis of glycosylation sites showed that GA733-2 antigen contained N-linked carbohydrate but that no O-linked carbohydrate groups were detected. Of the three potential N-linked glycosylation sites, Asn175 was not glycosylated, whereas Asn88 was completely glycosylated, and Asn51 was partially glycosylated. These data show that the extracellular domain of the GA733-2 antigen consists of three distinct domains; a novel cysteine-rich N-terminal domain (GA733 type 1 motif), a cysteine-rich thyroglobulin type 1A domain (GA733 type 2 motif), and a unique nonglycosylated domain without cysteines (GA733 type 3 motif).     

Localization of carbohydrate attachment sites and disulfide bridges in limulus alpha 2-macroglobulin. Evidence for two forms differing primarily in their bait region sequences

The primary structure determination of the dimeric invertebrate alpha(2)-macroglobulin (alpha(2)M) from Limulus polyphemus has been completed by determining its sites of glycosylation and disulfide bridge pattern. Of seven potential glycosylation sites for N-linked glycosylation, six (Asn(275), Asn(307), Asn(866), Asn(896), Asn(1089), and Asn(1145)) carry common glucosamine-based carbohydrates groups, whereas one (Asn(80)) carries a carbohydrate chain containing both glucosamine and galactosamine. Nine disulfide bridges, which are homologues with bridges in human alpha(2)M, have been identified (Cys(228)-Cys(269), Cys(456)-Cys(580), Cys(612)-Cys(799), Cys(657)-Cys(707), Cys(849)-Cys(876), Cys(874)-Cys(910), Cys(946)-Cys(1328), Cys(1104)-Cys(1155), and Cys(1362)-Cys(1475)). In addition to these bridges, Limulus alpha(2)M contains three unique bridges that connect Cys(361) and Cys(382), Cys(1370) and Cys(1374), respectively, and Cys(719) in one subunit with the same residue in the other subunit of the dimer. The latter bridge forms the only interchain disulfide bridge in Limulus alpha(2)M. The location of this bridge within the bait region is discussed and compared with other alpha-macroglobulins. Several peptides identified in the course of determining the disulfide bridge pattern provided evidence for the existence of two forms of Limulus alpha(2)M. The two forms have a high degree of sequence identity, but they differ extensively in large parts of their bait regions suggesting that they have different inhibitory spectra. The two forms (Limulus alpha(2)M-1 and -2) are most likely present in an approximately 2:1 ratio in the hemolymph of each animal, and they can be partially separated on a Mono Q column at pH 7.4 by applying a shallow gradient of NaCl.     

The disulfide bridges of the trypsin-kallikrein inhibitor K from snails (Helix pomatia). Thermal inactivation and proteolysis by thermolysin (author's transl)]

Isoinhibitor K is the main component of the complex mixture of isoinhibitors of broad specificity secreted into the mucus by the Roman snail (Helix pomatia). The disulfide pairing was determined after the amino acid sequence had been elucidated. Two cystine-containing peptides with the disulfide bridges Cys32-Cys53 and Cys32-Cys53 plus Cys7-Cys57 were obtained after thermolytic hydrolysis of the native inhibitor at 80 degrees C and chromatographic separation of the peptides using SE-Sephadex. The Cys16-Cys40 disulfide bridge could be reduced selectively by sodium borohydride with no loss in biological activity. This property and the covalent structure correspond to that of the intracellular inhibitor from bovine organs, which is largely homologous in its amino acid sequence to the secretory inhibitor from the snail. The complete covalent structure of isoinhibitor K will be presented. The snail inhibitor is less stable against proteolytic inactivation by thermolysin and against thermal denaturation at pH 8.0 than the inhibitor from bovine organs (Kunitz inhibitor).     

Two-step selective formation of three disulfide bridges in the synthesis of the C-terminal epidermal growth factor-like domain in human blood coagulation factor IX.

The 45-residue C-terminal EGF-like domain in human blood coagulation factor IX has been synthesized by a 2-step method to form selectively 3 disulfide bridges. Four out of 6 cysteines are blocked with either trityl or 4-methyl-benzyl, and the remaining 2 cysteines are blocked with acetamidomethyl (Acm). In the first step, 4 free cysteinyl thiols are released concurrently with the removal of all protecting groups except Acm and are oxidized to form 1 of the 3 possible isomers containing 2 pairs of disulfides. In the second step, iodine is used to remove the Acm groups to yield the third disulfide bridge. This approach reduces the number of possible disulfide bridging patterns from 15 to 3. To determine the optimal protecting group strategy, 3 peptides are synthesized, each with Acm blocking 1 of the 3 pairs of cysteines involved in disulfide bridges: Cys5 to Cys16 (Cys 1-3), Cys12 to Cys26 (Cys 2-4), or Cys28 to Cys41 (Cys 5-6). Only the peptide having the Cys 2-4 pair blocked with Acm forms the desired disulfide isomer (Cys 1-3/5-6) in high yield after the first step folding, as identified by proteolytic digestion in conjunction with mass spectrometric peptide mapping. Thus, the choice of which pair of cysteines to block with Acm is critically important. In the case of EGF-like peptides, it is better to place the Acm blocking groups on one of the pairs of cysteines involved in the crossing of disulfide bonds.     

Sites of interaction of thioredoxin with sorghum NADP-malate dehydrogenase

The activation pathway of the chloroplastic NADP-dependent malate dehydrogenase (MDH) by reduced thioredoxin has been examined using a method based on the mechanism of thiol/disulfide interchanges, i.e. the transient formation of a mixed disulfide between the target and the reductant. This disulfide can be stabilized when each of the partners is mutated in the less reactive cysteine of the disulfide/dithiol pair. As NADP-MDH has two regulatory disulfides per monomer, four different single cysteine mutants were examined, two for the C-terminal bridge and two for the N-terminal bridge. The results clearly show that the nucleophilic attack of thioredoxin on the C-terminal bridge proceeds through the formation of a disulfide with the most external Cys377. The results are less clear-cut for the N-terminal cysteines and suggest that the Cys24-Cys207 disulfide bridge previously proposed to be an intermediary step in MDH activation can form only when the C-terminal disulfide is reduced.     

Hemocyanins in spiders, XX. Sulfhydryl groups and disulfide bridges in subunit d of Eurypelma californicum hemocyanin

Subunit d of Eurypelma californicum hemocyanin contains after reduction 7 cysteine residues. Using 3,3'-dithiobis(6-nitrobenzoic acid) 3 mol cysteine/mol subunit were determined. The cysteine- and cystine-containing peptides of subunit d were obtained by cyanogen bromide cleavage and subsequent treatment with trypsin. The free cysteines were established at positions 102, 261, and 454 respectively. Cys205-Cys210 and Cys529-Cys579 are connected by disulfide bridges.     

Disulfide structures of highly bridged peptides: a new strategy for analysis.

A new approach is described for analyzing disulfide linkage patterns in peptides containing tightly clustered cystines. Such peptides are very difficult to analyze with traditional strategies, which require that the peptide chain be split between close or adjacent Cys residues. The water-soluble tris-(2-carboxyethyl)-phosphine (TCEP) reduced disulfides at pH 3, and partially reduced peptides were purified by high performance liquid chromatography with minimal thiol-disulfide exchange. Alkylation of free thiols, followed by sequencer analysis, provided explicit assignment of disulfides that had been reduced. Thiol-disulfide exchange occurred during alkylation of some peptides, but correct deductions were still possible. Alkylation competed best with exchange when peptide solution was added with rapid mixing to 2.2 M iodoacetamide. Variants were developed in which up to three alkylating agents were used to label different pairs of thiols, allowing a full assignment in one sequencer analysis. Model peptides used included insulin (three bridges, intra- and interchain disulfides; -Cys.Cys- pair), endothelin and apamin (two disulfides; -Cys.x.Cys- pair), conotoxin GI and isomers (two disulfides; -Cys.Cys- pair), and bacterial enterotoxin (three bridges within 13 residues; two -Cys.Cys- pairs). With insulin, all intermediates in the reduction pathway were identified; with conotoxin GI, analysis was carried out successfully for all three disulfide isomers. In addition to these known structures, the method has been applied successfully to the analysis of several previously unsolved structures of similar complexity. Rates of reduction of disulfide bonds varied widely, but most peptides did not show a strongly preferred route for reduction.     

Cysteine cross-linking defines part of the dimer and B/C domain interface of the Escherichia coli mannitol permease

Part of the dimer and B/C domain interface of the Escherichia coli mannitol permease (EII(mtl)) has been identified by the generation of disulfide bridges in a single-cysteine EII(mtl), with only the activity linked Cys(384) in the B domain, and in a double-cysteine EII(mtl) with cysteines at positions 384 and 124 in the first cytoplasmic loop of the C domain. The disulfide bridges were formed in the enzyme in inside-out membrane vesicles and in the purified enzyme by oxidation with Cu(II)-(1,10-phenanthroline)(3), and they were visualized by SDS-polyacrylamide gel electrophoresis. Discrimination between possible disulfide bridges in the dimeric double-cysteine EII(mtl) was done by partial digestion of the protein and the formation of heterodimers, in which the cysteines were located either on different subunits or on one subunit. The disulfide bridges that were identified are an intersubunit Cys(384)-Cys(384), an intersubunit Cys(124)-Cys(124), an intersubunit Cys(384)-Cys(124), and an intrasubunit Cys(384)-Cys(124). The disulfide bridges between the B and C domain were observed with purified enzyme and confirmed by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Mannitol did not influence the formation of the disulfide between Cys(384) and Cys(124). The close proximity of the two cysteines 124 was further confirmed with a separate C domain by oxidation with Cu(II)-(1,10-phenanthroline)(3) or by reactions with dimaleimides of different length. The data in combination with other work show that the first cytoplasmic loop around residue 124 is located at the dimer interface and involved in the interaction between the B and C domain.     

Molecular and biochemical basis of the interaction between tomato and its fungal pathogen Cladosporium fulvum

The interaction between the biotrophic fungal pathogen Cladosporium fulvum and tomato complies with the genefor-gene model. Resistance, expressed as a hypersensitive response (HR) followed by other defence responses, is based on recognition of products of avirulence genes from C. fulvum (race-specific elicitors) by receptors (putative products of resistance genes) in the host plant tomato. The AVR9 elicitor is a 28 amino acid (aa) peptide and the AVR4 elicitor a 106 aa peptide which both induce HR in tomato plants carrying the complementary resistance genes Cf9 and Cf4, respectively. The 3-D structure of the AVR9 peptide, as determined by 1H NMR, revealed that AVR9 belongs to a family of peptides with a cystine knot motif. This motif occurs in channel blockers, peptidase inhibitors and growth factors. The Cf9 resistance gene encodes a membrane-anchored extracellular glycoprotein which contains leucine-rich repeats (LRRs). 125I labeled AVR9 peptide shows the same affinity for plasma membranes of Cf9+ and Cf9- tomato leaves. Membranes of solanaceous plants tested so far all contain homologs of the Cf9 gene and show similar affinities for AVR9. It is assumed that for induction of HR, at least two plant proteins (presumably CF9 and one of his homologs) interact directly or indirectly with the AVR9 peptide which possibly initiates modulation and dimerisation of the receptor, and activation of various other proteins involved in downstream events eventually leading to HR. We have created several mutants of the Avr9 gene, expressed them in the potato virus X (PVX) expression system and tested their biological activity on Cf9 genotypes of tomato. A positive correlation was observed between the biological activity of the mutant AVR9 peptides and their affinity for tomato plasma membranes. Recent results on structure and biological activity of AVR4 peptides encoded by avirulent and virulent alleles of the Avr4 gene (based on expression studies in PVX) are also discussed as well as early defence responses induced by elicitors in tomato leaves and tomato cell suspensions.