Role of disulfide bridges in the folding, structure and biological activity of omega-conotoxin GVIA.

Omega-Conotoxin GVIA (GVIA), an N-type calcium channel blocker from the cone shell Conus geographus, is a 27 residue polypeptide cross-linked by three disulfide bonds. Here, we report the synthesis, structural analysis by (1)H NMR and bioassay of analogues of GVIA with disulfide bridge deletions and N- and C-terminal truncations. Two analogues that retain the crucial Lys-2 and Tyr-13 residues in loops constrained by two native disulfide bridges were synthesised using orthogonal protection of cysteine residues. In the first analogue, the Cys-15-Cys-26 disulfide bridge was deleted (by replacing the appropriate Cys residues with Ser), while in the second, this disulfide bridge and the eight C-terminal residues were deleted. No activity was detected for either analogue in a rat vas deferens assay, which measures N-type calcium channel activity in sympathetic nerve, and NMR studies showed that this was due to a gross loss of secondary and tertiary structure. Five inactive analogues that were synthesised without orthogonal protection of Cys residues as part of a previous study (Flinn et al. (1995) J. Pept. Sci. 1, 379-384) were also investigated. Three had single disulfide deletions (via Ser substitutions) and two had N- or C-terminal deletions in addition to the disulfide deletion. Peptide mapping and NMR analyses demonstrated that at least four of these analogues had non-native disulfide pairings, which presumably accounts for their lack of activity. The NMR studies also showed that all five analogues had substantially altered tertiary structures, although the backbone chemical shifts and nuclear Overhauser enhancements (NOEs) implied that native-like turn structures persisted in some of these analogues despite the non-native disulfide pairings. This work demonstrates the importance of the disulfides in omega-conotoxin folding and shows that the Cys-15-Cys-26 disulfide is essential for activity in GVIA. The NMR analyses also emphasise that backbone chemical shifts and short- and medium-range NOEs are dictated largely by local secondary structure elements and are not necessarily reliable monitors of the tertiary fold.     

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.     

Role of disulfide bonds in folding and activity of leiurotoxin I: just two disulfides suffice

The aim of this study is to investigate the contribution of each disulfide bond in the folding and function of leiurotoxin I, a short scorpion toxin that blocks small conductance K(+) channels. The structure of leiurotoxin I contains a motif conserved in all scorpion toxins, formed by a helix and a double-stranded beta-sheet and stabilized by three disulfide bridges. We synthesized three analogues, each presenting two alpha-aminobutyric acid (Abu) moieties replacing two bridged cysteine residues: LeTx1 ([Abu 3,21] Leiurotoxin I), LeTx2 ([Abu 8,26] Leiurotoxin I), and LeTx3 ([Abu 12,28] Leiurotoxin I). All three analogues fold into a major product containing two native disulfide bonds, while LeTx3 forms an additional isomer, containing non-native disulfides. In denaturing conditions, analogues LeTx2 and LeTx3 yield non-native isomers, while LeTx1 only forms the isomer with native disulfides. All isomers with native disulfides contain nativelike alpha-helical conformations and bind to synaptosomal membranes with affinities within a log of that shown by the native toxin. By contrast, the non-native LeTx3A analogue exhibits a disordered conformation and a decreased biological potency. Our results indicate that the "CxxxC, CxC" cysteine spacing, conserved in all scorpion toxins and preserved in LeTx1, may play an active role in folding, and that only two native disulfide bonds in leiurotoxin I are sufficient to preserve a nativelike and active conformation. Thus, in the scorpion toxin scaffold, modifications of conserved and interior cysteine residues may permit modulation of function, without significantly affecting folding efficiency and structure.     

Site-specific PEGylation of native disulfide bonds in therapeutic proteins

Native disulfide bonds in therapeutic proteins are crucial for tertiary structure and biological activity and are therefore considered unsuitable for chemical modification. We show that native disulfides in human interferon alpha-2b and in a fragment of an antibody to CD4(+) can be modified by site-specific bisalkylation of the two cysteine sulfur atoms to form a three-carbon PEGylated bridge. The yield of PEGylated protein is high, and tertiary structure and biological activity are retained.     

Dissecting a role of evolutionary-conserved but noncritical disulfide bridges in cysteine-rich peptides using ω-conotoxin GVIA and its selenocysteine analogs

Conotoxins comprise a large group of peptidic neurotoxins that use diverse disulfide-rich scaffolds. Each scaffold is determined by an evolutionarily conserved pattern of cysteine residues. Although many structure-activity relationship studies confirm the functional and structural importance of disulfide crosslinks, there is growing evidence that not all disulfide bridges are critical in maintaining activities of conotoxins. To answer the fundamental biological question of what the role of noncritical disulfide bridges is, we investigated function and folding of disulfide-depleted analogs of ω-conotoxin GVIA (GVIA) that belongs to an inhibitory cystine knot motif family and blocks N-type calcium channels. Removal of a noncritical Cys1-Cys16 disulfide bridge in GVIA or its selenopeptide analog had, as predicted, rather minimal effects on the inhibitory activity on calcium channels, as well as on in vivo activity following intracranial administration. However, the disulfide-depleted GVIA exhibited significantly lower folding yields for forming the remaining two native disulfide bridges. The disulfide-depleted selenoconotoxin GVIA analog also folded with significantly lower yields, suggesting that the functionally noncritical disulfide pair plays an important cooperative role in forming the native disulfide scaffold. Taken together, our results suggest that distinct disulfide bridges may be evolutionarily preserved by the oxidative folding or/and stabilization of the bioactive conformation of a disulfide-rich scaffold.     

Maurotoxin and the Kv1.1 channel: voltage-dependent binding upon enantiomerization of the scorpion toxin disulfide bridge Cys31-Cys34.

Maurotoxin (MTX) is a 34-amino acid polypeptide cross-linked by four disulfide bridges that has been isolated from the venom of the scorpion Scorpio maurus palmatus and characterized. Maurotoxin competed with radiolabeled apamin and kaliotoxin for binding to rat brain synaptosomes and blocked K+ currents from Kv1 channel subtypes expressed in Xenopus oocytes. Structural characterization of the synthetic toxin identified half-cystine pairings at Cys3-Cys24, Cys9-Cys29, Cys13-Cys19 and Cys31-Cys34 This disulfide bridge pattern is unique among known scorpion toxins, particularly the existence of a C-terminal '14-membered disulfide ring' (i.e. cyclic domain 31-34), We therefore studied structure-activity relationships by investigating the structure and pharmacological properties of synthetic MTX peptides either modified at the C-terminus ?i.e. MTX(1-29), [Abu31,34]-MTX and [Cys31,34, Tyr32]D-MTX) or mimicking the cyclic C-terminal domain [i.e. MTX(31-34)]. Unexpectedly, the absence of a disulfide bridge Cys31-Cys34 in [Abu 31,34]-MTX and MTX(1-29) resulted in MTX-unrelated half-cystine pairings of the three remaining disulfide bridges for the two analogs, which is likely to be responsible for their inactivity against Kv1 channel subtypes. Cyclic MTX(31-34) was also biologically inactive. [Cys31,34, Tyr32]D-MTX, which had a 'native', MTX-related, disulfide bridge organization, but a D-residue-induced reorientation of the C-terminal disulfide bridge, was potent at blocking the Kv1.1 channel. This peptide-induced Kv1.1 blockage was voltage-dependent (a property not observed for MTX), maximal in the low depolarization range and associated with on-rate changes in ligand binding. Thus, the cyclic C-terminal domain of MTX seems to be crucial for recognition of Kv1.3, and to a lesser extent, Kv1.2 channels and it may contribute to the stabilization and strength of the interaction between the toxin and the Kv1.1 channel.     

Characterization and 2D NMR study of the stable [9-21, 15-27] 2 disulfide intermediate in the folding of the 3 disulfide trypsin inhibitor EETI II.

The three disulfide Ecballium elaterium trypsin inhibitor II (EETI II) reduction with dithiothreitol (DTT) and reoxidation of the fully reduced derivative have been examined. A common stable intermediate has been observed for both processes. Isolation and sequencing of carboxymethylated material showed that the intermediate lacks the [2-19] bridge. The NMR study showed a very strong structural conservation as compared to the native EETI II, suggesting that the bridges are the [9-21] and [15-27] native ones. The differences occurred in sections 2-7 (containing the free cysteine 2 and the Arg 4-Ile 5 active site) and 19-21 (containing the second free cysteine). Distance geometry calculations and restrained molecular dynamics refinements were also in favor of a [9-21, 15-27] arrangement and resulted in a well-conserved (7-28) segment.     

Synthesis and evaluation of disulfide bond mimetics of amylin-(1-8) as agents to treat osteoporosis

Osteoporotic fracture is a significant public health problem, resulting in fractures in >50% of women and in almost one third of men age 65 and older. Most of the existing therapies act by slowing bone loss, through inhibiting the action of bone resorbing cells. However, more substantial reductions of fracture numbers will only result from treatments that can rebuild bone. Our own animal studies demonstrated the anabolic potential of the small but unstable octapeptide fragment of amylin-(1-37), namely amylin-(1-8) containing one disulfide bridge (Cys/2 and Cys/7) [Am. J. Physiol. Endocrinol. Metab.2000, 279, E730]. Herein, we describe the synthesis of amylin-(1-8) octapeptide and seven analogues thereof wherein the disulfide bridge is modified either via insertion of different linkers or bridges of a different nature in order to improve the stability and/or bone anabolic activity of the parent peptide. The peptide analogues were screened for proliferative activity in primary foetal rat bone-forming cells or osteoblasts at physiological concentrations. One such analogue showed promising biological activity.     

The covalent structure of the elastase inhibitor from Anemonia sulcata--a "non-classical" Kazal-type protein

The amino-acid sequence of the proteinase inhibitor specific for elastases from the sea anemone Anemonia sulcata was determined from performic-acid oxidized inhibitor and from three cyanogen bromide fragments of reduced and carboxymethylated inhibitor. The molecule consists of a single polypeptide chain formed from 48 amino-acid residues and is stabilized by three intramolecular disulfide bridges. After cyanogen bromide cleavage of the native protein at methionines 10 and 28 followed by chymotryptic cleavage two fragments each containing a single disulfide bridge were isolated. These indicated the location of three intramolecular disulfide linkages between Cys4 and Cys34 (part of A-loop), Cys8 and Cys27 (B-loop) and Cys16 and Cys48 (C-loop). The sequential homology and the disulfide pattern identified the elastase inhibitor as a Kazal-type inhibitor in which, however, not only the CysI-CysII segment is rather short but interestingly the Cys4-Cys34 disulfide anchoring point (i.e. CysI-CysV) in the C-loop is shifted by one turn in the alpha-helical segment towards the C-terminus. Thus, the elastase inhibitor is a non-classical Kazal-type inhibitor with respect to the positioning of the half-cystines. The inhibitor molecule was modelled based on the known three-dimensional structure of the silver pheasant ovomucoid third domain. The shortened amino-terminal segment was arranged in such a manner to allow disulfide bridge formation between the first cysteine Cys4 and the replaced Cys34 under maintenance of a suitable binding loop conformation. The characteristic ovomucoid scaffold consisting of a central alpha-helix, an adjacent three-stranded beta-sheet and the proteinase-binding loop cross-connected through disulfide bridges CysI-CysV and CysIII-CysVI was conserved.     

Structure-activity relationship studies of gomesin: importance of the disulfide bridges for conformation, bioactivities, and serum stability

Gomesin is an antimicrobial peptide isolated from hemocytes of the Brazilian spider Acanthoscurria gomesiana that contains two disulfide bridges Cys(2-15)/Cys(6-11) and presents a beta-hairpin structure. To investigate the role of the disulfide bridges on gomesin conformation, bioactivities, and serum stability, structure-activity relationship (SAR) studies were conducted. Initially, gomesin and variants lacking one or both disulfide bridges were synthesized. CD studies showed that the gomesin structure is very rigid independently of the solvent environment. On the other hand, the linearized analogues adopted secondary structures according to the environment, while the monocyclic disulfide-bridged peptides had a tendency to adopt a turn structure. The absence of one or both bridges resulted in a decrease in the antimicrobial and hemolytic activities. In addition, serum stability studies revealed that, contrasting to gomesin that was stable even after 48 h of incubation, the linearized analogues were rapidly degraded. The replacement of the disulfide bounds by lactam bridges led to monocyclic and bicyclic compounds. SAR studies indicated that the monocyclic lactam-bridged analogues tend to assume a alpha-helical structure being less potent, hemolytic, and serum stable than the wild-type gomesin. On the other hand, the bicyclic lactam/disulfide-bridged analogues displayed a similar conformation and degradation kinetics identical to gomesin. However, the antimicrobial activity appeared to be dependent on the lactam bridge position and size. These findings indicated that (i) the secondary structure plays a pivotal role for the full activity of gomesin; (ii) the antimicrobial and hemolytic activities of gomesin are correlated events; (iii) while at least one of the disulfide bridges is needed for the maintenance of a significant antimicrobial activity of gomesin, both bridges are required for high serum stability and optimal conformation; and finally (iv) the best analogue obtained was the bicyclo (2-15,6-11)[Glu2, Cys(6,11), Lys15]-Gm since it is as stable and potent as gomesin.     

Human β-Defensin 4 with Non-Native Disulfide Bridges Exhibit Antimicrobial Activity

Human defensins play multiple roles in innate immunity including direct antimicrobial killing and immunomodulatory activity. They have three disulfide bridges which contribute to the stability of three anti-parallel β-strands. The exact role of disulfide bridges and canonical β-structure in the antimicrobial action is not yet fully understood. In this study, we have explored the antimicrobial activity of human β-defensin 4 (HBD4) analogs that differ in the number and connectivity of disulfide bridges. The cysteine framework was similar to the disulfide bridges present in μ-conotoxins, an unrelated class of peptide toxins. All the analogs possessed enhanced antimicrobial potency as compared to native HBD4. Among the analogs, the single disulfide bridged peptide showed maximum potency. However, there were no marked differences in the secondary structure of the analogs. Subtle variations were observed in the localization and membrane interaction of the analogs with bacteria and Candida albicans, suggesting a role for disulfide bridges in modulating their antimicrobial action. All analogs accumulated in the cytosol where they can bind to anionic molecules such as nucleic acids which would affect several cellular processes leading to cell death. Our study strongly suggests that native disulfide bridges or the canonical β-strands in defensins have not evolved for maximal activity but they play important roles in determining their antimicrobial potency.     

Structure-function relationship in the binding of snake neurotoxins to the torpedo membrane receptor.

The Cys30-Cus34 bridge present in all long neutotoxins (71-74 amino acids, 5 disulfide bridges), but not in short toxins (60-63 amino acids, 4 disulfide bridges), is exposed at the surface since it can be reduced rapidly and selectively by sodium borohydride. Reduction and alkylation of the Cys30-Cys34 bridge of Naja haje neurotoxin III hardly alter the conformational properties of this model long toxin. Although alkylation by iodoacetic acid of th -SH groups liberated by reduction abolishes the toxicity, alkylation by iodoacetamide or ethylenimine does not affect the curarizing efficacy of the toxin. The Cys30-Cys34 bridge is not very important for the toxic activity of long neurotoxins. Reduction of the Cys30-Cys34 bridge followed by alkylation with radioactive iodoacetamide gave a labeled and active toxin which is a convenient derivative for binding experiments to the toxin receptor in membranes of the Torpedo electric organ. The binding capacity of these membrane is 1200 pmol of toxin/mg of membrane protein. The dissociation constant of the modified toxin-receptor complex at pH 7.4, 20 degrees is 10 minus 8m. Reduction with carbroxamidomethylation of the Cys30-Cys34 bridge decreases the affinity of the native Naja haje toxin only by a factor of 15. Carboxymethylation after reduction prevents binding to the membrane receptor. The binding properties of the derivative obtained by reduction and aminoethylation of Cys30-Cys34 are very similar to those of native neurotoxin III; the affinity is decreased only by a factor of 5. Binding properties to Toredo membrane of long neurotoxins (Naja haje neurotoxin III) and short neurotoxins (Naje haje toxin I and Naja mossambica toxin I) have been compared. Dissociation constants of receptor-long neurotoxin and receptor-short neurotoxin complexes are very similar (5.7 minus 8.2 times 10(-10) M at pH 7.4, 20degrees. However, the kinetics of complex formation and complex dissociation are quite different. Short neurotoxins associate 6-7 times faster with the toxin receptor and dissociate about 5-9 times faster that long neurotoxins. Acetylation and dansylation of Lys53 and Lys 27 decrease the affinity of long and short toxins for their receptor by a factor of about 200 at pH 7.4, 20 degrees, mainly because of the slower rate of association with the receptor.     

Designed peptide analogues of the potassium channel blocker ShK toxin.

ShK toxin, a potassium channel blocker from the sea anemone Stichodactyla helianthus, is a 35-residue polypeptide cross-linked by 3 disulfide bridges. In an effort to generate truncated peptidic analogues of this potent channel blocker, we have evaluated three analogues, one in which the native sequence was truncated and then stabilized by the introduction of additional covalent links (a non-native disulfide and two lactam bridges), and two in which non-native structural scaffolds stabilized by disulfide and/or lactam bridges were modified to include key amino acid residues from the native toxin. The effect of introducing a lactam bridge in the first helix of ShK toxin (to create cyclo14/18[Lys14,Asp18]ShK) was also examined to confirm that this modification was compatible with activity. All four analogues were tested in vitro for their ability to block Kv1.3 potassium channels in Xenopus oocytes, and their solution structures were determined using 1H NMR spectroscopy. The lactam bridge in full-length ShK is well tolerated, with only a 5-fold reduction in binding to Kv1.3. The truncated and stabilized analogue was inactive, apparently due to a combination of slight deviations from the native structure and alterations to side chains required for binding. One of the peptide scaffolds was also inactive because it failed to adopt the required structure, but the other had a K(d) of 92 microM. This active peptide incorporated mimics of Lys22 and Tyr23, which are essential for activity in ShK, and an Arg residue that could mimic Arg11 or Arg24 in the native toxin. Modification of this peptide should produce a more potent, low molecular weight peptidic analogue which will be useful not only for further in vitro and in vivo studies of the effect of blocking Kv1.3, but also for mapping the interactions with the pore and vestibule of this K(+) channel that are required for potent blockade.     

Denaturant-assisted formation of a stabilizing disulfide bridge from engineered cysteines in nonideal conformations

The engineered disulfide bridge A23C/L203C in human carbonic anhydrase II, inserted from homology modeling of Neisseria gonorrhoeae carbonic anhydrase, significantly stabilizes the native state of the protein. The inserted cysteine residues are placed in the interior of the structure, and because of the conformationally restrained localization, the protein is expressed in the reduced state and the cysteines are not readily oxidized. However, upon exposure to low concentrations of denaturant (0.6 M guanidine hydrochloride), corresponding to the lower part of the denaturation curve for the first unfolding transition, the oxidation rate of correctly formed disulfide bridges was markedly increased. By entropy estimations it appears that the increased flexibility, induced by the denaturant, enables the cysteines to find each other and hence to form the disulfide bridge. The outlined strategy of facilitating formation of disulfide bonds by addition of adjusted concentrations of a denaturant should be applicable to other proteins in which engineered cysteine residues are located in nonideal conformations. Moreover, a S99C/V242C variant was constructed, in which the cysteine residues are located on the surface. In this mutant the disulfide bridge was spontaneously formed and the native state was considerably stabilized (midpoint concentration of unfolding was increased from 1.0 to 1.4 M guanidine hydrochloride).     

About the structural role of disulfide bridges in serum albumins: evidence from protein simulated unfolding

The role of the 17 disulfide (S-S) bridges in preserving the native conformation of human serum albumin (HSA) is investigated by performing classical molecular dynamics (MD) simulations on protein structures with intact and, respectively, reduced S-S bridges. The thermal unfolding simulations predict a clear destabilization of the protein secondary structure upon reduction of the S-S bridges as well as a significant distortion of the tertiary structure that is revealed by the changes in the protein native contacts fraction. The effect of the S-S bridges reduction on the protein compactness was tested by calculating Gibbs free energy profiles with respect to the protein gyration radius. The theoretical results obtained using the OPLS-AA and the AMBER ff03 force fields are in agreement with the available experimental data. Beyond the validation of the simulation method, the results here reported provide new insights into the mechanism of the protein reductive/oxidative unfolding/folding processes. It is predicted that in the native conformation of the protein, the thiol (-SH) groups belonging to the same reduced S-S bridge are located in potential wells that maintain them in contact. The -SH pairs can be dispatched by specific conformational transitions of the peptide chain located in the neighborhood of the cysteine residues.     

Studies of structure-activity relationships of human interleukin-2

Human interleukin-2 (IL-2) has 3 cysteine residues; cysteines 58 and 105 form an intramolecular disulfide bridge, whereas cysteine 125 has a free sulfhydryl group. In this study, site-specific mutagenesis has been used to modify the cysteine residues of recombinant Escherichia coli-derived IL-2 (rIL-2) to evaluate the functional structure of IL-2. Substitution or deletion of cysteine 105 disrupted the disulfide bridge and yielded a mutant protein which was 8-10 times less active than wild type rIL-2. A similar modification at position 58, however, reduced the activity of rIL-2 by more than 250-fold. Although substitution of serine for cysteine 125 did not affect IL-2 activity, deletion of cysteine 125 or deletion of amino acids in the vicinity of this cysteine yielded mutant proteins with little, if any, activity. These results indicate that the protein structure in the vicinity of both positions 58 and 125 is more critical than that close to position 105. These findings may provide a clue to the understanding of the functional structure of human IL-2.     

Formation of disulfide bridges by a single-chain Fv antibody in the reducing ectopic environment of the plant cytosol

Disulfide bridge formation in the reducing environment of the cytosol is considered a rare event and is mostly linked to inactivation of protein activity. In this report the in vivo redox state of a single-chain Fv (scFv) antibody fragment in the plant cytosol was investigated. The scFv antibody fragment consists of the variable light and heavy chain domains from a mouse IgG antibody, which are connected by a flexible linker peptide. In each domain one disulfide bridge is present. The functionality of antibodies, which are normally secreted via the oxidizing environment of the endoplasmic reticulum, depends on the formation of intramolecular disulfide bridges. We demonstrate that a scFv can form intramolecular disulfide bridges and is functionally expressed in the cytosol of stably transformed plants. In addition, the formation of intermolecular disulfide bridges through a cysteine present in the linker peptide was observed. In contrast, transient expression in tobacco protoplasts resulted in a cytosolic scFv lacking disulfide bridges, which had a substantially reduced affinity for the antigen. This indicates that functionality rather than stability is determined by the presence of disulfide bridges in the in planta-expressed scFv antibody. The controversial observation of disulfide bond formation in the cytosol is discussed.     

Role of disulfide bridges in the activity and stability of a cold-active alpha-amylase

The cold-adapted alpha-amylase from Pseudoalteromonas haloplanktis unfolds reversibly and cooperatively according to a two-state mechanism at 30 degrees C and unfolds reversibly and sequentially with two transitions at temperatures below 12 degrees C. To examine the role of the four disulfide bridges in activity and conformational stability of the enzyme, the eight cysteine residues were reduced with beta-mercaptoethanol or chemically modified using iodoacetamide or iodoacetic acid. Matrix-assisted laser desorption-time of flight mass spectrometry analysis confirmed that all of the cysteines were modified. The iodoacetamide-modified enzyme reversibly folded/unfolded and retained approximately one-third of its activity. Removal of all disulfide bonds resulted in stabilization of the least stable region of the enzyme (including the active site), with a concomitant decrease in activity (increase in activation enthalpy). Disulfide bond removal had a greater impact on enzyme activity than on stability (particularly the active-site region). The functional role of the disulfide bridges appears to be to prevent the active site from developing ionic interactions. Overall, the study demonstrated that none of the four disulfide bonds are important in stabilizing the native structure of enzyme, and instead, they appear to promote a localized destabilization to preserve activity.     

Structural characterisation of human proteinosis surfactant protein A

Human surfactant protein-A (SP-A) has been purified from a proteinosis patient and characterised by a combination of automated Edman degradation and mass spectrometry. The complete protein sequence was characterised. The major part of SP-A was shown to consist of SP-A2 gene product, and only a small amount of SP-A1 gene product was shown to be present. A cysteine extension to the N-terminal was indicated by sequence data, but was not definitely proven. All proline residues in the Y position of Gly-X-Y in the collagen-like region were at least partially modified to hydroxy-proline, but no lysine residues were found to be modified. A complex N-linked glycosylation was found on Asn-187 showing great heterogeneity as variants from a mono-antennary to penta-antennary glycosylation with varying amounts of attached pentose were identified. The disulfide bridges in the carbohydrate recognition domain were identified to be in the 1-4, 2-3 pattern common for collectins. Interchain disulfide bridges were discovered between two Cys-48 residues and cysteine residues in the N-terminal region. However, the exact disulfide bridge connections within the bouquet-like ultrastructure could not be established.     

Disulfide bond structure of the AVR9 elicitor of the fungal tomato pathogen Cladosporium fulvum: evidence for a cystine knot

Disease resistance in plants is commonly activated by the product of an avirulence (Avr) gene of a pathogen after interaction with the product of a matching resistance (R) gene in the host. In susceptible plants, Avr products might function as virulence or pathogenicity factors. The AVR9 elicitor from the fungus Cladosporium fulvum induces defense responses in tomato plants carrying the Cf-9 resistance gene. This 28-residue beta-sheet AVR9 peptide contains three disulfide bridges, which were identified in this study as Cys2-Cys16, Cys6-Cys19, and Cys12-Cys26. For this purpose, AVR9 was partially reduced, and the thiol groups of newly formed cysteines were modified to prevent reactions with disulfides. After HPLC purification, the partially reduced peptides were sequenced to determine the positions of the modified cysteines, which originated from the reduced disulfide bridge(s). All steps involving molecules with free thiol groups were performed at low pH to suppress disulfide scrambling. For that reason, cysteine modification by N-ethylmaleimide was preferred over modification by iodoacetamide. Upon (partial) reduction of native AVR9, the Cys2-Cys16 bridge opened selectively. The resulting molecule was further reduced to two one-bridge intermediates, which were subsequently completely reduced. The (partially) reduced cysteine-modified AVR9 species showed little or no necrosis-inducing activity, demonstrating the importance of the disulfide bridges for biological activity. Based on peptide length and cysteine spacing, it was previously suggested that AVR9 isa cystine-knotted peptide. Now, we have proven that the bridging pattern of AVR9 is indeed identical to that of cystine-knotted peptides. Moreover, NMR data obtained for AVR9 show that it is structurally closely related to the cystine-knotted carboxypeptidase inhibitor. However, AVR9 does not show any carboxypeptidase inhibiting activity, indicating that the cystine-knot fold is a commonly occurring motif with varying biological functions.