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T E Creighton

Publications and source records attributed to T E Creighton.

At least 55 records · Page 3Linked to original sources
PubMed1993

Local conformations of peptides representing the entire sequence of bovine pancreatic trypsin inhibitor and their roles in folding.

The conformational properties of seven overlapping peptides, 9 to 16 residues long, that comprise the entire primary structure of bovine pancreatic trypsin inhibitor (BPTI) have been characterized by circular dichroism and 1H nuclear magnetic resonance. The peptides are largely disordered, although apparently with somewhat different average conformational propensities of the polypeptide backbone, similar to those indicated by methods to predict secondary structure. Reduced BPTI appears to be approximately the sum of the individual peptides. Several local interactions involving aromatic rings of side-chains interacting with groups nearby in the primary structure have been identified and verified by replacing the responsible side-chains. The roles of these interactions in folding of reduced BPTI could be determined, as the conformational and nuclear magnetic resonance properties of all the major disulphide intermediates are known. Two of these local interactions contribute to the folding process and to stability of the fully folded conformation, whereas the other two do not.

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

The reactive and destabilizing disulfide bond of DsbA, a protein required for protein disulfide bond formation in vivo.

The protein DsbA facilitates disulfide bond formation in the periplasm of Escherichia coli. It has only two cysteine residues that are separated in the sequence by two other residues and are shown to form a disulfide bond reversibly. Chemical modification studies demonstrate that only one of the cysteine residues has an accessible thiol group in the reduced protein. Equilibrium and kinetic characterization of thiol-disulfide exchange between DsbA and glutathione showed that the DsbA disulfide bond was 10(3)-fold more reactive than a normal protein disulfide. Similarly, the mixed disulfide between the accessible cysteine residue and glutathione was 10(4)-fold more reactive than normal. The overall equilibrium constant for DsbA disulfide bond formation from GSSG was only 8 x 10(-5) M. These properties indicate that disulfide-bonded DsbA is a potent oxidant and ideally suited for generating protein disulfide bonds. Disulfide bonds generally increase the stabilities of folded proteins, when the folded conformation reciprocally stabilizes the disulfide bonds. In contrast, the disulfide bond of DsbA was so unstable in the folded state that its stability increased by 4.5 +/- 0.1 kcal.mol-1 when the protein unfolded. This implies that the disulfide bond destabilizes the folded conformation of DsbA. This was confirmed by demonstrating that the reduced protein was 3.6 +/- 1.4 kcal.mol-1 more stable than that with the disulfide bond.

Amino Acid Sequence↗
PubMed1993

Pathway of disulfide-coupled unfolding and refolding of bovine alpha-lactalbumin.

alpha-Lactalbumin's four disulfide bonds have been used to probe the nature of its native, molten globule, and unfolded states. The Ca2+ concentration could be used to vary the stability of the native state because it binds a single calcium ion with high affinity. In the Ca(2+)-bound native state, the Cys6-120 and Cys28-111 disulfides were reduced sequentially, reflecting their relative accessibilities. The native disulfides do not stabilize the native conformation as much as in other proteins, nor was the unfolding process as cooperative. After two disulfides were reduced, only part of the protein molecule appeared to remain folded. In the absence of Ca2+, the native state was much less stable, and a molten globule-type conformation tended to be adopted. Formation of disulfide bonds in the reduced protein was initially noncooperative and nonspecific. The molten globule state increased the rate at which disulfides were formed, by tending to keep close in space cysteine residues that are distant in the sequence. A large number of nonnative disulfide pairings were preferred, and the disulfides were not stabilized to any great extent. The alpha-lactalbumin molten globule seems to be stabilized by nonspecific interactions and without a well-defined topology. The native protein was regenerated only when Ca2+ bound to and stabilized the three-disulfide intermediate lacking the Cys6-120 disulfide, which then rapidly formed the fourth disulfide bond. The rate-limiting intramolecular steps were rearrangement of disulfide bonds to native combinations in species with two and three disulfide bonds.

Animals↗
PubMed1993

Structural characterization of the disulfide folding intermediates of bovine alpha-lactalbumin.

Specific three- and two-disulfide intermediates that accumulate transiently during reduction of the disulfide bonds of Ca(2+)-bound bovine alpha-lactalbumin have been trapped, isolated, and characterized. The three-disulfide intermediate was shown to lack the Cys6-120 disulfide bond, confirming the observations of others. The newly-recognized two-disulfide form has been shown to lack the Cys6-120 and Cys28-111 native disulfide bonds. The remaining native disulfide bonds in the two partially reduced derivatives of alpha-lactalbumin are stable only when the proteins are in a Ca(2+)-bound state. Otherwise, they adopt an equilibrium between molten globule and unfolded conformations, and rapid thiol-disulfide interchange occurs, at a rate as high as when the proteins are fully unfolded in 8 M urea, to generate distinct mixtures of rearranged products. Urea gradient electrophoresis, circular dichroism, fluorescence, and ANS binding have been combined to give a detailed structural picture of alpha-lactalbumin, its derivatives with native and with nonnative disulfide bonds, and the fully reduced protein. The native structure of alpha-lactalbumin appears to be split by selective disulfide bond cleavage into at least one subdomain, which retains the Ca(2+)-binding site. The alpha-lactalbumin molten globule state is shown largely to result from nonspecific hydrophobic collapse, to be devoid of cooperative or specific tertiary interactions, and not to be stabilized substantially by the native or rearranged disulfide bonds.

Anilino Naphthalenesulfonates↗
PubMed1993

Local structure due to an aromatic-amide interaction observed by 1H-nuclear magnetic resonance spectroscopy in peptides related to the N terminus of bovine pancreatic trypsin inhibitor.

A synthetic peptide corresponding to the 15 N-terminal residues of bovine pancreatic trypsin inhibitor, with serine replacing the two cysteine residues, has been characterized by 1H-nuclear magnetic resonance spectroscopy. This peptide has a very disordered conformation that is essentially the same when it is part of the analogue of the (30-51) one-disulphide intermediate in folding. This confirms the conclusion of a previous paper, that the (30-51) intermediate is partially folded, with the N-terminal segment disordered. Local elements of non-random conformation were observed in the peptide. Especially prominent was an apparently electrostatic interaction between the face of the aromatic ring of Tyr10 and the amide group of Gly12, which caused the latter to have a very anomalous chemical shift. A similar interaction was observed in shorter peptides, especially in tetrapeptides with the sequences Tyr/Phe-X-Gly-Y. The local nature of this interaction indicates that it should be a general feature in peptides and in unfolded proteins with such a sequence.

Amides↗
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↗
PubMed1992

Protein folding pathways determined using disulphide bonds.

The best-characterized model pathway of protein folding, that of disulphide bond formation in the small protein BPTI, has been questioned recently. A reinvestigation of that pathway, using alternative methods, concluded that the intermediates with non-native disulphide bonds accumulated to lower levels than previously had been observed. On this basis, a revised pathway was proposed that simply omitted those intermediates. Even if totally correct, however, the new observations are not inconsistent with the important characteristics of the original pathway and even confirmed many of them. Certain crucial observations that were the experimental basis for the original pathway were ignored, and these observations invalidate the revised pathway.

Animals↗
PubMed1992

Folding in vitro of bovine pancreatic trypsin inhibitor in the presence of proteins of the endoplasmic reticulum.

The rates of folding and disulfide bond formation in reduced BPTI were measured in vitro in the presence and absence of total protein from the endoplasmic reticulum. The rates were increased substantially by the endoplasmic reticulum proteins, but only to the extent expected from the known content and activity of protein-disulfide-isomerase. No effects of added ATP or Ca2+ were observed, even though protein-disulfide-isomerase binds Ca2+ tightly.

Adenosine Triphosphate↗