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T E Creighton

Publications and source records attributed to T E Creighton.

At least 37 records · Page 2Linked to original sources
PubMed1995

Structural and functional characterization of DsbC, a protein involved in disulfide bond formation in Escherichia coli.

DsbC is a soluble protein of the bacterial periplasm that was identified genetically as being involved in protein disulfide formation. The gene sequence was corrected to include an additional proline residue and was then consistent with the molecular weight of the purified protein. Gel filtration and subunit hybridization indicate that DsbC is a stable dimer of identical subunits. Each subunit has a -Cys-Gly-Tyr-Cys- segment that forms an unstable and reactive disulfide bond; only the first cysteine residue is accessible, similar to thioredoxin and DsbA. The other two cysteine residues of DsbC form a buried, structural disulfide bond. The reactivities and stabilities of the active site disulfide bond of DsbC have been characterized and compared to that of DsbA. Both are very unstable and can be transferred rapidly to reduced proteins and peptides, although they differ somewhat in their kinetic reactivities. The two active sites of the DsbC dimer appear to function independently. DsbC is much more active than DsbA in catalyzing protein disulfide rearrangements, and this may be its main function in vivo.

Aprotinin↗
PubMed1995

Protein folding. An unfolding story.

A unified and coherent picture for the mechanism of protein folding is emerging. The crucial factor in folding is the cooperativity of multiple interactions that is required for stability of the folded state.

Protein Folding↗
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

The physical properties of local interactions of tyrosine residues in peptides and unfolded proteins.

Peptides and unfolded proteins with the sequence Xaa-Pro-Tyr or Xaa-Pro-Phe have a relatively strong local interaction, present about 64% of the time at 10 degrees C, of the aromatic ring with the side-chain of Pro and the C alpha H of residue Xaa, but only when the Xaa-Pro peptide bond is cis. With the sequence Tyr-Yaa-Gly (Yaa not equal to Pro), there is a somewhat present about 26% of the time, of the aromatic ring with the Gly residue. When present together, in the sequence Xaa-Pro-Tyr-Yaa-Gly, the two interactions of the Tyr side-chain compete and have the expected strengths. Both interactions have an enthalpic basis, with enthalpies of about -3 and -2.8 kcal/mol, respectively. The two interactions responded differently to the denaturants urea and guanidinium chloride; urea had little effect on either, but the second was weakened by guanidinium chloride, whereas the first interaction was strengthened slightly. This explains why such local interactions can be observed in unfolded proteins under strongly denaturing conditions. Interactions such as these are stronger than anticipated in an otherwise disordered peptide; they are probably important for determining the conformational tendencies of unfolded polypeptide chains and may play a role in protein folding. Similar interactions probably occur in protein-ligand interactions.

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

Phage tailspike protein. A fishy tale of protein folding.

The crystal structure of bacteriophage P22 tailspike protein reveals a striking fold with a distinctive, fish-like appearance, and helps explain many of the properties of this unusual molecule and its folding pathway.

Bacteriophage P22↗
PubMed1994

Electrophoretic characterization of the denatured states of staphylococcal nuclease.

The denatured state of staphylococcal (staph) nuclease has been shown to be altered by mutations, which complicates studies of stability of the folded state and may be important for the folding process. The relative hydrodynamic volumes as a function of urea concentration of staph nuclease, and of mutant forms in which the native state was substantially destabilized, have been examined by urea gradient gel electrophoresis. The native conformation is unique in its stabilization by specific ligands, which made it possible to distinguish between intermediate electrophoretic mobilities resulting from a partly populated native conformation and from other partly folded conformations. Four distinct conformational states were observed; two native-like: (1) the native, with and without bound ligands, and (2) an altered native-ligand complex at high urea concentrations; plus two denatured states: (3) a partly folded compact conformation that unfolded co-operatively and was sensitive to mutation of residues in the beta-barrel portion of the protein, and (4) the unfolded state, which appeared from its hydrodynamic volume to be as unfolded as reduced, alkylated ribonuclease A, even at very low urea concentrations, and was not altered by single mutations. The effects of mutations on the denatured state of staph nuclease may be due to the occurrence of the partly folded conformation (3).

Calcium↗
PubMed1994

Conformational specificity of the chaperonin GroEL for the compact folding intermediates of alpha-lactalbumin.

The chaperonin GroEL binds unfolded polypeptides, preventing aggregation, and then mediates their folding in an ATP-dependent process. To understand the structural features in non-native polypeptides recognized by GroEL, we have used alpha-lactalbumin (alpha LA) as a model substrate. alpha LA (14.2 kDa) is stabilized by four disulfide bonds and a bound Ca2+ ion, offering the possibility of trapping partially folded disulfide intermediates between the native and the fully unfolded state. The conformers of alpha LA with high affinity for GroEL are compact, containing up to three disulfide bonds, and have significant secondary structure, but lack stable tertiary structure and expose hydrophobic surfaces. Complex formation requires almost the complete alpha LA sequence and is strongly dependent on salts that stabilize hydrophobic interactions. Unfolding of alpha LA to an extended state as well as the burial of hydrophobic surface upon formation of ordered tertiary structure prevent the binding to GroEL. Interestingly, GroEL interacts only with a specific subset of the many partially folded disulfide intermediates of alpha LA and thus may influence in vitro the kinetics of the folding pathways that lead to disulfide bonds with native combinations. We conclude that the chaperonin interacts with the hydrophobic surfaces exposed by proteins in a flexible compact intermediate or molten globule state.

Animals↗
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

Reactivity and ionization of the active site cysteine residues of DsbA, a protein required for disulfide bond formation in vivo.

DsbA is a periplasmic protein of Escherichia coli that appears to be the immediate donor of disulfide bonds to proteins that are secreted. Its active site contains one accessible and one buried cysteine residue, Cys30 and Cys33, respectively, which can form a very unstable disulfide bond between them that is 10(3)-fold more reactive toward thiol groups than normal. The two cysteine residues have normal properties when in a short peptide. In DsbA, the Cys30 thiol group is shown to be reactive toward alkylating reagents down to pH 4 and to be fully ionized, on the basis of the UV absorbance of the thiolate anion at 240 nm. Its reactivity is altered by another, unknown group on the reduced protein titrating with a pKa of about 6.7. The other cysteine residue is buried and unreactive and has a high pKa value. The ionization properties of the DsbA thiol groups can explain, at least partly, the high reactivity of its disulfide bonds and thiol groups at both neutral and acidic pH values.

Amino Acid Sequence↗
PubMed1994

Effects of DsbA on the disulfide folding of bovine pancreatic trypsin inhibitor and alpha-lactalbumin.

DsbA is a protein found in the periplasm of Escherichia coli that is required for the formation of disulfide bonds in secreted proteins. It contains only two cysteine residues, which can form reversibly a very unstable disulfide bond that has been proposed to be the oxidant that introduces disulfide bonds into secreted proteins. The present study investigates the effect of DsbA on the well-characterized disulfide-coupled refolding processes of BPTI and of alpha-lactalbumin. Disulfide-bonded DsbA in stoichiometric amounts proved to be a very potent donor of disulfide bonds to reduced BPTI but showed little catalytic activity at neutral pH in the presence of a glutathione redox buffer. In contrast to the related eukaryotic enzyme protein disulfide isomerase, DsbA did not substantially catalyze the usual intramolecular disulfide bond rearrangements of quasi-native folding intermediates of BPTI. Neither did DsbA catalyze the intramolecular rearrangements observed in the three disulfide-bonded "molten globule" form of alpha-lactalbumin at neutral pH. Thiol-disulfide exchange is normally very slow at acidic pH but occurs rapidly with DsbA; consequently, DsbA catalyzed the disulfide folding of BPTI under acidic conditions. It was then possible to detect some increase in the rates of disulfide rearrangements, but only with stoichiometric amounts of DsbA and on the hour time scale. These results suggest that the primary role of DsbA in the bacterial periplasm is to introduce disulfide bonds into newly secreted proteins.

Amino Acid Sequence↗
PubMed1994

Replacement of the active-site cysteine residues of DsbA, a protein required for disulfide bond formation in vivo.

DsbA is a periplasmic protein of Escherichia coli that was identified genetically as being involved in the formation of disulfide bonds in secreted proteins. Its active site contains one accessible and one buried cysteine residue, separated in the primary structure by only two other residues. These cysteine residues can form a very unstable disulfide bond that is 10(3)-fold more reactive toward thiols than normal. Moreover, the mixed disulfide between the accessible cysteine residue and glutathione is 10(4)-fold more reactive than normal. Site-directed mutagenesis was carried out to replace either one or both cysteine residues by serine. Cys30 is shown to be the accessible thiol, while Cys33 is shielded from the solvent. Even though the thiol group of Cys30 is exposed and reactive, it formed a very unstable mixed disulfide with glutathione. This disulfide bond was 2.17 +/- 0.02 kcal mol-1 less stable in the native conformation than when DsbA was unfolded. If the native conformation destabilizes the mixed disulfide, the mixed disulfide must destabilize the folded conformation to the same extent. This was confirmed by demonstrating that the folded conformation of the mixed disulfide form of the mutant DsbA was 2.7 +/- 0.9 kcal mol-1 less stable than that of the reduced form; these stability effects originated almost exclusively in the folded conformation. Replacing the cysteine residues by serine destabilized the folded conformation of the reduced protein to varying extents. This suggests that the thiol groups are involved in interactions that stabilize the folded conformation, which would cause any disulfide bonds, either inter- or intramolecular, that involve these groups to be unstable.

Base Sequence↗
PubMed1994

Disulfide-rearranged molten globule state of alpha-lactalbumin.

A three-disulfide form of human alpha-lactalbumin, with free thiols on Cys6 and Cys120, can adopt the molten globule conformation. It then spontaneously rearranges its three disulfide bonds to many isomers that tend to maintain the molten globule conformation. The distribution of free thiol groups within the rearranged species has been determined quantitatively by chemical modification and peptide mapping. The protein's eight cysteine residues were modified with nearly equal frequencies, although there were significant departures from randomness. The results confirm that the molten globule state of alpha-lactalbumin does not maintain the native-like topology of the polypeptide backbone but is more like a collapsed form of an unfolded protein.

Calcium↗
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↗