Disulfide Interchange Research Articles

S-Alkylating Labeling Strategy for Site-Specific Identification of the S-Nitrosoproteome

S-nitrosylation, a post-translational modification of cysteine residues induced by nitric oxide, mediates many physiological functions. Due to the labile nature of S-nitrosylation, detection by mass spectrometry (MS) is challenging. Here, we developed an S-alkylating labeling strategy using the irreversible biotinylation on S-nitrosocysteines for site-specific identification of the S-nitrosoproteome by LC-MS/MS. Using COS-7 cells without endogenous nitric oxide synthase, we demonstrated that the S-alkylating labeling strategy substantially improved the blocking efficiency of free cysteines, minimized the false-positive identification caused by disulfide interchange, and increased the digestion efficiency for improved peptide identification using MS analyses. Using this strategy, we identified total 586 unique S-nitrosylation sites corresponding to 384 proteins in S-nitroso-N-acetylpenicillamine (SNAP)/l-cysteine-treated mouse MS-1 endothelial cells, including 234 previously unreported S-nitrosylated proteins. When the topologies of 84 identified transmembrane proteins were further analyzed, their S-nitrosylation sites were found to mostly face the cytoplasmic side, implying that S-nitrosylation occurs in the cytoplasm. In addition to the previously known acid/basic motifs, the ten deduced consensus motifs suggested that combination of local hydrophobicity and acid/base motifs in the tertiary structure contribute to the specificity of S-nitrosylation. Moreover, the S-nitrosylated cysteines showed preference on beta-strand, having lower relative surface accessibility at the S-nitrosocysteines.

Essential role of copper in the activity and regular periodicity of a recombinant, tumor-associated, cell surface, growth-related and time-keeping hydroquinone (NADH) oxidase with protein disulfide-thiol interchange activity (ENOX2)

ECTO-NOX proteins are growth-related cell surface proteins that catalyze both hydroquinone or NADH oxidation and protein disulfide interchange and exhibit time-keeping and prion-like properties. A bacterially expressed truncated recombinant 46kDa ENOX2 with full ENOX2 activity bound ca 2 moles copper and 2 moles of zinc per mole of protein. Unfolding of the protein in trifluoroacetic acid in the presence of the copper chelator bathocuproine resulted in reversible loss of both enzymatic activities and of a characteristic pattern in the Amide I to Amide II ratios determined by FTIR with restoration by added copper. The H546-V-H together with His 562 form one copper binding site and H582 represents a second copper site as determined from site-directed mutagenesis. Bound copper emerges as having an essential role in ENOX2 both for enzymatic activity and for the structural changes that underly the periodic alternations in activity that define the time-keeping cycle of the protein.

Topology of the disulfide bonds in the antiviral lectin scytovirin

The antiviral lectin scytovirin (SVN) contains a total of five disulfide bonds in two structurally similar domains. Previous reports provided contradictory results on the disulfide pairing in each individual domain, and we have now re-examined the disulfide topology. N-terminal sequencing and mass spectrometry were used to analyze proteolytic fragments of native SVN obtained at acidic pH, yielding the assignment as Cys7-Cys55, Cys20-Cys32, Cys26-Cys38, Cys68-Cys80, and Cys74-Cys86. We also analyzed the N-terminal domain of SVN (SD1, residues 1-48) prepared by expression/oxidative folding of the recombinant protein and by chemical synthesis. The disulfide pairing in the chemically synthesized SD1 was forced into predetermined topologies: SD1A (Cys20-Cys26, Cys32-Cys38) or SD1B (Cys20-Cys32, Cys26-Cys38). The topology of native SVN was found to be in agreement with the SD1B and the one determined for the recombinant SD1 domain. Although the two synthetic forms of SD1 were distinct when subjected to chromatography, their antiviral properties were indistinguishable, having low nM activity against HIV. Tryptic fragments, the "cystine clusters" [Cys20-Cys32/Cys26-Cys38; SD1] and [Cys68-Cys80/Cys74-C-86; SD2], were found to undergo rapid disulfide interchange at pH 8. This interchange resulted in accumulation of artifactual fragments in alkaline pH digests that are structurally unrelated to the original topology, providing a rational explanation for the differences between the topology reported herein and the one reported earlier (Bokesh et al., Biochemistry 2003;42:2578-2584). Our observations emphasize the fact that proteins such as SVN, with disulfide bonds in close proximity, require considerable precautions when being fragmented for the purpose of disulfide assignment.

Protein disulfide isomerase-immunopositive inclusions in patients with Alzheimer disease

Alzheimer disease (AD) is the most common neurodegenerative disease, but there is currently no effective treatment available because the etiology or mechanism of AD is still unclear. Many neurodegenerative diseases feature inclusions, which contain accumulations of misfolded, aggregated proteins. Amyloid plaques and neurofibrillary tangles (NFTs) are the major pathological hallmarks of AD. NFTs are composed of tubular filaments, and paired helical filaments containing polymerized hyperphosphorylated tau protein. Another feature is excessive generation of nitric oxide synthetase, reactive nitrogen species, and reactive oxygen species. Protein disulfide isomerase (PDI) is a member of the thioredoxin (TX) superfamily and is believed to accelerate the folding of disulfide-bonded proteins by catalyzing the disulfide interchange reaction, which is the rate-limiting step during protein folding in the luminal space of the endoplasmic reticulum (ER). Nitric oxide (NO)-induced S-nitrosylation of PDI inhibits its enzymatic activity, leading to the accumulation of polyubiquitinated proteins, and activates the unfolded protein response in neurodegenerative diseases. In this study, we found NFTs positive for anti-PDI-antibody in the brain of patients with AD. As far as we know, this is the first report of anti-PDI-antibody-immunopositive inclusions in AD. In AD, NO may inhibit PDI by inducing S-nitrosylation, which inhibits its enzymatic activity and thus allows protein misfolding to occur. Consequently, the accumulation of misfolded proteins induces ER stress. The ER stress can cause apoptosis of neuronal cells. These results suggest that PDI could be a therapeutic target to prevent ER stress in neuronal cells in AD.

Myeloperoxidase-dependent Inactivation of Surfactant Protein D in Vitro and in Vivo

Surfactant protein D (SP-D) plays diverse and important roles in innate immunity and pulmonary homeostasis. Neutrophils and myeloperoxidase (MPO) colocalized with SP-D in a murine bacterial pneumonia model of acute inflammation, suggesting that MPO-derived reactive species might alter the function of SP-D. Exposure of SP-D to the complete MPO-H(2)O(2)-halide system caused loss of SP-D-dependent aggregating activity. Hypochlorous acid (HOCl), the major oxidant generated by MPO, caused a similar loss of aggregating activity, which was accompanied by the generation of abnormal disulfide-cross-linked oligomers. A full-length SP-D mutant lacking N-terminal cysteine residues and truncation mutants lacking the N-terminal domains were resistant to the oxidant-induced alterations in disulfide bonding. Mass spectroscopy of HOCl-treated human SP-D demonstrated several modifications, but none involved key ligand binding residues. There was detectable oxidation of cysteine 15, but no HOCl-induced cysteine modifications were observed in the C-terminal lectin domain. Together, the findings localize abnormal disulfide cross-links to the N-terminal domain. MPO-deficient mice showed decreased cross-linking of SP-D and increased SP-D-dependent aggregating activity in the pneumonia model. Thus, MPO-derived oxidants can lead to modifications of SP-D structure with associated alterations in its characteristic aggregating activity.

2P102 In vitro selection for covalent binding via disulfide interchange with ribosome display(The 48th Annual Meeting of the Biophysical Society of Japan)

2P102 In vitro selection for covalent binding via disulfide interchange with ribosome display(The 48th Annual Meeting of the Biophysical Society of Japan)

ECTO-NOX (ENOX) proteins of the cell surface lack thioredoxin reductase activity.

This study was to determine if ENOX proteins of the cell surface act as cell surface thioredoxin reductases. To measure formation of thiols a turbimetric insulin assay was used. No turbidity was observed with insulin alone or with insulin plus DTT. However, the combination of insulin +DTT + recombinant his-tagged ENOX2 (tNOX) did result in increased turbidity. An ENOX1 (CNOX) preparation also resulted in turbidity changes. In contrast, we were unable to demonstrate ENOX2-dependent insulin reduction by high density SDS-PAGE. Inclusion of reduced serum albumin as a source of free thiols for the protein disulfide interchange activity catalyzed by ENOX2 failed to result in insulin reduction in the presence of ENOX2. A direct effect of ENOX2 on thioredoxin reduction in the presence of NADPH also was not observed. The DTNB assay for thioredoxin reductase activity also failed to reveal activity. Thus, ENOX proteins appear not to function as thioredoxin reductases at the cell surface nor do they appear to recognize reduced insulin as a substrate for protein disulfide-thiol interchange. The enhanced turbidity of insulin solutions resulting from ENOX presence was traced to ENOX-catalyzed insulin fibrillation either through nucleation enhancement or some other mechanism. Fibrillation was determined using Thioflavin T fluorescence which paralleled the turbimetric results and the formation of multimers (polymerization) observed on SDS-PAGE.

Thiol−Disulfide Interchange Mediated Reversible Dendritic Megamer Formation and Dissociation

We report the formation of dynamic, reversible cross-linked dendritic megamers and their dissociation to monomeric dendrimers, through a thiol−disulfide interchange reaction. For this study, poly(alkyl aryl ether) dendrimers up to three-generations presenting thiol functionalities, were prepared. The series from zero to three generations of dendrimers were installed with 3, 6, 12, and 24 thiol functionalities at their peripheries. Upon synthesis, cross-linking of the dendrimer was accomplished through disulfide bond formation. The cross-linking of dendrimers was monitored through optical density changes at 420 nm. Dense cross-linking led to visible precipitation of dendritic megamers and the morphologies of the megamers were characterized by transmission electron microscopy. The disulfide cross-links between megamer monomers could be dissociated readily upon reduction of disulfide bond by dithiothreitol reagent. Preliminary studies show that dendritic megamers encapsulate C60 and the efficiency of encapsulation increased with increasing generation of dendritic megamer.

Experimental and Computational Evaluation of Forces Directing the Association of Transmembrane Helices

The forces that define the interactions of transmembrane helices have been evaluated using a model membrane-soluble peptide (MS1), whose packing is modeled on the two-stranded coiled-coil from GCN4. The thermodynamic stability of water-soluble coiled-coils depends on the side chain at the buried "a" position of the repeat, favoring large hydrophobic residues over small side chains. Here we show that just the opposite is true for the membrane-soluble peptide. Analytical ultracentrifugation and equilibrium disulfide interchange show that the stability of MS1 is greatest when Gly is at each "a" position of the heptad repeat (MS1-Gly), followed by Ala > Val > Ile. Moreover, MS1-Gly has a strong tendency to form antiparallel dimers, MS1-Ala forms a mixture of parallel and antiparallel dimers, while MS1-Val and MS1-Ile have a preference to form parallel dimers. Calculations based on exhaustive conformational searching and rotamer optimization were in excellent agreement with experiments, in terms of the overall stability of the structures and the preference for parallel vs antiparallel packing. The MS1-Gly helices are able to achieve more favorable and uniform packing in an antiparallel dimer, while MS1-Val and MS1-Ile have more favorable van der Waals interactions in a parallel dimer. Finally, the electrostatic component arising from the partial charges of the backbones become significant in the antiparallel MS1-Gly and MS1-Ala conformations, due to close packing of the helices. Thus, van der Waals interactions and electrostatic interactions contribute to the stability and orientational preferences of the dimers.

Function of Glu-469′ in the Acid−Base Catalysis of Thioredoxin Reductase from Drosophila melanogaster

Thioredoxin reductase (TrxR) catalyzes the reduction of thioredoxin (Trx) by NADPH. Because dipteran insects such as Drosophila melanogaster lack glutathione reductase, their TrxRs are particularly important for antioxidant protection; reduced Trx reacts nonenzymatically with oxidized glutathione to maintain a high glutathione/glutathione disulfide ratio. Like other members of the pyridine nucleotide-disulfide oxidoreductase family, TrxR is a homodimer; in the enzyme from D. melanogaster (DmTrxR), each catalytically active unit consists of three redox centers: FAD and an N-terminal Cys-57-Cys-62 redox-active disulfide from one monomer and a Cys-489'-Cys-490' C-terminal redox-active disulfide from the second monomer. A dyad of His-464' and Glu-469' in TrxR acts as the acid-base catalyst of the dithiol-disulfide interchange reactions required in catalysis [Huang, H.-H., et al. (2008) Biochemistry 47, 1721-1731]. In this investigation, the role of Glu-469' in catalysis by DmTrxR has been studied. The E469'A and E469'Q DmTrxR variants retain 28 and 35% of the wild-type activity, respectively, indicating that this glutamate residue is important but not critical to catalysis. The pH dependence of V(max) for both glutamate variants yields pK(a) values of 6.0 and 8.7, compared to those in the wild-type enzyme of 6.4 and 9.3, respectively, indicating that the basicity of His-464' in TrxR in complex with its substrate, DmTrx-2, is significantly lower in the glutamate variants than in wild-type enzyme. The rates of some steps in the reductive half-reactions in both glutamate variants are much slower than those of the wild-type enzyme. On the basis of our observations, it is proposed that the function of Glu-469' is to facilitate the positioning of His-464' toward the interchange thiol, Cys-57, as suggested for the analogous residue in glutathione reductase.

Polyrotaxane Networks Formed via Rotaxanation Utilizing Dynamic Covalent Chemistry of Disulfide

Polyrotaxane networks were synthesized from a mixture of poly(crown ether)s (2−5) as a trunk polymer and dumbbell-shaped bifunctional secondary ammonium salt 6 bearing a disulfide linkage and bulky end caps as a cross-linking agent in the presence of a catalytic amount of benzenethiol via a reversible thiol−disulfide interchange reaction. Trunk polymers 2−4 were prepared by copolymerization of bis(hydroxymethyl)dibenzo-24-crown-8-ether 1 and comonomers. Poly(crown ether) 5 bearing a poly(tetrahydrofuran) (PTHF) spacer was also synthesized from 1 and PTHF with 4,4′-methylenebis(phenyl isocyanate) (MDI) in N,N-dimethylacetamide (DMAc) at room temperature. The treatment of these poly(crown ether)s (2−5) with axle component 6 in the presence of benzenethiol gave the corresponding polyrotaxane networks (7−10) in quantitative yields. We confirmed the cross-linking process by monitoring the 1H NMR spectral change during the polymerization. Thermal properties, swelling, and dynamic viscoelasticity of these polyrotaxane networks were evaluated. Polyrotaxane network 10 bearing 5 afforded higher swelling and elasticity than those of other networks because of the flexibility of the trunk polymer as wheel components.

Allochromatium vinosum DsrC: Solution-State NMR Structure, Redox Properties, and Interaction with DsrEFH, a Protein Essential for Purple Sulfur Bacterial Sulfur Oxidation

Sequenced genomes of dissimilatory sulfur-oxidizing and sulfate-reducing bacteria containing genes coding for DsrAB, the enzyme dissimilatory sulfite reductase, inevitably also contain the gene coding for the 12-kDa DsrC protein. DsrC is thought to have a yet unidentified role associated with the activity of DsrAB. Here we report the solution structure of DsrC from the sulfur-oxidizing purple sulfur bacterium Allochromatium vinosum determined with NMR spectroscopy in reducing conditions, and we describe the redox behavior of two conserved cysteine residues upon transfer to an oxidizing environment. In reducing conditions, the DsrC structure is disordered in the highly conserved carboxy-terminus. We present multiple lines of evidence that, in oxidizing conditions, a strictly conserved cysteine (Cys111) at the penultimate position in the sequence forms an intramolecular disulfide bond with Cys100, which is conserved in DsrC in all organisms with DsrAB. While an intermolecular Cys111-Cys111 disulfide-bonded dimer is rapidly formed under oxidizing conditions, the intramolecularly disulfide-bonded species (Cys100-Cys111) is the thermodynamically stable form of the protein under these conditions. Treatment of the disulfidic forms with reducing agent regenerates the monomeric species that was structurally characterized. Using a band-shift technique under nondenaturing conditions, we obtained evidence for the interaction of DsrC with heterohexameric DsrEFH, a protein encoded in the same operon. Mutation of Cys100 to serine prevented formation of the DsrC species assigned as an intramolecular disulfide in oxidizing conditions, while still allowing formation of the intermolecular Cys111-Cys111 dimer. In the reduced form, this mutant protein still interacted with DsrEFH. This was not the case for the Cys111Ser and Cys100Ser/Cys111Ser mutants, both of which also did not form protein dimers. Our observations highlight the central importance of the carboxy-terminal DsrC cysteine residues and are consistent with a role as a sulfur–substrate binding/transferring protein, as well as with an electron-transfer function via thiol–disulfide interchanges.

Efficient Site-Specific Labeling of Proteins via Cysteines

Methods for chemical modifications of proteins have been crucial for the advancement of proteomics. In particular, site-specific covalent labeling of proteins with fluorophores and other moieties has permitted the development of a multitude of assays for proteome analysis. A common approach for such a modification is solvent-accessible cysteine labeling using thiol-reactive dyes. Cysteine is very attractive for site-specific conjugation due to its relative rarity throughout the proteome and the ease of its introduction into a specific site along the protein's amino acid chain. This is achieved by site-directed mutagenesis, most often without perturbing the protein's function. Bottlenecks in this reaction, however, include the maintenance of reactive thiol groups without oxidation before the reaction, and the effective removal of unreacted molecules prior to fluorescence studies. Here, we describe an efficient, specific, and rapid procedure for cysteine labeling starting from well-reduced proteins in the solid state. The efficacy and specificity of the improved procedure are estimated using a variety of single-cysteine proteins and thiol-reactive dyes. Based on UV/vis absorbance spectra, coupling efficiencies are typically in the range 70-90%, and specificities are better than approximately 95%. The labeled proteins are evaluated using fluorescence assays, proving that the covalent modification does not alter their function. In addition to maleimide-based conjugation, this improved procedure may be used for other thiol-reactive conjugations such as haloacetyl, alkyl halide, and disulfide interchange derivatives. This facile and rapid procedure is well suited for high throughput proteome analysis.

ECTO‐NOX (ENOX) proteins of the cell surface lack thioredoxin reductase activity

AbstractThis study was to determine if ENOX proteins of the cell surface act as cell surface thioredoxin reductases. To measure formation of thiols a turbimetric insulin assay was used. No turbidity was observed with insulin alone or with insulin plus DTT. However, the combination of insulin + DTT + recombinant his‐tagged ENOX2 (tNOX) did result in increased turbidity. An ENOX1 (CNOX) preparation also resulted in turbidity changes. In contrast, we were unable to demonstrate ENOX2‐dependent insulin reduction by high density SDS‐PAGE. Inclusion of reduced serum albumin as a source of free thiols for the protein disulfide interchange activity catalyzed by ENOX2 failed to result in insulin reduction in the presence of ENOX2. A direct effect of ENOX2 on thioredoxin reduction in the presence of NADPH also was not observed. The DTNB assay for thioredoxin reductase activity also failed to reveal activity. Thus, ENOX proteins appear not to function as thioredoxin reductases at the cell surface nor do they appear to recognize reduced insulin as a substrate for protein disulfide‐thiol interchange. The enhanced turbidity of insulin solutions resulting from ENOX presence was traced to ENOX‐catalyzed insulin fibrillation either through nucleation enhancement or some other mechanism. Fibrillation was determined using Thioflavin T fluorescence which paralleled the turbimetric results and the formation of multimers (polymerization) observed on SDS‐PAGE.

Mechanism of Thiolate−Disulfide Interchange Reactions in Biochemistry

Both density functional theory (DFT) (B3LYP) and CCSD ab initio calculations were employed in a theoretical investigation of the mechanism of thiolate-disulfide exchange reactions. The reaction pathway for degenerate thiolate-disulfide exchange reactions with dimethyl disulfide has been shown to proceed through a SN2-like transition structure that is very close in energy to the corresponding trisulfur anionic intermediate ([delta-S-S-Sdelta(-)]). When relatively small substituents are involved, the level of theory must be increased to CCSD to make this rather subtle mechanistic distinction. With the more sterically hindered exchange reaction involving t-butyl mercaptide and di-t-butyl disulfide, the potential energy surface exhibits a distinct preference for the S(N)2 displacement pathway with an activation barrier of 9.8 kcal/mol. When corrections for solvent polarity are included (COSMO), an S(N)2 mechanism is also implicated in both polar and nonpolar solvents. DFT studies on thiolate-disulfide exchange, when the substituent is a model peptide, strongly support the intermediacy of a trisulfur intermediate that lies 10.7 kcal/mol below isolated reactants. A well depth of this magnitude should provide a sufficient lifetime of the intermediate to accommodate the requisite conformational adjustments that accompanies formation of the new disulfide bond.

A new series of schistosomicide drugs, the alkylaminoalkanethiosulfuric acids, partially inhibit the activity of Schistosoma mansoni ATP diphosphohydrolase

The effects of the alkylaminoalkanethiosulfuric acids (AAATs), new schistosomicidal drugs, on Schistosoma mansoni ATP diphosphohydrolase isoforms, members of the NTPDase family, were analyzed. Pre-incubation of worm adult tegument with AAATs derivatives, with small apolar alkyl groups and an apolar alkane portion of 6 or 8 carbon atoms linked to the amino group, inhibited ATPase activity with a Ki 100–1000 μM. Little inhibition (20%) was observed when ADP was the substrate. The 2-[( tert-butyl)amino]-1-ethanethiosulfuric acid (100 μM) which has a less lipophilic structure, inhibited 28% ATPase and 12% ADPase activities, suggesting that the lipophilicity, although important, is not the only requisite for enzyme activity inhibition. The N-( sec-butyl)-2-bromo-1-octanaminium bromide, which contains a bromide atom instead of thiosulphate, inhibited < 10% of the enzyme activity, suggesting the involvement of cysteine residue(s) from S. mansoni ATP diphosphohydrolase isoforms in a mixed disulfide formation. Treatment of parasite tegument with 5 mM iodoacetamide or 1 mM dithiothreitol protected ATPase and ADPase activities against inhibition by AAATs, corroborating the participation of disulfide interchange in the AAATs mechanism. Since S. mansoni ATP diphosphohydrolase isoforms and potato apyrase share structural similarities, the latter enzyme was also tested. ADPase activity from potato apyrase was inhibited by 55%, showing a higher sensitivity to 1 mM AAATs than that shown by ADPase activity from the tegument, while the ATPase activities from both samples showed similar inhibition levels. Furthermore, sulfhydryl reagents protected potato apyrase activity. Therefore, it is possible that both soluble S. mansoni ATP diphosphohydrolase and membrane-associated isoforms are targets for the AAATs.

Cellular thiol pools are responsible for sequestration of cytotoxic reactive aldehydes: Central role of free cysteine and cysteamine

Cellular thiol pools have been shown to be important in the regulation of the redox status of cells, providing a large antioxidant pool consisting of free thiols, thiols bound in the disulfide form and thiols bound to proteins. However, experimental studies with the thiol cysteamine and its disulfide cystamine have demonstrated dramatic cytoprotection in experimental models where antioxidants provide only minor protection. These data suggest that an alternate action of thiols is important in their cytoprotective actions. A common feature of the in vitro and in vivo models, where these thiol agents demonstrate cytoprotection, is the generation of cytotoxic aldehydes. We therefore studied the actions of cystamine, cysteamine and several reference thiol agents as cytoprotectants against cell death induced by increased “aldehyde load”. We found that all the thiol agents examined provided dramatic protection against aldehyde-induced cell death in SN56 cholinergic neurons, under conditions in which acrolein induced 100% cell death. With regard to mechanism of action, the reference thiols cysteine, N-acetylcysteine, 2-mercaptoethanesulfonic acid, mercapto-propionyglycine, and cysteamine can directly sequester aldehydes. In addition, these thiols were all found to augment intracellular cysteine levels via disulfide interchange reactions. Cysteamine and cystamine also augmented basal intracellular cysteamine levels. Our data, for the first time, demonstrate the importance of intracellular thiols in sequestering toxic reactive aldehyde products of lipid peroxidation and polyamine metabolism. In addition it appears that pharmacological manipulation of intracellular thiol pools might offer a new approach in the design of neuroprotective drug candidates.

Cysteine Reactivity and Thiol−Disulfide Interchange Pathways in AhpF and AhpC of the Bacterial Alkyl Hydroperoxide Reductase System

AhpC and AhpF from Salmonella typhimurium undergo a series of electron transfers to catalyze the pyridine nucleotide-dependent reduction of hydroperoxide substrates. AhpC, the peroxide-reducing (peroxiredoxin) component of this alkyl hydroperoxidase system, is an important scavenger of endogenous hydrogen peroxide in bacteria and acts through a reactive, peroxidatic cysteine, Cys46, and a second cysteine, Cys165, that forms an active site disulfide bond. AhpF, a separate disulfide reductase protein, regenerates AhpC every catalytic cycle via electrons from NADH which are transferred to AhpC through a tightly bound flavin and two disulfide centers, Cys345-Cys348 and Cys129-Cys132, through putative large domain movements. In order to assess cysteine reactivity and interdomain interactions in both proteins, a comprehensive set of single and double cysteine mutants (replacing cysteine with serine) of both proteins were prepared. Based on 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) and AhpC reactivity with multiple mutants of AhpF, the thiolate of Cys129 in the N-terminal domain of AhpF initiates attack on Cys165 of the intersubunit disulfide bond within AhpC for electron transfer between proteins. Cys348 of AhpF has also been identified as the nucleophile attacking the Cys129 sulfur of the N-terminal disulfide bond to initiate electron transfer between these two redox centers. These findings support the modular architecture of AhpF and its need for domain rotations for function, and emphasize the importance of Cys165 in the reductive reactivation of AhpC. In addition, two new constructs have been generated, an AhpF-AhpC complex and a "twisted" form of AhpF, in which redox centers are locked together by stable disulfide bonds which mimic catalytic intermediates.

Allosteric Disulfide Bonds

Disulfide bonds have been generally considered to be either structural or catalytic. Structural bonds stabilize a protein, while catalytic bonds mediate thiol-disulfide interchange reactions in substrate proteins. There is emerging evidence for a third type of disulfide bond that can control protein function by triggering a conformational change when it breaks and/or forms. These bonds can be thought of as allosteric disulfides. To better define the properties of allosteric disulfides, we have analyzed the geometry and dihedral strain of 6874 unique disulfide bonds in 2776 X-ray structures. A total of 20 types of disulfide bonds were identified in the dataset based on the sign of the five chi angles that make up the bond. The known allosteric disulfides were all contained in 1 of the 20 groups, the -RHStaple bonds. This bond group has a high mean potential energy and narrow energy distribution, which is consistent with a functional role. We suggest that the -RHStaple configuration is a hallmark of allosteric disulfides. About 1 in 15 of all structurally determined disulfides is a potential allosteric bond.

Characterization of a Membrane Protein Folding Motif, the Ser Zipper, Using Designed Peptides

Polar residues play important roles in the association of transmembrane helices and the stabilities of membrane proteins. Although a single Ser residue in a transmembrane helix is unable to mediate a strong association of the helices, the cooperative interactions of two or more appropriately placed serine hydroxyl groups per helix has been hypothesized to allow formation of a "serine zipper" that can stabilize transmembrane helix association. In particular, a heptad repeat Sera Xxx Xxx Leud Xxx Xxx Xxx (Xxx is a hydrophobic amino acid) appears in both antiparallel helical pairs of polytopic membrane proteins as well as the parallel helical dimerization motif found in the murine erythropoietin receptor. To examine the intrinsic conformational preferences of this motif independent of its context within a larger protein, we synthesized a peptide containing three copies of a SeraLeud heptad motif. Computational results are consistent with the designed peptide adopting either a parallel or antiparallel structure, and conformational search calculations yield the parallel dimer as the lowest energy configuration, which is also significantly more stable than the parallel trimer. Analytical ultracentrifugation indicated that the peptide exists in a monomer-dimer equilibrium in dodecylphosphocholine micelles. Thiol disulfide interchange studies showed a preference for forming parallel dimers in micelles. In phospholipid vesicles, only the parallel dimer was formed. The stability of the SerZip peptide was studied in vesicles prepared from phosphatidylcholine (PC) lipids of different chain length: POPC (C16:0C18:1 PC) and DLPC (C12:0PC). The stability was greater in POPC, which has a good match between the length of the hydrophobic region of the peptide and the bilayer length. Finally, mutation to Ala of the Ser residues in the SerZip motif gave rise to a relatively small decrease in the stability of the dimer, indicating that packing interactions rather than hydrogen-bonding provided the primary driving force for association.