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PubMed2002

Complete disulfide bond assignment of a recombinant immunoglobulin G4 monoclonal antibody.

Recombinant monoclonal antibodies (mAbs) are an emerging therapeutic area. However, there are few reports on disulfide bond assignment of recombinant mAbs. This work describes the complete disulfide bond assignment of a recombinant immunoglobulin G4 (IgG4) mAb. N-ethylmaleimide (NEM) was used to mask free sulfhydryl groups present in the mAb. Digestion of the mAb with endoproteinase Lys-C without disulfide scrambling was achieved by denaturing the mAb in the presence of NEM in guanidine hydrochloride (GuHCl). The Lys-C digest was subsequently reduced with dithiothreitol (DTT). Native and reduced Lys-C digests were mass analyzed by on-line reversed-phase-high-performance liquid chromatography mass spectrometry (RP-HPLC/MS). Disulfide-containing peptides were sequenced by off-line nanoelectrospray quadrupole time-of-flight mass spectrometry (nanoESI-QTOF MS) and N-terminal Edman sequencing for verifying connectivities. The recombinant IgG4 mAb was found to contain the expected disulfide linkages with the proposed method. The NEM alkylating reagent was critical in minimizing disulfide scrambling during the denaturation and digestion of the mAb. This integrated approach, combining MS and N-terminal Edman sequencing, was capable of assigning the disulfide pattern of the IgG4 mAb rapidly and completely, and should be applicable for disulfide bond assignment and structural analysis of other mAbs and large proteins with multiple disulfide bonds.

Amino Acid Sequence↗

PubMed1989

Disulfide cross-linking of urea-insoluble proteins in rabbit lenses treated with hyperbaric oxygen.

In vivo exposure of human patients and experimental animals to hyperbaric O2 has been shown by other investigators to lead to opacification of the lens especially in the nuclear region. In the present study, cultured rabbit lenses were treated with hyperbaric O2 in order to investigate possible formation of disulfide-cross-linked proteins in the urea-insoluble fraction of lens cortex and nucleus. When lenses were treated with 100 atmospheres of 100% O2 for 24 hr. intermolecular disulfide-linked proteins formed in both the cortical and nuclear regions. Under these conditions the level of reduced glutathione and the activity of glyceraldehyde-3 phosphate dehydrogenase (G-3PD) were depleted by greater than 95% in both regions. The lenses were hazy in appearance but not opaque. Two-dimensional diagonal electrophoresis followed by immunoblotting indicated that the majority of the cross-linked proteins were beta- and gamma-crystallins. Also involved in the cross-linking was the enzyme G-3PD but not the main intrinsic membrane protein. MIP26 kDa. Treatment of the nuclear urea-insoluble fraction of O2-treated lenses with sodium borohydride showed a nearly fourfold increase in the level of protein disulfide compared to that present in the same fraction of either fresh lenses or N2-treated controls. It was determined that an increase of approximately one disulfide group per 10(5) Da molecular weight corresponded to cross-linking of nearly 20% of the urea-insoluble protein present in the O2-treated lenses. Experiments carried out at 8 atmospheres O2 were used to determine the region of the lens in which urea-insoluble disulfide first formed after exposure to O2. After 8 hr of treatment of lenses with 8 atmospheres O2 an increase in protein disulfide was observed in the urea-insoluble proteins of the lens nucleus but not of the cortex. Under these conditions, the level of glutathione had decreased by 62% in the nucleus compared to only 13% in the cortex. Increasing the culture time to 16 hr under 8 atmospheres O2 produced a further increase in protein disulfide in the nuclear region. The formation of a small amount of cross-linked protein in the cortex and a significantly greater decrease of G-3PD activity in the lens nucleus compared to the cortex. The overall results of the study demonstrate that exposure of lenses to hyperbaric O2 leads to disulfide-cross-linking of crystallins in the urea-insoluble fraction and that the initial formation of protein disulfide as well as the initial loss of glutathione occurs first in the lens nucleus.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1997

Synthesis, solution structure, binding activity, and cGMP activation of human guanylin and its disulfide isomer.

Guanylin is a recently isolated peptide consisting of 15 amino acid residues with four cysteines, which may form two intramolecular disulfide bridges, and stimulates intestinal membrane guanylate cyclase. The position of the disulfide linkages of guanylin was predicted from its structural similarity to a heat stable enterotoxin which is thought to be responsible for secretory diarrhoea. Both guanylin, with disulfide positions 4-12 and 7-15, and its disulfide isomer, with disulfides positions 4-15 and 7-12, were chemically synthesized by the solid-phase method and purified. Two specific disulfides were selectively formed and confirmed by sequencing, mass spectrometry and high-performance liquid chromatography in combination with enzymatic cleavage. The structure of both isomers has been investigated in solution by 1H nuclear magnetic resonance spectroscopy. Guanylin exists as a mixture of two stable conformations which have compact spiral structures, from comparison with literature data. In contrast, the disulfide isomer of guanylin shows only a single conformation with an elongated curved plate-like structure. Binding assays were performed using labelled guanylin with membranes obtained from rat jejunum. Both disulfide isomers were investigated by the cGMP assay. Both binding and cGMP assays indicated that the relevant form of disulfide bridges in the intact guanylin was as predicted.

Amino Acid Sequence↗

PubMed2003

Structural stability of human alpha-thrombin studied by disulfide reduction and scrambling.

Human alpha-thrombin is a very important plasma serine protease, which is involved in physiologically vital processes like hemostasis, thrombosis, and activation of platelets. Knowledge regarding the structural stability of alpha-thrombin is essential for understanding its biological regulation. Here, we investigated the structural and conformational stability of alpha-thrombin using the techniques of disulfide reduction and disulfide scrambling. alpha-Thrombin is composed of a light A-chain (36 residues) and a heavy B-chain (259 residues) linked covalently by an inter-chain disulfide bond (Cys(1)-Cys(122)). The B-chain is stabilized by three intra-chain disulfide bonds (Cys(42)-Cys(58), Cys(168)-Cys(182), and Cys(191)-Cys(220)) (Chymotrypsinogen nomenclature). Upon reduction with dithiothreitol (DTT), alpha-thrombin unfolded in a 'sequential' manner with sequential reduction of Cys(168)-Cys(182) within the B-chain followed by the inter-chain disulfide, generating two distinct partially reduced intermediates, I-1 and I-2, respectively. Conformational stability of alpha-thrombin was investigated by the technique of disulfide scrambling. alpha-Thrombin denatures by scrambling its native disulfide bonds in the presence of denaturant [urea, guanidine hydrochloride (GdmCl) or guanidine thiocyanate (GdmSCN)] and a thiol initiator. During the process, cleavage of the inter-chain disulfide bond and release of the A-chain from B-chain was the foremost event. The three disulfides in the B-chain subsequently scrambled to form three major isomers (designated as X-Ba, X-Bb, and X-Bc). Complete denaturation of alpha-thrombin was observed at low concentrations of denaturants (0.5 M GdmSCN, 1.5 M GdmCl, or 3 M urea) indicating low conformational stability of the protease.

Disulfides↗

PubMed1995

Disulfide bridges of a cysteine-rich repeat of the LDL receptor ligand-binding domain.

The low density lipoprotein (LDL) receptor is the prototype of a family of structurally related cell surface receptors that mediate the endocytosis of multiple ligands in mammalian cells. Its ligand-binding domain consists of seven cysteine-rich ligand-binding repeats, each approximately 40 amino acid residues long. Ligand-binding repeats occur in other members of the LDL receptor (LDLR) gene family and in a number of functionally unrelated proteins. As a first step toward an understanding of the structure and function of LB repeats, we have expressed the amino-terminal ligand-binding repeat (LB1) of the human LDLR as a recombinant peptide (rLB1) and have determined its disulfide-pairing scheme. Oxidative folding of rLB1 yielded a single isomer which contained three disulfide bonds. This isomer reacted with a conformation-specific monoclonal antibody (IgG-C7) made to LB1 in the native LDLR, suggesting that rLB1 was correctly folded. rLB1 was resistant to digestion with trypsin, chymotrypsin, and V8 protease, consistent with a tightly folded structure. Disulfide bond connections were established using two separate approaches. Digestion with the nonspecific proteolytic enzyme proteinase K yielded an 8 amino acid peptide with a single disulfide bond which connected Cys(IV) and Cys(VI). In the second approach, disulfide bonds were sequentially reduced with tris(2-carboxyethyl)phosphine and the resulting cysteine residues alkylated with iodoacetamide. An analysis of peptides which contained two cysteinylacetamide residues, derived from a single reduced disulfide bond, showed that Cys(I) and Cys(III) were disulfide-bonded and confirmed the presence of a disulfide bond between Cys(IV) and Cys(VI). We infer that the remaining disulfide bond bridges Cys(II) and Cys(V).(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗

PubMed1993

Disulfide arrangement and functional domains of beta-1,4-endoglucanse E5 from Thermomonospora fusca.

Thermomonospora fusca cellulase E5 contains six cysteine residues. The number and location of the disulfide bonds and the effect of reduction of the disulfides and modification of the resulting half-cystine residues on enzymatic activity were determined. No free sulfhydryl groups were found in E5. Reduction and subsequent labeling with iodoacetamide of E5 and of an enzymatically active 32-kDa proteolytic derivative of E5 (E5cd) showed that one of the three disulfides is accessible to reduction under nondenatured conditions while the other two are not accessible. Full reduction of the disulfides and complete carboxymethylation of the six cysteines decrease the specific activity of E5 on CMC by more than half, but reduction of only the exposed disulfide bond does not affect enzymatic activity or binding of E5 to cellulose. A 14-kDa proteolytic fragment of E5 containing 120 amino acids from the N-terminus of the protein was shown to bind to crystalline cellulose. This confirms earlier evidence that the cellulose binding domain of E5 is located at the N-terminus of the protein. This 14-kDa fragment contains the accessible disulfide bond involving Cys93 and Cys100. The location of the two disulfide bonds in the other fragment (E5cd) was determined by cleaving it with cyanogen bromide under conditions that left the disulfide bonds intact. The resulting peptides were separated under both nonreducing and reducing conditions using RP-HPLC. Amino acid analysis of peptide peaks indicated that one disulfide linkage in E5cd joins Cys138 to Cys143 while the other joins Cys166 to Cys406.

Actinomycetales↗

PubMed1993

Bacterial protein disulfide isomerase: efficient catalysis of oxidative protein folding at acidic pH.

Periplasmic protein disulfide isomerase (DsbA) is essential for disulfide formation in Escherichia coli. The tryptophan fluorescence of DsbA measures the redox state of the enzyme during catalysis of the oxidative folding of hirudin, a thrombin inhibitor containing three disulfide bonds and lacking tryptophan. With stoichiometric amounts of DsbA, reduced hirudin is rapidly oxidized in a process initially leading to random disulfides. Disulfide reshuffling involving reduced DsbA yields completely native inhibitor within 1 h, even at pH 4. Catalytic amounts of DsbA become essential for hirudin folding in the presence of redox buffers at acidic pH. The second-order rate constants of disulfide exchange reactions involving DsbA are several orders of magnitude above the known values for alkyl dithiols and disulfide oxidoreductases. DsbA preferably reacts with reduced, unfolded polypeptides. The reduction of DsbA by hirudin is faster by 1 order of magnitude than its reduction by the strong reductant dithiothreitol. Together, unusually fast disulfide interchange reactions and a preference for folding polypeptides appear to be responsible for the catalytic efficiency of DsbA and for disulfide formation in vivo at acidic pH.

Amino Acid Sequence↗

PubMed1992

Cooperative disulfide bond formation in apamin.

Apamin is being studied as a model for the folding mechanism of proteins whose structures are stabilized by disulfide bonds. Apamin consists of 18 amino acid residues and forms a stable structure consisting of a C-terminal alpha-helix and two reverse turns. This structure is stabilized by two disulfide bonds connecting Cys-1 to Cys-11 and Cys-3 to Cys-15. We used glutathione and dithiothreitol as reference thiols to measure the stabilities of the two disulfide bonds as a function of urea concentration and temperature in order to understand what contributes to the stability of the native structure. The results demonstrate modest contributions from secondary structure to the overall stability of the two disulfide bonds. The equilibrium constants for disulfide bond formation between the fully reduced peptide and the native structure with two disulfide bonds at 25 degrees C and pH 7.0 are 0.42 M2 using glutathione and 2.7 x 10(-5) using dithiothreitol. The equilibrium constant decreases by a factor of approximately 4 in 8 M urea and decreases by a factor of 3 between 0 and 60 degrees C. At least three one-disulfide intermediates are found at low concentrations in the equilibrium mixture. Using glutathione, the equilibrium constants for forming the one-disulfide intermediates with respect to the reduced peptide are approximately 0.025 M. The second disulfide bond forms with an equilibrium constant of approximately 17 M. Thus, apamin folding is very cooperative, but the native structure is only modestly stabilized by urea- or temperature-denaturable secondary structure.

Amino Acid Sequence↗

PubMed1994

Assignment of disulfide bond location in prothoracicotropic hormone of the silkworm, Bombyx mori: a homodimeric peptide.

The disulfide bond location of a homodimeric peptide, prothoracicotropic hormone (PTTH) of the silkworm, Bombyx mori, was determined by a combination of partial reduction and sequence analysis of peptide fragments generated through a partial reduction of PTTH followed by alkylation and enzyme digestion. The partial reduction and S-alkylation broke the interchain disulfide bond but did not affect the intrachain disulfide bonds, generating monomeric PTTH whose intrachain disulfide bonds were kept intact. This monomeric PTTH has about one-half the biological activity of intact PTTH. Sequence analysis of the fragments generated by lysyl endopeptidase digestion of this monomeric PTTH after complete reduction and S-alkylation by another S-alkylating reagent showed that only the Cys15 residue was reduced and S-alkylated by the foregoing partial reduction, indicating that this residue formed the interchain disulfide bond. The other disulfide bonds which formed intrachain bridgings were determined by sequence and mass analyses of the fragments generated by two successive enzyme digestions of the monomeric PTTH. In conclusion, the disulfide bond location of PTTH was assigned to Cys15-Cys15' as an interchain disulfide linkage and Cys17-Cys54, Cys40-Cys96, and Cys48-Cys98 as intrachain disulfide linkages.

Amino Acid Sequence↗

PubMed1989

Protein engineering of disulfide bonds in subtilisin BPN'.

Five single-disulfide mutants were studied in subtilisin BPN', a cysteine-free, secreted serine protease from Bacillus amyloliquefaciens. The disulfides were engineered between residues 26-232, 29-119, 36-210, 41-80, and 148-243. These bonds connected a variety of secondary structural elements, located in buried or exposed positions at least 10 A from the catalytic Ser-221, and linked residues that were separated by 39 up to 206 amino acids. All disulfide bonds formed in the enzyme when the expressed protein was secreted from Bacillus subtilis, and the disulfides had only minor effects on the enzyme kinetics. Although these disulfide bonds varied by over 50-fold in their equilibrium constants for reduction with dithiothreitol, there was no correlation between the strength of the disulfide bond and the stability it imparted to the enzyme to irreversible inactivation. In some cases, the disulfide-bonded protein was stabilized greatly relative to its reduced counterpart. However, no disulfide mutant was substantially more stable than wild-type subtilisin BPN'. Some of these results can be rationalized by destabilizing effects of the cysteine mutations that disrupt interactions present in the folded enzyme structure. It is also possible that the rate of irreversible inactivation depends upon the kinetics and not the thermodynamics of unfolding and so the entropically stabilizing effect expected from a disulfide bond may not apply.

Bacillus↗

PubMed2002

Pathway of oxidative folding of alpha-lactalbumin: a model for illustrating the diversity of disulfide folding pathways.

The pathway of oxidative folding of alpha-lactalbumin (alpha LA) (four disulfide bonds) has been characterized by structural and kinetic analysis of the acid-trapped folding intermediates. In the absence of calcium, oxidative folding of alpha LA proceeds through highly heterogeneous species of one-, two-, three-, and four-disulfide (scrambled) intermediates to reach the native structure. In the presence of calcium, the folding intermediates of alpha LA comprise two predominant isomers (alpha LA-IIA and alpha LA-IIIA) adopting exclusively native disulfide bonds, including the two disulfide bonds (Cys(61)-Cys(77) and Cys(73)-Cys(91)) located within the beta-sheet calcium binding domain. alpha LA-IIA is a two-disulfide species consisting of Cys(61)-Cys(77) and Cys(73)-Cys(91) disulfide bonds. alpha LA-IIIA contains Cys(61)-Cys(77), Cys(73)-Cys(91), and Cys(28)-Cys(111) disulfide bonds. The underlying mechanism of the contrasting folding pathways of calcium-bound and calcium-depleted alpha LA is congruent with the cause of diversity of disulfide folding pathways observed among many well-characterized three-disulfide proteins, including bovine pancreatic trypsin inhibitor and hirudin. Our study also reveals novel aspects of the folding mechanism of alpha LA that have not been described previously.

Animals↗

PubMed2003

Shifting the competition between the intramolecular Reshuffling reaction and the direct oxidation reaction during the oxidative folding of kinetically trapped disulfide-insecure intermediates.

The oxidative folding pathway(s) of single-domain proteins can be characterized by the existence, stability, and structural nature of the intermediates that populate the regeneration pathway. Structured intermediates can be disulfide-secure in that they are able to protect their existing (native) disulfide bonds from SH/SS reshuffling and reduction reactions, and thereby form the native protein directly, i.e., by oxidation of their exposed (or locally exposable) thiols. Alternatively, they can be disulfide-insecure, usually requiring global unfolding to expose their free thiols. However, such an unfolding event also exposes the existing native disulfide bonds. Thus, the subsequent oxidation reaction to form the native protein in a disulfide-insecure intermediate competes with the intramolecular attack by the thiols of the macromolecule on its own native disulfide bonds, resulting in a large population of intermediates that are reshuffled instead of being oxidized. Under stabilizing conditions, disulfide-insecure species become long-lived kinetically trapped intermediates that slowly and only indirectly convert to the native protein through reshuffling reactions. In this study, trans-[Pt(en)(2)Cl(2)](2+), a strong oxidizing agent which has not traditionally been used in oxidative folding, was applied to shift the competition between reshuffling and oxidation reactions in des [58-110] and des [26-84], two long-lived disulfide-insecure intermediates of RNase A, and oxidize them directly under stable conditions to form the native protein. Such a successful direct conversion of kinetically trapped intermediates to the native molecule by trans-[Pt(en)(2)Cl(2)](2+) may be helpful in facilitating the oxidative folding processes of multi-disulfide-containing proteins in general.

Animals↗

PubMed2004

Disulfide bond rearrangement during formation of the chorionic gonadotropin beta-subunit cystine knot in vivo.

The intracellular kinetic folding pathway of the human chorionic gonadotropin beta-subunit (hCG-beta) reveals the presence of a disulfide between Cys residues 38-57 that is not detected by X-ray analysis of secreted hCG-beta. This led us to propose that disulfide rearrangement is an essential feature of cystine knot formation during CG-beta folding. To test this, we used disulfide bond formation to monitor progression of intracellular folding intermediates of a previously uncharacterized protein, the CG-beta subunit of cynomolgous macaque (Macaca fascicularis). Like its human counterpart hCG-beta with which it shares 81% identity, macaque (m)CG-beta is a cystine knot-containing subunit that assembles with an alpha-subunit common to all glycoprotein hormone members of its species to form a biologically active heterodimer, mCG, which, like hCG, is required for pregnancy maintenance. An early mCG-beta folding intermediate, mpbeta1, contained two disulfide bonds, one between Cys34 and Cys88 and the other between Cys38 and Cys57. The subsequent folding intermediate, mpbeta2-early, was represented by an ensemble of folding forms that, in addition to the two disulfides mentioned above, included disulfide linkages between Cys9 and Cys57 and between Cys38 and Cys90. These latter two disulfides are those contained within the beta-subunit cystine knot and reveal that a disulfide exchange occurred during the mpbeta2-early folding step leading to formation of the mCG-beta knot. Thus, while defining the intracellular kinetic protein folding pathway of a monkey homologue of CG-beta, we detected the previously predicted disulfide exchange event crucial for CG-beta cystine knot formation and attainment of CG-beta assembly competence.

Alkylation↗

PubMed2006

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

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

Amino Acid Sequence↗

PubMed1996

Flavin-dependent alkyl hydroperoxide reductase from Salmonella typhimurium. 2. Cystine disulfides involved in catalysis of peroxide reduction.

The two-component alkyl hydroperoxide reductase enzyme system from Salmonella typhimurium catalyzes the pyridine nucleotide-dependent reduction of alkyl hydroperoxide and hydrogen peroxide substrates. This system is composed of a flavoenzyme, AhpF, which is related to the disulfide-reducing enzyme thioredoxin reductase, and a smaller protein, AhpC, which lacks a chromophoric cofactor. We have demonstrated that NADH-linked reduction of AhpF under anaerobic conditions converts two cystine disulfide centers to their dithiol forms. The AhpC cystine disulfide center, shown to exist as an intersubunit disulfide bond, is stoichiometrically reducible by NADH in the presence of a catalytic amount of AhpF and can be reoxidized by ethyl hydroperoxide. Disulfide bridges within oxidized AhpF form between Cys129 and Cys132 and between Cys345 and Cys348; the two C-terminal half-cystine residues, Cys476 and Cys489, exist as free thiol groups in oxidized AhpF and play no role in catalysis. Removal of the N-terminal 202-amino acid segment containing the Cys129-Cys132 disulfide center obliterates the ability of AhpF to transfer electrons to 5,5'-dthiobis(2-nitrobenzoic acid) (DTNB) and AhpC. NADH added anaerobically to AhpF causes spectral changes consistent with preferential reduction of both disulfides relative to flavin reduction; the reduction potentials of the disulfide centers are thus appropriately poised for electron transfer from NADH and flavin to disulfide-containing substrates (AhpC or DTNB), and ultimately to peroxides from AhpC. Blue, neutral flavin semiquinone is also generated in high yields during reductive titrations (91% yield during dithionite titrations), although the relatively slow formation of this species indicates its catalytic incompetence. A long wavelength absorbance band beyond 900 nm attributable to an FADH2-->NAD+ charge transfer interaction is generated during NADH, but not dithionite, titrations and may be indicative of a species directly involved in the catalytic cycle. A catalytic mechanism including the transient formation of cysteine sulfenic acid within AhpC is proposed.

Amino Acid Sequence↗

PubMed1999

Engineering out motion: a surface disulfide bond alters the mobility of tryptophan 22 in cytochrome b5 as probed by time-resolved fluorescence and 1H NMR experiments.

In the accompanying paper [Storch et al. (1999) Biochemistry 38, 5054-5064] equilibrium denaturation studies and molecular dynamics (MD) simulations were used to investigate localized dynamics on the surface of cytochrome b5 (cyt b5) that result in the formation of a cleft. In those studies, an S18C:R47C disulfide mutant was engineered to inhibit cleft mobility. Temperature- and urea-induced denaturation studies revealed significant differences in Trp 22 fluorescence between the wild-type and mutant proteins. On the basis of the results, it was proposed that wild type populates a conformational ensemble that is unavailable to the disulfide mutant and is mediated by cleft mobility. As a result, the solvent accessibility of Trp 22 is decreased in S18C:R47C, suggesting that the local environment of this residue is less mobile due to the constraining effects of the disulfide on cleft dynamics. To further probe the structural effects on the local environment of Trp 22 caused by inhibition of cleft formation, we report here the results of steady-state and time-resolved fluorescence quenching, differential phase/modulation fluorescence anisotropy, and 1H NMR studies. In Trp fluorescence experiments, the Stern-Volmer quenching constant increases in wild type versus the oxidized disulfide mutant with increasing temperature. At 50 degrees C, KSV is nearly 1.5-fold greater in wild type compared to the oxidized disulfide mutant. In the reduced disulfide mutant, KSV was the same as wild type. The bimolecular collisional quenching constant, kq, for acrylamide quenching of Trp 22 increases 2.7-fold for wild type and only 1.8-fold for S18C:R47C, upon increasing the temperature from 25 to 50 degrees C. The time-resolved anisotropy decay at 25 degrees C was fit to a double-exponential decay for both the wild type and S18C:R47C. Both proteins exhibited a minor contribution from a low-amplitude fast decay, consistent with local motion of Trp 22. This component was more prevalent in the wild type, and the fractional contribution increased significantly upon raising the temperature. The fast rotational component of the S18C:R47C mutant was less sensitive to increasing temperature. A comparison of the 1H NMR monitored temperature titration of the delta-methyl protons of Ile 76 for wild type and oxidized disulfide mutant, S18C:R47C, showed a significantly smaller downfield shift for the mutant protein, suggesting that Trp 22 in the mutant protein experiences comparatively decreased cleft dynamics in core 2 at higher temperatures. Furthermore, comparison of the delta'-methyl protons of Leu 25 in the two proteins revealed a difference in the ratio of the equilibrium heme conformers of 1.2:1 for S18C:R47C versus 1.5:1 for wild type at 40 degrees C. The difference in equilibrium heme orientations between wild type and S18C:R47C suggests that the disulfide bond affects heme binding within core 1, possibly through damped cleft fluctuations. Taken together, the NMR and fluorescence studies support the proposal that an engineered disulfide bond inhibits the formation of a dynamic cleft on the surface of cyt b5.

Amino Acid Substitution↗

PubMed2000

Disulfide bonds and protein folding.

The applications of disulfide-bond chemistry to studies of protein folding, structure, and stability are reviewed and illustrated with bovine pancreatic ribonuclease A (RNase A). After surveying the general properties and advantages of disulfide-bond studies, we illustrate the mechanism of reductive unfolding with RNase A, and discuss its application to probing structural fluctuations in folded proteins. The oxidative folding of RNase A is then described, focusing on the role of structure formation in the regeneration of the native disulfide bonds. The development of structure and conformational order in the disulfide intermediates during oxidative folding is characterized. Partially folded disulfide species are not observed, indicating that disulfide-coupled folding is highly cooperative. Contrary to the predictions of "rugged funnel" models of protein folding, misfolded disulfide species are also not observed despite the potentially stabilizing effect of many nonnative disulfide bonds. The mechanism of regenerating the native disulfide bonds suggests an analogous scenario for conformational folding. Finally, engineered covalent cross-links may be used to assay for the association of protein segments in the folding transition state, as illustrated with RNase A.

Animals↗

PubMed2002

Formation of transitory intrachain and interchain disulfide bonds accompanies the folding and oligomerization of simian virus 40 Vp1 in the cytoplasm.

Pentamer formation by Vp1, the major capsid protein of simian virus 40, requires an interdigitation of structural elements from the Vp1 monomers [Liddington, R. C., Yan, Y., Moulai, J., Sahli, R., Benjamin, T. L. & Harrison, S. C. (1991) Nature (London) 354, 278-284]. Our analyses reveal that disulfide-linked Vp1 homooligomers are present in the simian virus 40-infected cytoplasm and that they are derived from a 41-kDa monomeric intermediate containing an intrachain disulfide bond(s). The 41-kDa species, emerging within 5 min of pulse labeling with [(35)S]methionine, is converted into a 45-kDa, disulfide-free Vp1 monomer and disulfide-bonded dimers through pentamers. The covalent oligomer formation is blocked in the presence of a sulfhydryl-modifying reagent. We propose that there are two stages in this Vp1 disulfide bonding. First, the newly synthesized Vp1 monomers acquire intrachain bonds as they fold and begin to interact. Next, these bonds are replaced with intermolecular bonds as the monomers assemble into pentamers. This sequential appearance of transitory disulfide bonds is consistent with a role for sulfhydryl-disulfide redox reactions in the coordinate folding of Vp1 chains into pentamers. The cytoplasmic Vp1 does not colocalize with marker proteins of the endoplasmic reticulum. This paper demonstrates in vivo disulfide formations and exchanges coupled to the folding and oligomerization of a mammalian protein in the cytoplasm, outside the secretory pathway. Such disulfide dynamics may be a general phenomenon for other cysteine-bearing mammalian proteins that fold in the cytoplasm.

Animals↗