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PubMed1987

Characterization of disulfide bonds in recombinant proteins: reduced human interleukin 2 in inclusion bodies and its oxidative refolding.

Cloned cDNA of human interleukin 2 (IL-2) was expressed in Escherichia coli cells in which IL-2 formed insoluble inclusion bodies. Human IL-2 has three Cys residues, namely, Cys-58, Cys-105, and Cys-125, and native IL-2 has an intramolecular disulfide bond between Cys-58 and Cys-105. Since the formation of inclusion bodies was thought to be due to disorder in the oxidation state of these Cys residues, all intramolecular disulfide bond isomers of IL-2 were prepared by denaturation of native IL-2 to characterize the state of a disulfide bond in IL-2 in the inclusion bodies. These isomers can be separated from native IL-2, reduced IL-2, and IL-2's with intermolecular disulfide bonds by means of reversed-phase high-performance liquid chromatography. Human IL-2 produced in inclusion bodies in E. coli carrying a recombinant DNA was analyzed by HPLC and was proved to be a fully reduced form with no intra- and intermolecular disulfide bonds. Refolding of reduced IL-2 in the presence of reduced and oxidized glutathione and a low concentration of guanidine hydrochloride resulted in the formation of the biologically active IL-2 quantitatively. Further purification provided a practically pure IL-2 preparation without contamination of any disulfide bond isomers.

Amino Acid Sequence↗
PubMed1990

Denaturant-dependent folding of bovine pancreatic trypsin inhibitor mutants with two intact disulfide bonds.

The equilibrium and kinetic behavior of the guanidine hydrochloride (Gdn-HCl) induced unfolding/refolding of four bovine pancreatic trypsin inhibitor (BPTI) mutants was examined by using ultraviolet difference spectroscopy. In three of the mutants, we replaced the buried 30-51 disulfide bond with alanine at position 51 and valine (Val30/Ala51), alanine (Ala30/Ala51), or threonine (Thr30/Ala51) at position 30. For the fourth mutant, the solvent-exposed 14-38 disulfide was substituted by a pair of alanines (Ala14/Ala38). All mutants retained the 5-55 disulfide. Experiments were performed under oxidizing conditions; thus, both the unfolded and folded forms retained two native disulfide bonds. Equilibrium experiments demonstrated that all four mutants were destabilized relative to wild-type BPTI. However, the stability of the 30-51 mutants increased with the hydrophobicity of the residue substituted at position 30. Kinetic experiments showed that all four mutants contained two minor slow refolding phases with characteristics of proline isomerization. The specific behavior of the phases depended on the location of the disulfide bonds. The major unfolding/refolding phase for each of the 30-51 mutants was more than an order of magnitude slower than for Ala14/Ala38 or for BPTI in which the 14-38 disulfide bond was specifically reduced and blocked with iodoacetamide [Jullien, M., & Baldwin, R. L. (1981) J. Mol. Biol. 145, 265-280]. Since this effect is independent of the stability of the protein, it is consistent with a model in which the proper docking of the interior residues of the protein is the rate-limiting step in the folding of these mutants.

Amino Acid Sequence↗
PubMed2001

Probing the dark state tertiary structure in the cytoplasmic domain of rhodopsin: proximities between amino acids deduced from spontaneous disulfide bond formation between cysteine pairs engineered in cytoplasmic loops 1, 3, and 4.

To probe proximities between amino acids in the cytoplasmic domain by using mutants containing engineered cysteine pairs, three sets of rhodopsin mutants have been prepared. In the first two sets, a cysteine was placed, one at a time, at positions 311-314 in helix VIII, while the second cysteine was fixed at position 246 (set I) and at position 250 (set II) at the cytoplasmic end of helix VI. In the third set, one cysteine was fixed at position 65 while the second cysteine was varied between amino acid positions 306 and 321 located at the cytoplasmic end of helix VII and throughout in helix VIII. Rapid disulfide bond formation in the dark was found between the cysteine pairs in mutants A246C/Q312C,A246C/K311C and in mutants H65C/C316, H65C/315C and H65C/312C. Disulfide bond formation at much lower rates was found in mutants A246C/F313C, V250C/Q312C, H65C/N310C, H65C/K311C, H65C/F313C, and H65C/R314C; the remaining mutants showed no significant disulfide bond formation. Comparisons of the results from disulfide bond formation in solution with the distances observed in the rhodopsin crystal structure showed that the rates of disulfide bond formation in most cases were consistent with the amino acid proximities as revealed in crystal structure. However, deviations were also found, in particular, in the set containing fixed cysteine at position Cys246 and cysteines at positions 311-314. The results implicate significant effects of structural dynamics on disulfide bond formation in solution.

Amino Acid Sequence↗
PubMed2002

Solution structures of the cis- and trans-Pro30 isomers of a novel 38-residue toxin from the venom of Hadronyche Infensa sp. that contains a cystine-knot motif within its four disulfide bonds.

The primary sequence and three-dimensional structure of a novel peptide toxin isolated from the Australian funnel-web spider Hadronyche infensa sp. is reported. ACTX-Hi:OB4219 contains 38 amino acids, including eight-cysteine residues that form four disulfide bonds. The connectivities of these disulfide bonds were previously unknown but have been unambiguously determined in this study. Three of these disulfide bonds are arranged in an inhibitor cystine-knot (ICK) motif, which is observed in a range of other disulfide-rich peptide toxins. The motif incorporates an embedded ring in the structure formed by two of the disulfides and their connecting backbone segments penetrated by a third disulfide bond. Using NMR spectroscopy, we determined that despite the isolation of a single native homologous product by RP-HPLC, ACTX-Hi:OB4219 possesses two equally populated conformers in solution. These two conformers were determined to arise from cis/trans isomerization of the bond preceding Pro30. Full assignment of the NMR spectra for both conformers allowed for the calculation of their structures, revealing the presence of a triple-stranded antiparallel beta sheet consistent with the inhibitor cystine-knot (ICK) motif.

Amino Acid Sequence↗
PubMed2002

A protein caught in a kinetic trap: structures and stabilities of insulin disulfide isomers.

Proinsulin contains six cysteines whose specific pairing (A6-A11, A7-B7, and A20-B19) is a defining feature of the insulin fold. Pairing information is contained within A and B domains as demonstrated by studies of insulin chain recombination. Two insulin isomers containing non-native disulfide bridges ([A7-A11,A6-B7,A20-B19] and [A6-A7,A11-B7,A20-B19]), previously prepared by directed chemical synthesis, are metastable and biologically active. Remarkably, the same two isomers are preferentially formed from native insulin or proinsulin following disulfide reassortment in guanidine hydrochloride. The absence of other disulfide isomers suggests that the observed species exhibit greater relative stability and/or kinetic accessibility. The structure of the first isomer ([A7-A11,A6-B7,A20-B19], insulin-swap) has been described [Hua, Q. X., Gozani, S. N., Chance, R. E., Hoffmann, J. A., Frank, B. H., and Weiss, M. A. (1995) Nat. Struct. Biol. 2, 129-138]. Here, we demonstrate that the second isomer (insulin-swap2) is less ordered than the first. Nativelike elements of structure are retained in the B chain, whereas the A chain is largely disordered. Thermodynamic studies of guanidine denaturation demonstrate the instability of the isomers relative to native insulin (DeltaDeltaG(u) > 3 kcal/mol). In contrast, insulin-like growth factor I (IGF-I) and the corresponding isomer IGF-swap, formed as alternative products of a bifurcating folding pathway, exhibit similar cooperative unfolding transitions. The insulin isomers are similar in structure and stability to two-disulfide analogues whose partial folds provide models of oxidative folding intermediates. Each exhibits a nativelike B chain and less-ordered A chain. This general asymmetry is consistent with a hierarchical disulfide pathway in which nascent structure in the B chain provides a template for folding of the A chain. Structures of metastable disulfide isomers provide probes of the topography of an energy landscape.

Amino Acid Motifs↗
PubMed2002

Interactions that favor the native over the non-native disulfide bond among residues 58-72 in the oxidative folding of bovine pancreatic ribonuclease A.

In the initial stages of the oxidative folding of both bovine pancreatic ribonuclease A (RNase A) and a 58-72 fragment thereof from the fully reduced, denatured state, the 65-72 correctly paired disulfide bond forms in preponderance over the incorrectly paired 58-65 disulfide bond. Since both disulfide-bonded loops contain the same number of amino acid residues, the question arises as to whether the native pairing results from interactions within the 58-72 segment that lead to a nativelike structure even in its fully reduced form. To answer this question, the chain buildup procedure, based on ECEPP, including a solvation treatment, was used to generate the low-energy structures for the 58-72 RNase segment, beginning with residue 72 and building back to residue 58; in this fragment, all three Cys residues (at positions 58, 65, and 72) initially exist in the reduced (CysH) state. After the open-chain energy minima of the 65-72 peptide were generated, these conformations were allowed to form the 65-72 disulfide bond, and the energies of the resulting oxidized conformations were reminimized and rehydrated. The global minimum of the loop-closed 65-72 structure and many of the low-lying loop-closed minima could be superimposed on the energy-minimized X-ray structure for residues 65-72. The low-energy structures for the full open chain 58-72 peptide were then computed and were allowed to form disulfide bonds either between residues 65 and 72 (native) or between residues 58 and 65 (non-native), and their energies were reminimized and rehydrated in the loop-closed state. Although the overall fold of the 65-72 loop-closed global minimum was the same as for the energy-minimized X-ray structure of these residues, the overall rms deviation was 3.9 A because of local deviations among residues 58-64. In contrast, the 65-72 segment of the global minimum of the 58-72 fragment could be superimposed on the corresponding residues of the energy-minimized X-ray structure. The lowest-energy structure for the 58-65 non-native paired 58-72 sequence was 6 kcal/mol higher in energy than that for the 58-72 peptide with the 65-72 disulfide bond formed. These results suggest that the native pairing of the 65-72 peptide arises from energetic determinants (adoption of left-handed single-residue conformations by Gly 68, and side chain interactions involving Gln 69) contained within this peptide sequence.

Animals↗
PubMed2005

Characterization of disulfide bonds in human nucleoside triphosphate diphosphohydrolase 3 (NTPDase3): implications for NTPDase structural modeling.

Cell-surface nucleotidases (NTPDases) contain 10 invariant cysteine residues in their extracellular regions. To investigate disulfide structure in human NTPDase3, we made single and double mutants of these 10 cysteines, and analyzed their enzymatic activity, glycosylation pattern, trafficking to the cell membrane, and sensitivity to reduction. The mutants constituted five distinct phenotypes, thus, strongly suggesting disulfide bonds between C92-C116 (first bond), C261-C308 (second bond), C289-C334 (third bond), C347-C353 (fourth bond), and C399-C422 (fifth bond). Due to conservation of the 10 cysteines, the identified five disulfide bonds are likely to exist in all cell-surface NTPDases. The third and fifth bonds are also present in the soluble NTPDases and are critical for processing, trafficking, and enzymatic activity. The fourth bond has minimal effect on processing and function, while the first and second bonds are of intermediate importance. Most of the N-linked glycosylation sites in the wild-type enzyme are processed to complex oligosaccharides, but at least one site is high-mannose or hybrid in structure. Interestingly, disruption of the first disulfide bond resulted in some enzyme that lost sensitivity to endoglycosidase H, suggesting that the first disulfide bond in the wild-type enzyme shields some high-mannose glycans from terminal glycosylation. Comparative modeling by threading and homology modeling of the NTPDase3 sequence revealed a high degree of structural fold similarity with a bacterial exopolyphosphatase (PDB ). The resultant theoretical 3-D model of the extracellular portion of NTPDase3, based on homology with this exopolyphosphatase, is consistent with the assignment of the disulfide bonds occurring in regions of good fold similarity between NTPDase3 and the exopolyphosphatase. The 3-D model obtained for NTPDase3 also suggests the structural basis for the importance of several apyrase conserved regions for the nucleotidase activities of the NTPDases.

Amino Acid Sequence↗
PubMed2005

Disulfide bond mutagenesis and the structure and function of the head-to-tail macrocyclic trypsin inhibitor SFTI-1.

SFTI-1 is a novel 14 amino acid peptide comprised of a circular backbone constrained by three proline residues, a hydrogen-bond network, and a single disulfide bond. It is the smallest and most potent known Bowman-Birk trypsin inhibitor and the only one with a cyclic peptidic backbone. The solution structure of [ABA(3,11)]SFTI-1, a disulfide-deficient analogue of SFTI-1, has been determined by (1)H NMR spectroscopy. The lowest energy structures of native SFTI-1 and [ABA(3,11)]SFTI-1 are similar and superimpose with a root-mean-square deviation over the backbone and heavy atoms of 0.26 +/- 0.09 and 1.10 +/- 0.22 A, respectively. The disulfide bridge in SFTI-1 was found to be a minor determinant for the overall structure, but its removal resulted in a slightly weakened hydrogen-bonding network. To further investigate the role of the disulfide bridge, NMR chemical shifts for the backbone H(alpha) protons of two disulfide-deficient linear analogues of SFTI-1, [ABA(3,11)]SFTI-1[6,5] and [ABA(3,11)]SFTI-1[1,14] were measured. These correspond to analogues of the cleavage product of SFTI-1 and a putative biosynthetic precursor, respectively. In contrast with the cyclic peptide, it was found that the disulfide bridge is essential for maintaining the structure of these open-chain analogues. Overall, the hydrogen-bond network appears to be a crucial determinant of the structure of SFTI-1 analogues.

Benzamides↗
PubMed2004

NMR characterization of three-disulfide variants of lysozyme, C64A/C80A, C76A/C94A, and C30A/C115A--a marginally stable state in folded proteins.

Our earlier NMR study showed that a two-disulfide variant of hen lysozyme containing intra-alpha-domain disulfide bridges, C6-C127 and C30-C115, is partially folded, with the alpha domain tightly folded to the nativelike conformation and the beta domain flexible or unfolded. With a view that the formation of a third disulfide bridge is a key for the accomplishment of the overall chain fold, three-dimensional structures of three-disulfide variants of hen lysozyme lacking one disulfide bridge (C64A/C80A, C76A/C94A, and C30A/C115A) were studied in detail using NMR spectroscopy. Amide hydrogen exchange rates were measured to estimate the degree of conformational fluctuation in a residue-specific manner. The structure of C76A/C94A was found to be quite similar to that of the wild type, except for the peptide segment of residues 74-78. The structure of C64A/C80A was considerably disordered in the entire region of the loop (residues 62-79). Further, it was found that a network of hydrogen bonds within the beta sheet and the 3(10) helix in the beta domain were disrupted and fluctuating. In C30A/C115A, the D helix was unstructured and the interface of the B helix with the D helix was significantly perturbed. However, the structural disorder generated in the hydrophobic core of the alpha domain was prevented by the C helix from propagating toward the beta domain. A marginally stable state in folded proteins is discussed based on the structures remaining in each three-disulfide variant.

Amides↗
PubMed2006

Use of an in situ disulfide cross-linking strategy to study the dynamic properties of the cytoplasmic end of transmembrane domain VI of the M3 muscarinic acetylcholine receptor.

The ligand-induced activation of G protein-coupled receptors (GPCRs) is predicted to involve pronounced conformational changes on the intracellular surface or the receptor proteins. A reorientation of the cytoplasmic end of transmembrane domain VI (TM VI) is thought to play a key role in GPCR activation and productive receptor/G protein coupling. Disulfide cross-linking studies with solubilized, Cys-substituted mutant versions of bovine rhodopsin and the M3 muscarinic acetylcholine receptor suggested that the cytoplasmic end of TM VI is conformationally highly flexible, even in the absence of activating ligands (Farrens, D. L., et al. (1996) Science 274, 768-770; Zeng, F. Y., et al. (1999) J. Biol. Chem. 274, 16629-16640). To test the hypothesis that the promiscuous disulfide cross-linking pattern observed in these studies was caused by the use of solubilized receptor proteins endowed with increased conformational flexibility, we employed a recently developed in situ disulfide cross-linking strategy that allows the detection of disulfide bonds in Cys-substituted mutant M3 muscarinic receptors present in their native membrane environment. Specifically, we used membranes prepared from transfected COS-7 cells to analyze a series of double Cys mutant M3 receptors containing one Cys residue within the sequence K484(6.29) to S493(6.38) at the cytoplasmic end of TM VI and a second Cys residue at the cytoplasmic end of TM III (I169C(3.54)). This analysis revealed a disulfide cross-linking pattern that was strikingly more restricted than that observed previously with solubilized receptor proteins, both in the absence and in the presence of the muscarinic agonist, carbachol. Carbachol stimulated the formation of disulfide bonds in only two of the 10 analyzed mutant muscarinic receptors, I169C(3.54)/K484C(6.29) and I169C(3.54)/A488C(6.33), consistent with an agonist-induced rotation of the cytoplasmic end of TM VI. These findings underline the usefulness of analyzing the structural and dynamic properties of GPCRs in their native lipid environment.

Animals↗
PubMed2005

Disulfide structure of the leucine-rich repeat C-terminal cap and C-terminal stalk region of Nogo-66 receptor.

Nogo-66 receptor (NgR1) is a leucine-rich repeat (LRR) protein that forms part of a signaling complex modulating axon regeneration. Previous studies have shown that the entire LRR region of NgR1, including the C-terminal cap of the LRR, LRRCT, is needed for ligand binding, and that the adjacent C-terminal region (CT stalk) of the NgR1 contributes to interaction with its coreceptors. To provide structure-based information for these interactions, we analyzed the disulfide structure of full-length NgR1. Our analysis revealed a novel disulfide structure in the C-terminal region of the NgR1, wherein the two Cys residues, Cys-335 and Cys-336, in the CT stalk are disulfide-linked to Cys-266 and Cys-309 in the LRRCT region: Cys-266 is linked to Cys-335, and Cys-309 to Cys-336. The other two Cys residues, Cys-264 and Cys-287, in the LRRCT region are disulfide-linked to each other. The analysis also showed that Cys-419 and Cys-429, in the CT stalk region, are linked to each other by a disulfide bond. Although published crystal structures of a recombinant fragment of NgR1 had revealed a disulfide linkage between Cys-266 and Cys-309 in the LRRCT region and we verified its presence in the corresponding fragment, this is artificially caused by the truncation of the protein, since this linkage was not detected in intact NgR1 or a slightly larger fragment containing Cys-335 and Cys-336. A structural model of the LRRCT with extended residues 311-344 from the CT stalk region is proposed, and its function in coreceptor binding is discussed.

Alkylation↗
PubMed1996

Flavins inhibit human cytomegalovirus UL80 protease via disulfide bond formation.

Among the most potent inhibitors of human cytomegalovirus protease identified by random screening of a chemical library was 1,4-dihydro-7,8-dimethyl 6H-pyrimido[1,2-b]-1,2,4,5-tetrazin-6-one (1) (PTH2). The oxidized form (2), PT, which is present in solutions of PTH2, was shown to be the actual inhibitory species which irreversibly inactivates the protease; recycling of PTH2 by dissolved oxygen results in complete inhibition of the protease at substoichiometric amounts of compound. No evidence for a covalent adduct between the protease and the inhibitor was obtained, and protease activity was restored by incubation of the inactivated enzyme with the reducing agent bismercaptoethyl sulfone, suggesting that disulfide bond formation was responsible for the observed inhibition. The five cysteines of the protease are normally in the reduced state; analysis of tryptic peptides from inhibited protease indicated that disulfide bonds Cys84-Cys87 and Cys138-Cys161 were formed. Using site-directed mutagenesis, the disulfide pair induced between Cys138 and Cys161 disulfide is dependent upon interaction of PT with the protease and does not form spontaneously, unlike that of the Cys84-Cys87 pair which can form in the absence of inhibitor. The inhibitor's redox chemistry is analogous to that of flavin, and, in fact, flavin inhibits the protease by the same mechanism, causing formation of a disulfide bond between Cys138 and Cys161. That the cysteines are dispensable, but can regulate protease activity by formation of a unique disulfide pair, suggests a plausible mechanism for control of proteolysis during the viral life cycle.

Base Sequence↗
PubMed1996

Disulfide structure and N-glycosylation sites of an extracellular domain of granulocyte-colony stimulating factor receptor.

An extracellular domain containing 603 amino acid residues of human granulocyte-colony stimulating factor receptor was expressed in Chinese hamster ovary cells. The affinity-purified material has previously been shown to dimerize when combined with the ligand. In this paper we have characterized the primary structure of this active receptor. Laser desorption mass spectrometry of the purified receptor showed a broad peak at a molecular weight of 84,000, ranging from 77,000 to 91,000. The molecular weight heterogeneity is due to glycosylation. Since the molecular weight based on the amino acid sequence is 67,322, by subtraction the carbohydrate content is approximately 17,000. Disulfide structure of the receptor was determined by peptide mapping in the absence and presence of reducing agent. Sequence and mass spectral analyses of these peptides showed the receptor to contain eight disulfide bonds and three free cysteines. These disulfide bonds are consistent with the known domain motifs of the receptor in that no interdomain disulfides were present. One of the three free cysteines is reactive with alkylating agents, while the others are less reactive, probably being buried in the interior of the molecule. Blocking the free cysteines did not affect the ligand binding. Carbohydrate moieties are somewhat evenly spaced throughout the molecule, at eight different N-glycosylation sites, some of which show heterogeneity in their compositions. Glycosylation seems necessary for stabilizing the molecule against disulfide-linked oligomerization of the receptor, indicating that the free cysteine residues become reactive for oxidation and disulfide exchange upon deglycosylation.

Amino Acid Sequence↗
PubMed1997

Effects of the intramolecular disulfide bond on ligand binding dynamics in myoglobin.

In order to investigate the effects of an intramolecular disulfide bond on protein structure and ligand binding dynamics in myoglobin, we prepared a mutant myoglobin having a disulfide bond at the EF corner by introducing two cysteine at the position of Ile 75 and Glu 85. On the basis of the spectral features of the mutant, the formation of the disulfide bond only affected minor structural deviations of the heme environmental structure in the carbonmonoxy form, whereas more substantial structural alterations were induced in the deoxygenated form. Laser photolysis experiments for carbon monoxide rebinding clearly showed that the artificial S-S bond accelerates the bimolecular rebinding rate from 1.0 to 1.8 microM-1 s-1 and increases the geminate yield from 0.072 to 0.092. The ligand migration rate from the solvent to the heme pocket and the bond formation rate from the heme pocket to the heme iron also increased. The free energy diagram for the mutants indicates that the energy barrier for the bond formation was raised as well as that for the ligand migration by introduction of the disulfide bond. However, the effects of the disulfide linkage at the EF corner on the kinetic parameter is much smaller than those of the amino acid substitutions located in the heme cavity. We can conclude that the perturbation of the protein fluctuations by formation of the disulfide bond would be localized at the mutation site or the contributions from other regions and motions might be more important for the ligand binding dynamics.

Binding Sites↗
PubMed1997

Determination of tumor necrosis factor binding protein disulfide structure: deviation of the fourth domain structure from the TNFR/NGFR family cysteine-rich region signature.

Tumor necrosis factor binding protein is a soluble molecule derived from the extracellular domain of the 55 kDa human tumor necrosis factor receptor, which can block the biological function of tumor necrosis factor by binding to the growth factor. This cysteine-rich molecule is subdivided into four domains, each containing six conserved cysteines that form three intrachain disulfide linkages known as the tumor necrosis factor receptor/nerve growth factor receptor family cysteine-rich region signature structure. In an effort to elucidate the molecular integrity of the molecule, we performed detailed analysis and searched for strategies to elucidate the complete disulfide structure of the E. coli-derived tumor necrosis factor binding protein and to determine the disulfide arrangement in the fourth domain of Chinese hamster ovary cell-derived molecule. The methods employed included various proteolytic digestions, peptide mapping, partial reduction, and assignment of disulfides by N-terminal sequencing and matrix-assisted laser desorption ionization mass spectrometry with post-source decay. The first three domains of the molecule were confirmed to have disulfide structures identical to the cysteine-rich region signature structure found in the above-mentioned receptor superfamily. The fourth domain has a different structure from the first three domains where the last four cysteines form two disulfide bonds in opposite positions.

Amino Acid Sequence↗
PubMed1998

Regeneration of bovine pancreatic ribonuclease A: identification of two nativelike three-disulfide intermediates involved in separate pathways.

During the regeneration of bovine pancreatic ribonuclease A (RNase A) from the reduced to the native form with mixtures of oxidized and reduced dithiothreitol at 25 degrees C, pH 8.0, the disulfide-containing protein intermediates achieve a steady-state distribution. By manipulating the redox conditions after the attainment of the steady-state condition, it has been possible to kinetically trap and, thereby, isolate and identify the disulfide-bonded species that follow the rate-determining step in the regeneration pathway. Two three-disulfide species have been identified by peptide mapping. Both species contain three native disulfide-bond pairings, one lacks the 65-72 disulfide bond (des-[65-72]), and the other lacks the 40-95 disulfide bond (des-[40-95]). These species are the same as those identified during the reduction of RNase A. By restarting the regeneration process from isolated des-[65-72] and des-[40-95], it is shown that both intermediates lie directly on regeneration pathways.

Acrylamides↗
PubMed1998

Role of the [Fe4S4] cluster in mediating disulfide reduction in spinach ferredoxin:thioredoxin reductase.

Thioredoxin reduction in plant chloroplasts is catalyzed by a unique class of disulfide reductases which use a one-electron donor, [Fe2S2]2+,+ ferredoxin, and has an active site involving a disulfide in close proximity to a [Fe4S4]2+ cluster. In this study, spinach ferredoxin:thioredoxin reductase (FTR) reduced with stoichiometric amounts of reduced benzyl viologen or frozen under turnover conditions in the presence of thioredoxin is shown to exhibit a slowly relaxing S = 1/2 resonance (g = 2.11, 2.00, 1.98) identical to that of a modified form of the enzyme in which one of the cysteines of the active-site disulfide is alkylated with N-ethylmaleimide (NEM-FTR). Hence, in accord with the previous proposal [Staples, C.R., Ameyibor, E., Fu, W., Gardet-Salvi, L., Stritt-Etter, A.-L., Schürmann, P., Knaff, D.B., and Johnson, M.K. (1996) Biochemistry 35, 11425-11434], NEM-FTR is shown to be a stable analogue of a one-electron-reduced enzymatic intermediate. The properties of the Fe-S cluster in NEM-FTR have been further investigated by resonance Raman and electron nuclear double resonance spectroscopies; the results, taken together with the previous UV-visible absorption, variable temperature magnetic circular dichroism, and resonance Raman data, indicate the presence of a novel type of [Fe4S4]3+ cluster that is coordinated by five cysteinates with little unpaired spin density delocalized onto the cluster-associated cysteine of the active-site disulfide. While the ligation site of the fifth cysteine remains undefined, the best candidate is a cluster bridging sulfide. On the basis of the spectroscopic and redox results, mechanistic schemes are proposed for the benzyl viologen-mediated two-electron-reduction of FTR and the catalytic mechanism of FTR. The catalytic mechanism involves novel S-based cluster chemistry to facilitate electron transfer to the active-site disulfide resulting in covalent attachment of the electron-transfer cysteine and generation of the free interchange cysteine that is required for the thiol-disulfide interchange reaction with thioredoxin.

Benzyl Viologen↗
PubMed1999

Comparison of local and global stability of an analogue of a disulfide-folding intermediate with those of the wild-type protein in bovine pancreatic ribonuclease A: identification of specific regions of stable structure along the oxidative folding pathway.

We have identified specific regions of the polypeptide chain of bovine pancreatic ribonuclease A (RNase A) that are critical for stabilizing the oxidative folding intermediate des-[40-95] (with three native disulfide bonds but lacking the fourth native Cys40-Cys95 disulfide bond) in an ensemble of largely disordered three-disulfide precursors (3S if des-[40-95]). A stable analogue of des-[40-95], viz., [C40A, C95A] RNase A, which contains three out of four native disulfide pairings, was previously found to have a three-dimensional structure very similar to that of the wild-type protein. However, it is determined here from GdnHCl denaturation experiments to have significantly reduced global stability, i.e., = 4.5 kcal /mol at 20 degrees C and pH 4.6. The local stability of [C40A, C95A] RNase A was also examined using site-specific amide (2)H/(1)H exchange measurements at pD 5.0 to determine the individual unfolding free energy of specific residues under both strongly native (12 degrees C) and more destabilizing (20 degrees C) conditions. Comparison of the relative stabilities at specific amide sites of [C40A, C95A] RNase A at both temperatures with the corresponding values for the wild-type protein at 35 degrees C corroborates previous experimental evidence that unidentified intramolecular contacts in the vicinity of the preferentially formed native one-disulfide (C65-C72) loop are crucial for stabilizing early folding intermediates, leading to des-[40-95]. Moreover, values of for residues at or near the third alpha-helix, and in part of the second beta-sheet of [C40A, C95A] RNase A, indicate that these two regions of regular backbone structure contribute to stabilizing the global chain fold of the des-[40-95] disulfide-folding intermediate in the wild-type protein. More significantly, we have identified numerous specific residues in the first alpha-helix and the first beta-sheet of the protein that are stabilized in the final step of the major oxidative regeneration pathway of RNase A (des-[40-95] --> N).

Amides↗