Biomedical subjects

P M Horowitz

Publications and source records attributed to P M Horowitz.

At least 37 records · Page 2Linked to original sources

PubMed1998

Photoincorporation of fluorescent probe into GroEL: defining site of interaction.

We have elucidated conditions for the covalent incorporation of a nonspecific hydrophobic probe, bisANS, into various proteins. Using this method, we are able to map hydrophobic surfaces in proteins. In addition, we have shown that for GroEL, we are able to use the fluorescence of the incorporated bisANS to monitor conformational changes in a defined region of the protein in response to various effectors. This method should be useful for studying both protein structure and dynamics.

Affinity Labels↗

PubMed1997

Conditions for nucleotide-dependent GroES-GroEL interactions. GroEL14(groES7)2 is favored by an asymmetric distribution of nucleotides.

A still unresolved question regarding the mechanism of chaperonin-assisted protein folding involves the stoichiometry of the GroEL-GroES complex. This is important, because the activities of the Escherichia coli chaperonin GroEL are modulated by the cochaperonin GroES. In this report, the binding of GroES to highly purified GroEL in the presence of ATP, ADP, and the nonhydrolyzable ATP analogue, 5'-adenylyl beta,gamma-imidodiphosphate (AMP-PNP), was investigated by using the fluorescence anisotropy of succinimidyl-1-pyrenebutyrate-labeled GroES. In the presence of Mg2+-ATP and high [KCl] (10 mM), two GroES7 rings bind per one GroEL14. In contrast, in the presence of ADP or AMP-PNP only one molecule of oligomeric GroES can be tightly bound by GroEL. With AMP-PNP, binding of a small amount (<20%) of a second GroES can be detected. In the presence of ADP alone, a second GroES ring can bind to GroEL weakly and with negative cooperativity. Strikingly, addition of AMP-PNP to the solution containing preformed GroEL14(GroES7) complexes formed in the presence of ADP results in an increase in the fluorescence anisotropy. Analysis of this effect indicates that 2 mol of GroES oligomer can be bound in the presence of mixed nucleotides. A similar conclusion follows from studies in which ADP is added to an GroEL14 (GroES7) complex formed in the presence of AMP-PNP. This is the first demonstration of an asymmetric distribution of nucleotides bound on the 1:2 GroEL14 (GroES7)2 complex. The relation of the observed phenomena to the proposed mechanism of the GroEL function is discussed.

Adenine Nucleotides↗

PubMed1997

ATP hydrolysis is critical for induction of conformational changes in GroEL that expose hydrophobic surfaces.

The degree of hydrophobic exposure in the molecular chaperone GroEL during its cycle of ATP hydrolysis was analyzed using 1,1'-bis(4-anilino)naphthalene-5,5'disulfonic acid (bisANS), a hydrophobic probe, whose fluorescence is highly sensitive to the environment. In the presence of 10 mM MgCl2 and 10 mM KCl the addition of ATP, but not ADP or AMP-PNP, resulted in a time-dependent, linear increase in the bisANS fluorescence. The rate of the increase in the bisANS fluorescence depended on the concentrations of both GroEL and the probe. The effect could be substantially inhibited by addition of excess ADP or by converting ATP to ADP using hexokinase, showing that the increase in the bisANS fluorescence was correlated with ATP hydrolysis. The rate of ATP hydrolysis catalyzed by GroEL was uncompetitively inhibited in the presence of bisANS. This uncompetitive inhibition suggests that the probe can interact with the GroEL-ATP complex. The inability of the nonhydrolyzable ATP analog, AMP-PNP, to cause a similar effect is explained by the interaction of bisANS with a transient conformational state of GroEL formed consequent to ATP hydrolysis. It is suggested that this short lived hydrophobic exposure reflects a conformational shift in GroEL that results from electrostatic repulsion between the bound products of ATP hydrolysis, and it plays an important role in the mechanism of the chaperonin cycle.

Adenosine Triphosphate↗

PubMed1997

Model peptide studies demonstrate that amphipathic secondary structures can be recognized by the chaperonin GroEL (cpn60).

The molecular chaperone cpn60 binds many unfolded proteins and facilitates their proper folding. Synthetic peptides have been used to probe the question of how cpn60 might recognize such a diverse set of unfolded proteins. Three hybrid peptides were synthesized encompassing portions of the bee venom peptide, apamin, and the sequence KWLAESVRAGK from an amphipathic helix in the NH2-terminal region of bovine rhodanese. Two disulfides connecting cysteine residues hold the peptides in stable helical conformations with unobstructed faces oriented away from the disulfides. Peptides were designed to present either a hydrophobic or hydrophilic face of the amphipathic helix that is similar to the one near the amino terminus of rhodanese. Aggregation of these peptides was detected by measuring 1,1'-bis(4-anilino)napthalene-5,5'-disulfonic acid (bisANS) fluorescence at increasing peptide concentrations, and aggregation was not apparent below 2 microM. Thus, all experiments with the peptides were performed at a concentration of 1 microM. Reducing agents cause these helical peptides to form random coils. Fluorescence anisotropy measurements of fluorescein-labeled peptide with the exposed hydrophobic face yielded a Kd = approximately 106 microM for binding to cpn60, whereas there was no detectable binding of the reduced form. The peptide with the exposed hydrophilic face did not bind to cpn60 in either the oxidized or reduced states. Fluorescence experiments utilizing bisANS as a probe showed that binding of the helical hydrophobic peptide could induce the exposure of hydrophobic surfaces on cpn60, whereas the same peptide in its random coil form had no effect. Thus, binding to cpn60 is favored by a secondary structure that organizes and exposes a hydrophobic surface, a feature found in amphipathic helices. Further, the binding of a hydrophobic surface to cpn60 can induce further exposure of complementary surfaces on cpn60 complexes, thus amplifying interactions available for target proteins.

Amino Acid Sequence↗

PubMed1997

Conditions of forming protein complexes with GroEL can influence the mechanism of chaperonin-assisted refolding.

The interaction of GroEL with urea-unfolded dihydrofolate reductase (DHFR) has been studied in the presence of DHFR substrates by investigating the ability of GroES to release enzyme under conditions where a stable GroES-GroEL-DHFR ternary complex can be formed. In these circumstances, GroES could only partially discharge the DHFR if ADP was present in the solution and approximately half of the DHFR remained bound on the chaperonin. This bound DHFR could be rescued by addition of ATP and KCl into the refolding mixture. The stable ternary complex did not show any significant protection of bound DHFR against proteolysis by Proteinase K. These results are in contrast to those observed with the GroEL-DHFR complex formed by thermal inactivation of DHFR at 45 degrees C in which GroES addition leads to partial protection of bound DHFR. Thus, the method of presentation influences the properties of the bound intermediates. It is suggested that the ability of GroES to bind on the same side of the GroEL double toroid as the target protein and displace it into the central cavity depends on the way the protein-substrate is presented to the GroEL molecule. Therefore, the compact folding intermediate formed by thermal unfolding can be protected against proteolysis after GroES binds to form a ternary complex. In addition, structural changes within GroEL induced by the experimental conditions may contribute to differences in the properties of the complexes. The more open urea-unfolded DHFR binds on the surface of chaperonin and can be displaced into solution by the tighter binding GroES molecule. It is suggested that the state of the unfolded protein when it is presented to GroEL determines the detailed mechanism of its assisted refolding. It follows that individual proteins, having characteristic folding intermediates, can have different detailed mechanisms of chaperonin-assisted folding.

Adenosine Triphosphate↗

PubMed1997

The influence of chaotropic reagents on neuronal nitric oxide synthase and its flavoprotein module. Urea and guanidine hydrochloride stimulate NADPH-cytochrome c reductase activity of both proteins.

Changes in flavin and protein fluorescence of neuronal nitric oxide synthase (nNOS) and its flavoprotein module were studied in the presence of urea and compared with those previously reported for cytochrome P450 reductase (CPR) [R. Narayanasami, P. M. Horowitz, and B. S. S. Masters (1995) Arch. Biochem. Biophys. 316, 267-274]. As in the case of CPR, FMN was relatively loosely bound to nNOS and the flavoprotein module, but FAD remained bound at concentrations of up to 2 M urea Protein fluorescence increased progressively with increasing urea concentration, but could not be correlated with changes in flavin binding. NADPH-cytochrome c reductase activity of both nNOS and the flavoprotein module, but not that of CPR, was stimulated at early time points by both urea and guanidine hydrochloride (GnHCl), with levels of initial activity returning to baseline values within 60 min after addition of the chaotropic agent. Thus, at 3-4 M urea, enhancements of reductase activities of 20- and 5-fold with nNOS and the flavoprotein module, respectively, were obtained. Comparable enhancements of 12- and 6- to 7-fold, respectively, were obtained with calmodulin (CaM)/ CaCl2 and 0.5 M GnHCl. Thus, the effects of urea and GnHCl mimicked the stimulating effects of CaM. Separate preincubations of nNOS and cytochrome c with urea or GnHCl prior to initiation of the reductase assay showed that sensitivity to chaotropic agent under these conditions was a property of nNOS and not of cytochrome c. Moreover, when the nonprotein electron acceptor 2,6-dichlorophenolindophenol was employed in place of cytochrome c, comparable stimulation of reductase activity was observed in the presence of either urea or GnHCl. Fluorescence of 4,4'-dianilino-1,1'-binaphthyl-5,5'-disulfate in the presence of either nNOS or the flavoprotein module was increased optimally between 3 and 4 M urea, consistent with simultaneous exposure of hydrophobic regions of both proteins to solvent and optimization of reductase activity. FMN release from nNOS, but not from the flavoprotein module, was enhanced by CaM. Addition of FMN or FMN + FAD to nNOS, in the presence or absence of urea, brought about a doubling of initial cytochrome c reductase activity, but did not prevent the eventual decline in activity to basal levels. These data are consistent with conformational changes which favor increased electron transfer similar to that achieved with nNOS in the presence of CaM.

Base Sequence↗

PubMed1997

Preformed GroES oligomers are not required as functional cochaperonins.

We have previously shown that the C-terminal sequence of GroES is required for oligomerization [Seale and Horowitz (1995), J. Biol. Chem. 270, 30268-30270]. In this report, we have generated a C-terminal deletion mutant of GroES with a significantly destabilized oligomer and have investigated its function in the chaperonin-assisted protein folding cycle. Removal of the two C-terminal residues of GroES results in a cochaperonin [GroESD(96-97)] that is monomeric at concentrations where GroES function is assessed. Using equilibrium ultracentrifugation, we measured the dissociation constant for the oligomer-monomer equilibrium to be 7.3 x 10(-34)M6. The GroESD(96-97) is fully active as a cochaperonin. This mutant is able to inhibit the ATPase activity of GroEL to levels comparable to wild-type GroES. It is also able to assist the refolding of urea-denatured rhodanese by GroEL. While GroESD(96-97) can function at levels comparable to wild-type GroES, higher concentrations of mutant are required to produce the same effect. These results support the idea that the performed GroES heptamer is not required for function, but they suggest that the oligomeric cochaperonin is most efficient.

Adenosine Triphosphatases↗

PubMed1996

Intrinsic fluorescence studies of the chaperonin GroEL containing single Tyr --> Trp replacements reveal ligand-induced conformational changes.

Two mutants of GroEL containing the single tyrosine to tryptophan replacement of either residue 203 or 360 in the apical domain have been purified, characterized, and used for fluorescence studies. Both mutants can facilitate the in vitro refolding of rhodanese in an ATP- and GroES-dependent manner, producing yields of recoverable activity comparable to the wild-type chaperonin. Y203W shows some increased hydrophobic exposure and easier urea-induced disassembly compared with wild-type or Y360W, although the unfolding of all the species was similar at high concentrations of urea. Intrinsic fluorescence studies of the two mutants reveal that nucleotide binding (ADP or AMP-PNP (adenosine 5'-(beta,gamma-imino)triphosphate)) induces conformational changes in the tetradecamer that are independent of the presence of the co-chaperonin, GroES. The K1/2 for this transition is approximately 5 microM for both mutants. Energy transfer experiments show that the tryptophan fluorescence of the Y360W mutant is partially quenched ( approximately 50%) upon binding of the fluorescent, hydrophobic probe 4,4'-bis(1-anilino-8-naphthalenesulfonic acid), while the fluorescence of the Y203W mutant is significantly quenched ( approximately 75%). These results are discussed in relation to the molecular mechanism for GroEL function.

Adenosine Diphosphate↗

PubMed1996

Nucleotides reveal polynucleotide phosphorylase activity from conventionally purified GroEL.

GroEL, as conventionally purified, can be incubated with nucleotides to produce high molecular weight material with an absorption maximum at 260 nm. This material is most clearly demonstrated when samples are subjected to gel filtration under conditions where GroEL is monomeric. There is a time-dependent increase in the high molecular weight material that occurs on incubation with ADP or, more slowly, with ATP. This material is generated during incubation, and none is present in the initial samples. Experiments with nucleases, proteases, radiolabeled nucleotides, and chemical cleavage reagents demonstrate that the high molecular weight material is polyadenylic acid whose formation is inhibited by phosphate. These results are consistent with the GroEL samples containing polynucleotide phosphorylase activity. Nondenaturing gels stained with acridine orange, after incubation in ADP, reveal that the activity producing the poly(A) coelectrophoreses with authentic polynucleotide phosphorylase. Conditions that remove the tryptophan-like fluorescence from preparations of GroEL also remove the PNPase activity. Thus, this activity is not associated with GroEL itself. The results are consistent with reports that GroEL can associate with RNase E and with other studies showing that RNase E and PNPase can form complexes. Thus, the present experiments support suggestions that GroEL can participate in multiprotein complexes that are involved in mRNA processing and degradation.

Adenosine Diphosphate↗

PubMed1996

Reversible oligomerization and denaturation of the chaperonin GroES.

The chaperonin GroEL can assist protein folding and normally acts with the co-chaperonin GroES. These Escherichia coli proteins are encoded on the same operon, with GroES positioned first. In this report, we have investigated the reversible folding of GroES. Using fluorescence anisotropy of dansyl-labeled GroES, intrinsic fluorescence, bis-ANS binding, sedimentation velocity, and limited proteolysis, we show that GroES unfolds in a single, two-state transition. Importantly, intrinsic fluorescence and sedimentation velocity analyses show that GroES is capable of refolding and reassembling from a urea denatured state. The refolded GroES is fully active as shown by its ability to assist GroEL in the refolding of rhodanese. These results indicate that chaperonins may not require other chaperonins for successful folding/assembly. We also show that GroES is capable of assisting in the refolding/reassembly of fully denatured GroEL. The reversible folding of GroES coupled with the ability of GroES to assist the refolding/reassembly of GroEL suggest that the groE operon may be organized in a manner that provides a structural role in GroES/GroEL assembly as well as a functional role.

Chaperonin 10↗

PubMed1996

Ligand-induced conformational changes in the apical domain of the chaperonin GroEL.

Although the role of nucleotides in the catalytic cycle of the GroESL chaperonin system has been extensively studied, the molecular effects of nucleotides in modulating exposure of sites on GroEL has not been thoroughly investigated. We report here that nucleotides (ATP, ADP, or adenosine 5'-(beta, gamma-imino)triphosphate) in the presence of Mg2+ make the oligomer selectively sensitive to trypsin proteolysis in a fashion suggesting conformational changes in the monomers of one heptameric ring. The site of proteolysis in the monomer that is exposed upon nucleotide binding by the oligomer is in the apical domain (Arg-268). Further, complexes of GroEL with GroES or rhodanese display the same sensitivity to proteolysis, unlike the GroEL-GroES-rhodanese complex, which is protected from proteolysis. The influence of various cations on trypsin proteolysis is investigated to elucidate the differential effects that monovalent and divalent cations have on the oligomeric structure of the chaperonin. These results are discussed in relation to the molecular basis for the chaperonin activity.

Adenine Nucleotides↗

PubMed1995

The C-terminal sequence of the chaperonin GroES is required for oligomerization.

The Escherichia coli protein GroES is a co-chaperonin that is able to assist GroEL in the refolding of proteins. GroES is a heptamer of seven identical subunits. Recent work has focused on the structural aspects of GroES. We have investigated the role of the C-terminal portion of GroES on its oligomerization. Limited proteolysis of GroES by carboxypeptidase Y gives a product in which the C-terminal 7 amino acid residues have been removed. Sedimentation velocity analysis reveals that the truncated form of GroES is unable to reassemble. The results presented here implicate the C-terminal sequence in intermonomer actions within the GroES oligomer. In addition, this work provides experimental verification of predictions implied in the recent x-ray determination of the GroES structure (Hunt, J. F., Weaver, A. J., Landry, S. J., Gierasch, L. M., and Deisenhofer, J. Nature, in press).

Amino Acid Sequence↗

PubMed1995

The chaperonin GroEL is destabilized by binding of ADP.

The urea-induced dissociation and subsequent conformational transitions of the nucleotide-bound form of GroEL were studied by light scattering, 4,4'-bis(1-anilino-8- naphthalenesulfonic acid) binding, and intrinsic tyrosine fluorescence. Magnesium ion alone (10 mM) stabilizes GroEL and leads to coordination of the structural transitions monitored by the different parameters. The midpoint of the light-scattering transition that monitored dissociation of the 14-mer with bound magnesium was raised to approximately 3 M, which is considerably higher than the ligand-free form of the protein, which exhibits a transition with a midpoint at approximately 2 M urea. Binding of ADP results in destabilization of the GroEL oligomeric structure, and complete dissociation of the 14-mer in the presence of 5 mM ADP occurs at about 2 M urea with the midpoint of the transition at approximately 1 M urea. The same destabilization by ADP and stabilization by Mg2+ were seen when the conformation was followed by the intrinsic fluorescence. Complexation with the nonhydrolyzable ATP analog, 5'-adenylimidodiphosphate gave an apparent stability of the quaternary structure that was between that observed with Mg2+ and that with ADP. The ADP-bound form of the protein demonstrated increased hydrophobic exposure at lower urea concentrations than the uncomplexed GroEL. In addition, the GroEL-ADP complex is more accessible for proteolytic digestion by chymotrypsin than the uncomplexed protein, consistent with a more open, flexible form of the protein. The implication of the conformational changes to the mechanism of the GroEL function is discussed.

Adenosine Diphosphate↗

PubMed1995

Residual structure in urea-denatured chaperonin GroEL.

The urea denaturation of the chaperonin GroEL has been studied by circular dichroism, intrinsic tyrosine fluorescence and fluorescence of the hydrophobic probe, 1,1'-bis(4-anilino)naphthalene-5,5'-disulfonic acid (bisANS). It is shown that GroEL denaturation, monitored by CD and intrinsic fluorescence measurements, can be well described by a two-state transition that is complete by 3-3.1 M urea. The beginning of this transition overlaps the urea concentrations where the oligomeric protein starts to dissociate into individual monomers. Subsequent addition of the denaturant leads to complete unfolding of the monomers. Monomers unfolded at urea concentrations higher than 3.1 M are not competent to form their native conformations under the conditions employed here, and they are not able to reassemble to oligomers upon dilution of urea. In contrast to the CD and intrinsic fluorescence measurements, bisANS bound to GroEL exhibits considerable fluorescence intensity under conditions where the CD and intrinsic fluorescence signals have already reached their minimum values (> 3.1 M urea). This binding of bisANS, under conditions where the majority of the secondary structure of GroEL has already unfolded, indicates the existence of hydrophobic residual structure. This structure cannot be detected by CD measurements, but it can be unfolded by raising further the urea concentration. The existence of this structure does not depend on the source or method of the protein preparation. Intrinsic fluorescence and trypsin digestion demonstrate no difference between the bisANS-bound form of GroEL and the free form of the protein, showing that the GroEL structure is not greatly affected by the interaction with bisANS.(ABSTRACT TRUNCATED AT 250 WORDS)

Anilino Naphthalenesulfonates↗

PubMed1995

Inactive GroEL monomers can be isolated and reassembled to functional tetradecamers that contain few bound peptides.

For the first time, it has been shown that GroEL can be converted from tetradecamers (14-mers) to monomers under conditions commonly used for the preparation of this chaperonin. The essential requirements are the simultaneous presence of nucleotides such as MgATP or MgADP and a solid-phase anion-exchange medium. The monomers that are formed are metastable in that they only reassemble to a small degree in the absence of additives. These results are in keeping with previous studies on high pressure dissociation that showed the separated monomers display conformational plasticity and can undergo conformational relaxation when relieved of the constraints of the quaternary structure in the oligomer (Gorovits, B., Raman, C. S., and Horowitz, P. M. (1995) J. Biol. Chem. 270, 2061-2066). The monomers display greatly enhanced hydrophobic exposure to the probe 1,1'-bis(4-anilino)naphthalene-5,5'-disulfonic acid, although they are not active in folding functions, and they are unable to form complexes with partially folded rhodanese. The monomers can be completely reassembled to 14-mers by incubation in 1 M ammonium sulfate. There is no evidence of intermediates in the reassembly process. Compared with the original oligomers, the reassembled 14-mers have (a) very low levels of polypeptide contaminants and tryptophan-like fluorescence, two problems that previously hampered spectroscopic studies of GroEL structure and function; (b) functional properties that are very similar to the original material; (c) considerably decreased hydrophobic exposure in the native state; and (d) a similar triggered exposure of hydrophobic surfaces after treatment with urea or spermidine. This study demonstrates that the quaternary structure of GroEL is more labile than previously thought. These results are consistent with suggestions that nucleotides can loosen subunit interactions and show that changes in quaternary structure can operate under conditions where GroEL function has been demonstrated.

Adenosine Diphosphate↗

PubMed1995

Mutation in the interdomain tether influences the stability and refolding of the enzyme rhodanese.

Rhodanese is a single polypeptide chain of 293 amino acids that is folded into two globular domains of nearly equal size that are connected by a 16 amino acid tether. Two amino acids, Val-Asp (VD), were inserted into the interdomain tether through site-directed mutagenesis to produce the new interdomain sequence, E145PSRPEPAIFKAVDTLNR. The purified mutant protein, when unperturbed, was virtually indistiguishable in all properties tested and gave a specific activity that was at least 90% of the WT. However, the tether mutant was considerably less stable to perturbation compared with the WT enzyme. The interdomain hydrophobic surfaces in the mutant were more easily exposed, and the formation of intermediate folding states was facilitated. The rate of unassisted refolding was slightly less for the mutant, and the yield of active enzyme was somewhat reduced. The mutation introduced a new V8 proteinase cleavage site, but this site was not accessible in the native mutant which was as resistant to proteolysis as the WT enzyme. However, perturbation with low concentrations of urea that could form folding intermediate(s), allowed facile cleavage of the mutant to give fragments that appeared to represent the individual domains. In addition, the perturbed mutant could be proteolyzed close to one end of the polypeptide, a position that is far from the site of mutation, and which was not readily cleaved in the WT enzyme or the native form of the mutant. These results indicate that mutation in the interdomain tether can have dramatic effects on the stability and conformational transitions of rhodanese.

Amino Acid Sequence↗

PubMed1995

Refolding and reassembly of active chaperonin GroEL after denaturation.

Conditions are reported that, for the first time, permit the folding and assembly of active chaperonin, GroEL, following denaturation in 8 m urea. The folding could be achieved by dilution or dialysis, and the best yields required the simultaneous presence of ammonium sulfate and the Mg2+ complexes of ATP or ADP. Ammonium sulfate was the key to this particular protocol, since there was a small recovery of oligomer in its presence, but no detectable recovery was induced by ATP or ADP without ammonium sulfate. The refolded/reassembled GroEL could arrest the spontaneous folding of rhodanese, and it could participate in the chaperonin-assisted refolding of rhodanese as effectively as GroEL that had never been unfolded. The results demonstrate that the primary sequence of GroEL contains the information required for its folding, assembly, and function. Thus, in contrast to previous studies, although chaperonins may facilitate GroEL folding, they are not necessary for the acquisition of the functional oligomeric state of this chaperone. This ability to fold denatured GroEL in vitro will facilitate studies of the influences that determine the interesting folding pattern adopted by the native protein.

Ammonium Sulfate↗

PubMed1995

Rhodanese conformational changes permit oxidation to give disulfides that form in a kinetically determined sequence.

When the structure of the monomeric enzyme rhodanese is perturbed by urea or SDS and heat, sulfhydryl assays combined with SDS gel analyses reveal that intrachain disulfides are formed rapidly. Two intrachain disulfide bonded species can be distinguished. One contains a single disulfide and comigrates on SDS gels with fully reduced rhodanese (Band I), while a second species contains two disulfides and migrates faster than the reduced enzyme (Band II). The kinetic path and identity of the participating sulfhydryl groups are suggested by the results with sulfhydryl mutants. On mild oxidation or perturbation, a single disulfide forms that involves two of the three sulfhydryl groups in C-terminal domain of the protein, i.e., two of the sulfhydryl groups from among the three residues: the active-site Cys-247, Cys-254 and Cys-263. These disulfides are the same as those that are formed upon oxidation of the native enzyme. The remaining sulfhydryl group of these three, in a kinetically slower process, can form a disulfide with Cys-63 which is in the N-terminal domain in native rhodanese. The resulting looped structure is so conformationally constrained that its shape and/or altered SDS binding gives rise to the 'fast' Band II on the SDS gels. The conformationally constrained species with two disulfides may be related to oxidized rhodanese species that are difficult to reduce.

Cysteine↗