Biomedical subjects

N J Darby

Publications and source records attributed to N J Darby.

At least 19 recordsLinked to original sources

PubMed1997

The folding catalyst protein disulfide isomerase is constructed of active and inactive thioredoxin modules.

BACKGROUND: Protein disulfide isomerase (PDI), a multifunctional protein of the endoplasmic reticulum, catalyzes the formation, breakage and rearrangement of disulfide bonds during protein folding. Dissection of this protein into its individual domains has confirmed the presence of the a and a' domains, which are homologous to thioredoxin, having related structures and activities. The a and a' domains both contain a -Cys-Gly-His-Cys- active-site sequence motif. The remainder of the molecule consists primarily of two further domains, designated b and b' which are thought to be sequence repeats on the basis of a limited sequence similarity. The functions of the b and b' domains are unknown and, until now, the structure of neither domain was known. RESULTS: Heteronuclear nuclear magnetic resonance (NMR) methods have been used to determine the global fold of the PDI b domain. The protein has an alpha/beta fold with the order of the elements of secondary structure being beta1-alpha1-beta2-alpha2-beta3-alpha3-beta4-beta5+ ++-alpha4. The strands are all in a parallel arrangement with respect to each other, except for beta4 which is antiparallel. The arrangement of the secondary structure elements of the b domain is identical to that found in the a domain of PDI and in the ubiquitous redox protein thioredoxin; the three-dimensional folding topology of the b domain is also very similar to that of these proteins. CONCLUSIONS: Our determination of the global fold of the b domain of PDI by NMR reveals that, like the a domain, the b domain contains the thioredoxin motif, even though the b domain has no significant amino-acid sequence similarities to any members of the thioredoxin family. This observation, together with indications that the b' domain adopts a similar fold, suggests that PDI consists of active and inactive thioredoxin modules. These modules may have been adapted during evolution to provide PDI with its complete spectrum of enzymatic activities.

Amino Acid Sequence↗

PubMed1996

Electrostatic interactions in the active site of the N-terminal thioredoxin-like domain of protein disulfide isomerase.

Proteins with the thioredoxin fold have widely differing stabilities of the disulfide bond that can be formed between the two cysteines at their active site sequence motif Cys1-Xaa2-Yaa3-Cys4. This is believed to be regulated not by varying the disulfide bond itself, but by modulating the stability of the dithiol form of the protein through interactions with the ionized form of the Cys1 thiol group. A consistent relationship between disulfide bond stability and Cys1 thiol pKa value is found here for DsbA, thioredoxin, and the N-terminal thioredoxin-like domain of protein disulfide isomerase (PDI a), which has a very low thiol pKa value of 4.5. This thiolate anion is stabilized by 5.7 kcal/mol in the dithiol form, giving rise to the corresponding instability of the disulfide bond and the oxidizing properties of PDI a. Electrostatic interactions in the active site of the PDI a-domain have been characterized in order to understand the physical basis of this stabilization. Linkage with the ionization of the imidazole group of His3 in the active site demonstrates that this charge-charge interaction contributes 1.1 kcal/mol. The remainder of the stabilization is believed to be due primarily to interactions with the partial positive charges at the N-terminus of an alpha-helix, which are exceedingly sensitive to charges of surrounding residues.

Binding Sites↗

PubMed1996

Identifying and characterizing a structural domain of protein disulfide isomerase.

Protein disulfide isomerase (PDI) appears on the basis of its primary structure to be a multidomain protein, but the number and nature of the domains has been uncertain. Two of the domains, a and a', which are homologous to thioredoxin and active in catalysis of disulfide bond formation, have been identified and characterized previously. Sections of the N-terminal half of the PDI sequence have been expressed and the limits of their folded structures delineated by limited proteolysis. In addition to the a-domain, the boundaries of a domain with no activity on thiol/disulfide groups, designated b, have been identified. This domain has been produced independently; its cooperative unfolding transition and its CD and NMR spectra confirm that it is an autonomously folded structure in isolation and when part of PDI. Fusion of the b-domain to the a-domain, as occurs naturally in the first half of PDI, did not alter substantially the catalytic activity of the a-domain. It still catalyzes only a subset of the thiol/disulfide exchange reactions of intact PDI and has a reduced ability to catalyze protein disulfide rearrangements. The a- and b-domains account structurally for virtually all of the first half of the PDI polypeptide chain, and it is very unlikely that there exists a proposed third domain homologous to the estrogen receptor. The b-domain exhibits some sequence homology to calsequestrin, a calcium binding protein from the sarcoplasmic reticulum of muscle.

Amino Acid Sequence↗

PubMed1996

Structure determination of the N-terminal thioredoxin-like domain of protein disulfide isomerase using multidimensional heteronuclear 13C/15N NMR spectroscopy.

As a first step in dissecting the structure of human protein disulfide isomerase (PDI), the structure of a fragment corresponding to the first 120 residues of its sequence has been determined using heteronuclear multidimensional NMR techniques. As expected from its primary structure homology, the fragment has the thioredoxin fold. Similarities and differences in their structures help to explain why thioredoxins are reductants, whereas PDI is an oxidant of protein thiol groups. The results confirm that PDI has a modular, multidomain structure, which will facilitate its structural and functional characterization.

Amino Acid Sequence↗

PubMed1996

The roles of partly folded intermediates in protein folding.

Proteins can fold very rapidly, undoubtedly because they do not do so simply by random searching. The stable, partly folded species that can be detected during protein refolding are, however, of uncertain kinetic significance. The available kinetic evidence indicates that the intermediates that are most responsible for the rapidity of folding are extremely unstable and not populated detectably; they are less extreme versions of the transition state for folding. Protein folding is most readily studied when it is coupled to disulfide formation, which has the advantages that the intermediates can be characterized in detail and their kinetic roles determined unambiguously. The most important aspects of the disulfide folding pathway of BPTI are understood to at least a first approximation, and several other protein disulfide folding pathways are known in outline. These pathways demonstrate that disulfide folding is not intrinsically different from that not involving disulfide formation. Partly folded conformations can increase the rate of folding somewhat by causing productive disulfide bonds to be populated preferentially, but the most important folding intermediates are not detectable. The essence of folding is to build up the cooperativity between the individual interactions that is necessary for a stable conformation.

Amino Acid Sequence↗

PubMed1995

Characterization of the active site cysteine residues of the thioredoxin-like domains of protein disulfide isomerase.

The dithiol/disulfide active sites of each of the two isolated thioredoxin-like domains of protein disulfide isomerase (PDI) expressed in Escherichia coli have been characterized in order to understand their catalytic mechanisms and their functions in PDI. In each of the folded domains, as in other proteins of the thioredoxin family, only one of the cysteine residues of the active site sequence -Cys-Gly-His-Cys- is accessible, and its thiol group is highly reactive and has a low pKa value. The kinetics and equilibria have been measured of the reactions between the active site cysteine residues and glutathione, the predominant thiol/disulfide reagent of the endoplasmic reticulum. A disulfide bond can be formed very rapidly between the pair of cysteine residues of each domain, but each disulfide bond is very unstable and reacts rapidly with reduced glutathione. The very low stabilities of these disulfide bonds, which destabilize the protein structures, account for the efficiency with which PDI and each of the isolated domains can introduce disulfide bonds into proteins. These kinetics and equilibrium data go far in helping to understand the catalytic mechanism of PDI and its individual domains.

Amino Acid Sequence↗

PubMed1995

Functional properties of the individual thioredoxin-like domains of protein disulfide isomerase.

The two thioredoxin-like domains of human protein disulfide isomerase (PDI) have been produced in bacteria as individual soluble, folded protein molecules, and their functional properties have been compared to those of intact PDI. The two individual domains were very similar in their functional properties, and there were no indications of synergy between them, so it is unlikely that they have intrinsically different functions in PDI. Both domains efficiently introduced disulfide bonds into unfolded model proteins and peptides but were less efficient than PDI with folded substrate protein molecules. Relative to PDI, neither domain had substantial activity in catalyzing disulfide bond isomerization. This pattern of activities is very similar to that of the bacterial catalyst DsbA and probably reflects similarities in the catalytic mechanisms of these proteins. The differences in activity between PDI and its thioredoxin-like domains suggest that other features of the PDI molecule are also required for its complete range of thiol-disulfide exchange activities.

Amino Acid Sequence↗

PubMed1995

Refolding of bovine pancreatic trypsin inhibitor via non-native disulphide intermediates.

The disulphide folding pathway of bovine pancreatic trypsin inhibitor (BPTI), especially at the two-disulphide stage, has been dissected by replacing one or two particular cysteine residues by serine. This restricts which disulphide species are possible, and the observed kinetics of disulphide-coupled folding reveal the roles of the remaining species. The results obtained confirm the kinetic roles in the original BPTI pathway of the two specific two-disulphide intermediates with non-native second disulphide bonds, (30-51, 5-14) and (30-51, 5-38). Moreover, the rates of folding through each of these intermediates are shown to account quantitatively for the rate of folding of the normal protein; therefore, essentially all the molecules refold through these two particular intermediates. They are amongst the most productive on the folding pathway, and their roles are readily explicable on the basis of their conformations.

Amino Acid Sequence↗

PubMed1995

Catalytic mechanism of DsbA and its comparison with that of protein disulfide isomerase.

The mechanism of action of the bacterial periplasm protein DsbA in introducing disulfide bonds into proteins was studied by its action on a model disordered peptide containing only two cysteine residues. Most of the reactions between the various thiol and disulfide forms of the peptide and of DsbA could be measured directly. All those involving DsbA occurred 10(2)-10(6) times more rapidly than is normally observed between other typical thiols and disulfides; DsbA apparently stabilizes the transition state of thiol-disulfide exchange. The reactions between DsbA and the peptide were even more rapid, and they were constrained to occur at only one sulfur atom of disulfide bonds involving the peptide. Both observations indicate that noncovalent binding interactions also occur between DsbA and the peptide, and the expected effect of binding between reactants on rates of reaction was quantified. Small quantities of DsbA had catalytic effects on the reaction between the peptide and glutathione, similar to those observed previously with the eukaryotic catalyst protein disulfide isomerase. The known reactions of DsbA could account quantitatively for these effects and indicated that the apparent catalysis was the result of the separate and sequential rapid reactions of the peptide and of glutathione at the active site of DsbA. DsbA did not catalyze the conformational changes involved in forming an intramolecular disulfide bond in the peptide; its catalytic effects were simply due to its rapid participation in thiol-disulfide exchange reactions. Protein disulfide isomerase is likely to function very similarly to DsbA.

Amino Acid Sequence↗

PubMed1995

Nuclear magnetic resonance characterization of the N-terminal thioredoxin-like domain of protein disulfide isomerase.

A genetically engineered protein consisting of the 120 residues at the N-terminus of human protein disulfide isomerase (PDI) has been characterized by 1H, 13C, and 15N NMR methods. The sequence of this protein is 35% identical to Escherichia coli thioredoxin, and it has been found also to have similar patterns of secondary structure and beta-sheet topology. The results confirm that PDI is a modular, multidomain protein. The last 20 residues of the N-terminal domain of PDI are some of those that are similar to part of the estrogen receptor, yet they appear to be an intrinsic part of the thioredoxin fold. This observation makes it unlikely that any of the segments of PDI with similarities to the estrogen receptor comprise individual domains.

Amino Acid Sequence↗

PubMed1995

Mechanisms and catalysts of disulfide bond formation in proteins.

The formation of disulphide bonds is an important co- and post-translational event in the biosynthesis of many extracellular proteins that is often coupled to protein folding. Progress in understanding how disulphide bonds form in model proteins provides insight into how the process can be manipulated to optimize the production of engineered proteins.

Amino Acid Sequence↗

PubMed1994

Dissecting the mechanism of protein disulfide isomerase: catalysis of disulfide bond formation in a model peptide.

As a model for understanding how protein disulfide isomerase (PDI) catalyzes disulfide bond formation in proteins, its action on a 28-residue disordered peptide containing only two cysteine residues has been examined. Disulfide formation in the peptide using the chemical reaction with small molecule thiol/disulfide reagents, such as oxidized and reduced glutathione or cystamine and cysteamine, occurs in two steps, via two alternative intermediate mixed disulfides between the reagent and either peptide cysteine residue. All thiol/disulfide forms of the peptide could be trapped and quantified, so the rates of their interconversion could be measured. Catalytic amounts of PDI increased the rates of these reactions. All rate enhancements were independent of the concentration of the peptide, indicating that it bound to PDI with an apparent Km of less than 3 microM. In the presence of glutathione, PDI accelerated the formation of both single mixed disulfide species, plus their subsequent rearrangement to form the peptide disulfide bond, but not interchange of the mixed disulfide glutathione between the two cysteine residues. In contrast, PDI did not catalyze the reaction of the reagent cystamine with the reduced peptide to form the mixed disulfide, nor the interchange of this mixed disulfide between cysteine residues, but did catalyze the subsequent intramolecular step of peptide disulfide bond formation to a similar extent as with the glutathione mixed disulfide. These effects on the two steps involving the mixed disulfides with glutathione or cystamine accounted for much of the overall catalytic effect of PDI on disulfide bond formation in the peptide, indicating that direct transfer of disulfide bonds from PDI to the peptide occurred less frequently. These findings demonstrate the utility of using such peptides as PDI substrates and have implications for the mechanism of action of PDI.

Amino Acid Sequence↗

PubMed1994

1H NMR analysis of the partly-folded non-native two-disulphide intermediates (30-51,5-14) and (30-51,5-38) in the folding pathway of bovine pancreatic trypsin inhibitor.

The conformational properties of analogues of the (30-51,5-14) and (30-51,5-38) disulphide intermediates in refolding of reduced BPTI, with non-native second disulphide bonds, have been characterized in detail by 1H NMR analysis. They are shown to have partly-folded conformations, very similar to that of the (30-51) one-disulphide intermediate from which they arise during folding. The non-native disulphide bonds are formed in flexible or unfolded parts of the polypeptide chain; they do not disrupt the folded portion nor do they introduce substantial non-native conformation. The conformational properties of these intermediates explain their important roles in the folding pathway.

Amino Acid Sequence↗

PubMed1993

On the biosynthesis of bovine pancreatic trypsin inhibitor (BPTI). Structure, processing, folding and disulphide bond formation of the precursor in vitro and in microsomes.

The natural gene for bovine pancreatic trypsin inhibitor (BPTI) was expressed by in vitro transcription/translation systems as the 100-residue pre-proBPTI, with a signal peptide for translocation into the endoplasmic reticulum. Expression in the presence of microsomes defined the site of co-translational cleavage of the signal peptide. The resulting proBPTI in the microsomes consists of the 58 residues of mature BPTI, plus an additional 13 residues at the N terminus, including a cysteine residue at position -10, and seven residues at the C terminus. ProBPTI remained in the unfolded, reduced form within microsomes when synthesized under reducing conditions, but folded and formed disulphide bonds rapidly when the disulphide form of glutathione was added. Complete folding could occur within about one minute, even when residue Cys10 was replaced by Ser. The structure of proBPTI was determined by circular dichroism and two-dimensional NMR and found to be that of mature BPTI with flexible extensions on both termini. Its inhibition of the activity of alpha-chymotrypsin was indistinguishable from that of the mature protein. The extensions of the precursor appeared to play only very minor roles in refolding in vitro under conditions where folding and disulphide bond formation are coupled. Under pH and redox conditions thought to reflect those in vivo, complete folding and disulphide bond formation required several hours. Addition of protein disulphide isomerase to in vitro folding experiments caused substantial and similar increases in the rate of formation of the fully folded state for both mature BPTI and proBPTI; the half time for folding to the native state was reduced to approximately two minutes, which is comparable to that occurring in microsomes. The absence of substantial effects of the N and C-terminal extensions on the protein structure, inhibitor activity and refolding leaves their functional roles to be discovered.

Amino Acid Sequence↗

PubMed1993

Dissecting the disulphide-coupled folding pathway of bovine pancreatic trypsin inhibitor. Forming the first disulphide bonds in analogues of the reduced protein.

The kinetics of disulphide bond formation and breakage have been measured in five analogues of the single-disulphide intermediates that occur in folding of bovine pancreatic trypsin inhibitor (BPTI), in which the cysteine residues not involved in disulphide bonds have been replaced by serine residues. Only a single disulphide bond can be made in each analogue, allowing the rates and equilibrium constants of the corresponding microscopic steps to be determined. These steps cannot be characterized in normal BPTI with six cysteine residues, as only the total overall rate of forming all 15 possible disulphide bonds can be measured. The intramolecular rate of forming each disulphide bond was found in the dithiol forms of reduced BPTI to be approximately proportional inversely to the size of the disulphide loop formed. On this basis, the 30-51 disulphide bond is not formed preferentially in the fully reduced protein, even though it predominates amongst the one-disulphide intermediates as a result of rapid intramolecular disulphide rearrangements. It is found to be more stable than the other one-disulphide intermediates because its disulphide is more stable to reduction than the others. This is undoubtedly due to the stable partially folded structure of (30-51), as the other intermediates are much less structured under folding conditions. Urea (8 M) uniformly decreased by a factor of 5 the effective concentrations of cysteine thiol groups in the analogues and in reduced BPTI, suggesting that reduced BPTI under normal refolding conditions is not an ideal random coil; any non-random conformations do not, however, favour formation of any specific disulphide bonds. Findings from these studies have led to further refinement and development of various approaches to measuring and interpreting the intramolecular rates of disulphide bond formation and breakage in proteins.

Animals↗

PubMed1993

Partially folded conformation of the (30-51) intermediate in the disulphide folding pathway of bovine pancreatic trypsin inhibitor. 1H and 15N resonance assignments and determination of backbone dynamics from 15N relaxation measurements.

An analogue of the important folding intermediate of BPTI with only the disulphide bond between Cys30 and Cys51 has been characterized by 1H and 15N NMR techniques. In particular, the dynamics of the polypeptide backbone were characterized using (1H)-15N NOE and 15N T1 and T2 relaxation data. The intermediate is partially folded, with part of the polypeptide chain stably folded and the remainder flexible or unfolded. The folded portion consists of the major elements of native-like secondary structure interacting through the hydrophobic core of the molecule. The 15N relaxation data show that the N-terminal 15 residues are very flexible, and the (1H, 1H) NOESY data show that these residues have no NOE interactions with the remainder of the molecule. The segment of residues 37 to 41 is also flexible. These observations explain why during folding this intermediate most readily forms any of the possible disulphide bonds between Cys5, Cys14 and Cys38, including the non-native 5-14 and 5-38 bonds. The native-like folded portion of the molecule limits the possible disulphide bonds that can be formed to those in the remainder of the polypeptide chain. Also, forming the non-native disulphide bonds need not involve any disruption of that folded structure, as the Cys residues involved are in flexible regions of the molecule.

Animals↗

PubMed1992

The partially folded conformation of the Cys-30 Cys-51 intermediate in the disulfide folding pathway of bovine pancreatic trypsin inhibitor.

The best-characterized protein folding pathway is that of bovine pancreatic trypsin inhibitor, which folds from the reduced form through a series of disulfide bond intermediates. The crucial one-disulfide intermediate of bovine pancreatic trypsin inhibitor with the disulfide bond between Cys-30 and Cys-51 is shown here to have a partially folded conformation in which the major elements of secondary structure interact via a core of apolar side chains, which resembles part of the native conformation. The stability of this structure can account for the predominance of this one-disulfide intermediate during folding. Much of the remaining one-third of the polypeptide chain, in particular the N-terminal 14 residues, is largely disordered; this accounts for the ability of this intermediate to form readily any of the three possible second disulfide bonds involving Cys-5, -14, and -38. The partially folded conformation of this intermediate provides direct evidence for the importance of native-like interactions between elements of secondary structure in directing protein folding, which is assumed in many studies.

Amino Acid Sequence↗

PubMed1992

Kinetic roles and conformational properties of the non-native two-disulphide intermediates in the refolding of bovine pancreatic trypsin inhibitor.

The most productive folding pathway of reduced bovine pancreatic trypsin inhibitor (BPTI) proceeds through the disulphide intermediates (30-51), (30-51, 5-14), and (30-51, 5-38); these are important kinetic intermediates in folding, even though the latter pair contain non-native disulphide bonds. Analogues of these intermediates have been prepared by protein engineering methods and their conformational properties examined by circular dichroism and 1H-nuclear magnetic resonance. The (30-51), (30-51, 5-14) and (30-51, 5-38) analogues exhibit comparable degrees of stable structure, which cannot include those portions of the polypeptide chain involving Cys5, Cys14 and Cys38. These properties are consistent with the roles of (30-51, 5-14) and (30-51, 5-38) in the folding pathway of BPTI, which demand that they exhibit a considerable degree of conformational flexibility in part of the molecule.

Animals↗