Conformational restrictions on the pathway of folding and unfolding of the pancreatic trypsin inhibitor

The kinetic roles of the partially folded, intermediate protein species with two disulphide bonds in folding and unfolding of the pancreatic trypsin inhibitor have been investigated further. Formation of a second disulphide bond between Cys5 and Cys55 during refolding of the reduced inhibitor, which would yield the species with the 30–51 and 5–55 disulphide bonds and, possibly, the native-like conformation of the protein, is not significant. Instead, three other second disulphide bonds (5–14, 5–38 and 14–38) are formed approximately 10⁵ times more readily, but each of these two-disulphide species then rearranges intramolecularly to the native-like, two-disulphide intermediate. Therefore, the reduced protein does not simply form sequentially the three disulphide bonds of the native state. Unfolding of the native state takes place by the reverse of this process.The kinetic importance for folding and unfolding of this transition between two-disulphide intermediates under the conditions used here was illustrated experimentally by a modified form of the inhibitor in which the thiols of Cys14 and Cys38 were blocked irreversibly. In the folded conformation, this modified protein is more stable to unfolding than normal, but after unfolding cannot readily regain the native-like conformation, because Cys14 or Cys38 are required to be involved in disulphide bonds during the interconversion of the two-disulphide intermediates.Some conception of the conformational transitions that take place at each stage of the folding transition may be inferred from the relative propensities of the six cysteine residues to make or rearrange disulphide bonds. It is concluded that the inhibitor probably does not refold by sequential adoption of the native conformation by the unfolded polypeptide chain. Instead, it appears that essentially all elements of the native conformation are attained simultaneously in the final stage of folding, within an unstable and flexible, yet relatively compact, form of the entire polypeptide chain produced by weak interactions between groups distant in the primary structure.     

Intermediates in the refolding of reduced ribonuclease A.

The intermediates with one, two, three or four disulphide bonds which accumulate during unfolding of native ribonuclease and refolding of the reduced protein have been trapped by rapid alkylation with iodoacetate and separated by ionexchange chromatography. They have been characterized to varying extents by their enzymic activity, electrophoretic mobility through polyacrylamide gels, disulphide bonds between cysteine residues, the environments of the six tyrosine residues as indicated by ultraviolet absorption and fluorescence spectra, interaction with antibodies directed against either the trapped unfolded reduced protein or the native folded protein, and for the disruption by urea of any stable conformation producing a change in molecular shape.Correctly refolded ribonuclease was indistinguishable from the original native protein, but virtually all the intermediates with up to four disulphide bonds formed directly from the reduced protein were enzymically inactive and unfolded by these criteria. Unfolding of native ribonuclease was an all-or-none transition to the fully reduced protein, with no accumulation of disulphide intermediates. The intermediates in refolding are separated from the fully folded state by the highest energy barrier in the folding transition; they may be considered rapidly interconvertible, relatively unstable microstates of the unfolded protein. The measured elements of the final conformation are not acquired during formation of the first three disulphide bonds, but appear simultaneously with formation of the fourth native disulphide bond.These observations with ribonuclease are qualitatively similar to those made previously in greater detail with pancreatic trypsin inhibitor and suggest a possible general pattern for the kinetic process of protein unfolding and refolding.     

The single-disulphide intermediates in the refolding of reduced pancreatic trypsin inhibitor

The intermediate species with one disulphide bond in the renaturation of reduced pancreatic trypsin inhibitor have been trapped, isolated, and the Cys residues involved in the disulphide bonds determined. Approximately half the intermediate species had the disulphide bond between Cys-30 and 51, a disulphide bond also present in the native inhibitor. The next most predominant species, representing one-quarter of the total, had a disulphide bond between Cys-5 and 30, and two more minor species involving Cys-30 and 55 and Cys-5 and 51 were detected; these disulphide bonds are not present in the native inhibitor.The nature of the disulphide bonds present are concluded to reflect primarily the conformational forces acting at this stage of folding, which may be primarily interactions between segments with propensities for secondary structure, either helices or β-sheet. The general importance of such interactions in protein folding is discussed.     

Intermediates in the refolding of reduced pancreatic trypsin inhibitor

The thiol-disulphide exchange reaction used to renature the reduced pancreatic trypsin inhibitor has been rapidly quenched by acidification or by the addition of iodoacetate or iodoacetamide. The species so trapped at various times during the renaturation of the inhibitor have been analysed by acrylamide gel electrophoresis, which resolved the reduced and renatured inhibitors and several transient species with intermediate electrophoretic mobilities. The intermediates have been tentatively assigned contents of either one or two disulphide bonds by their relative electrophoretic mobilities when free Cys residues were carboxymethylated.The kinetic properties of the species have been determined during renaturation with varying concentrations of several disulphide reagents. The three separable intermediate species with one disulphide bond appear to be in rapid equilibrium via an intramolecular transition and fulfil the kinetic criteria for alternative intermediates on the folding pathway. Several species with two disulphide bonds accumulate under some circumstances. Their kinetic roles have not been fully elucidated, but at least some of them seem to be kinetically trapped species not on the main folding pathway. They appear to be particularly unstable species, one disulphide bond being readily broken.     

Kinetics of refolding of reduced ribonuclease

The kinetics of disulphide bond formation in reduced ribonuclease have been determined by following electrophoretically the appearance and disappearance of protein molecules with one, two, three or four intramolecular disulphide bonds. Each successive protein disulphide bond was observed to be formed much less readily than the preceding one, and the resulting species are increasingly unstable to reduction of their disulphide bonds. Most of the species formed directly, even those with four disulphide bonds, do not have the electrophoretic mobility of native protein.Protein molecules apparently refolded correctly are formed by slow intramolecular interconversion of molecules with three disulphide bonds and by thiolcatalyzed interchange of incorrect disulphide bonds in three-or four-disulphide species.These observations are compared with the properties of the folding pathway elucidated for pancreatic trypsin inhibitor under the same conditions and are contrasted with those often envisaged as to how proteins might fold.     

Estimation of parameters in a multi-affinity-state model for haemoglobin from oxygen binding data in whole blood and in concentrated haemoglobin solutions.

The reduced fragment of pancreatic trypsin inhibitor lacking the six C-terminal residues, which is produced by cyanogen bromide cleavage, formed a seemingly random mixture of disulphide bonds under refolding conditions where normal pancreatic trypsin inhibitor refolds correctly and quantitatively. This illustrates the importance of the C-terminal residues in folding of the normal protein, the uniqueness of the normal folded conformation, and the apparently central role in protein folding of long-range interactions between residues distant in the primary structure.The intact polypeptide chain of reduced pancreatic trypsin inhibitor in which the methionine residue normally at position 52 had been converted to homoserine refolded slightly less readily than the normal reduced compound. This was observed to be due to an altered spectrum of single-disulphide intermediates: the normally predominant intermediate with the 30–51 disulphide bond was less stable by about 0.8 kcal/mol relative to the other normal single-disulphide intermediates. The other steps in refolding appeared to be normal, although the refolded protein was observed to be susceptible to an unexplained reaction with iodoacetate.     

ERp57 is essential for efficient folding of glycoproteins sharing common structural domains

ERp57 is a member of the protein disulphide isomerase family of oxidoreductases, which are involved in native disulphide bond formation in the endoplasmic reticulum of mammalian cells. This enzyme has been shown to be associated with both calnexin and calreticulin and, therefore, has been proposed to be a glycoprotein-specific oxidoreductase. Here, we identify endogenous substrates for ERp57 by trapping mixed disulphide intermediates between enzyme and substrate. Our results demonstrate that the substrates for this enzyme are mostly heavily glycosylated, disulphide bonded proteins. In addition, we show that the substrate proteins share common structural domains, indicating that substrate specificity may involve specific structural features as well as the presence of an oligosaccharide side chain. We also show that the folding of two of the endogenous substrates for ERp57 is impaired in ERp57 knockout cells and that prevention of an interaction with calnexin or calreticulin perturbs the folding of some, but not all, substrates with multiple disulphide bonds. These results suggest a specific role for ERp57 in the isomerisation of non-native disulphide bonds in specific glycoprotein substrates.     

Dissecting the roles of individual interactions in protein stability: lessons from a circularized protein

A circular form of bovine pancreatic trypsin inhibitor (BPTI) has been prepared by introducing a peptide bond between the N- and C-termini, which are in close proximity in the native conformation. The pathway and energetics of the disulphide-coupled folding transition of the circular protein have been studied using methods applied previously to the unmodified protein. The cross-link between the termini was found not to significantly stabilize the native state in spite of the expected reduction in entropy of the unfolded protein. This unexpected result has led to a reexamination of the stabilization expected from a cross-link, considering effects on the native, as well as unfolded, states of the protein. The greatest stabilization is expected when the cross-linked groups are held rigidly in the native protein in the optimum orientation for forming the cross-link. Similar analyses, utilizing thermodynamic cycles, can be applied to other interactions that stabilize native proteins, including disulphide bonds, salt bridges, and hydrogen bonds and to modifications to the protein that remove them. In general, the contribution of an individual interaction to the stability of the native state depends on the extent to which the interaction is favored in the native conformation, which can vary greatly depending on the local environment of the interacting groups.     

Reactivities of the cysteine residues of the reduced pancreatic trypsin inhibitor.

The six cysteine residues of the reduced pancreatic trypsin inhibitor have been found to be equally reactive toward iodoacetate under the conditions used for refolding of the protein. The rates of reaction of each residue were comparable to those observed with model thiol compounds. It is concluded that the reduced inhibitor has no stable conformational properties that affect the cysteine residues. The results corroborate the previous conclusion that all six cysteine residues participate in forming the first disulphide bond during refolding of the reduced inhibitor and confirm that disulphide bond formation is an accurate probe of the conformational transitions that occur during protein folding.     

Analycys: a database for conservation and conformation of disulphide bonds in homologous protein domains

Disulphide bonds in proteins are known to play diverse roles ranging from folding to structure to function. Thorough knowledge of the conservation status and structural state of the disulphide bonds will help in understanding of the differences in homologous proteins. Here we present a database for the analysis of conservation and conformation of disulphide bonds in SCOP structural families. This database has a wide range of applications including mapping of disulphide bond mutation patterns, identification of disulphide bonds important for folding and stabilization, modeling of protein tertiary structures and in protein engineering. The database can be accessed at: http://bioinformatics.univ-reunion.fr/analycys/.     

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.     

Selective loss of cysteine residues and disulphide bonds in a potato proteinase inhibitor II family

Disulphide bonds between cysteine residues in proteins play a key role in protein folding, stability, and function. Loss of a disulphide bond is often associated with functional differentiation of the protein. The evolution of disulphide bonds is still actively debated; analysis of naturally occurring variants can promote understanding of the protein evolutionary process. One of the disulphide bond-containing protein families is the potato proteinase inhibitor II (PI-II, or Pin2, for short) superfamily, which is found in most solanaceous plants and participates in plant development, stress response, and defence. Each PI-II domain contains eight cysteine residues (8C), and two similar PI-II domains form a functional protein that has eight disulphide bonds and two non-identical reaction centres. It is still unclear which patterns and processes affect cysteine residue loss in PI-II. Through cDNA sequencing and data mining, we found six natural variants missing cysteine residues involved in one or two disulphide bonds at the first reaction centre. We named these variants Pi7C and Pi6C for the proteins missing one or two pairs of cysteine residues, respectively. This PI-II-7C/6C family was found exclusively in potato. The missing cysteine residues were in bonding pairs but distant from one another at the nucleotide/protein sequence level. The non-synonymous/synonymous substitution (Ka/Ks) ratio analysis suggested a positive evolutionary gene selection for Pi6C and various Pi7C. The selective deletion of the first reaction centre cysteine residues that are structure-level-paired but sequence-level-distant in PI-II illustrates the flexibility of PI-II domains and suggests the functionality of their transient gene versions during evolution.     

Accessibilities and reactivities of cysteine thiols during refolding of reduced bovine pancreatic trypsin inhibitor

The experimental basis of the pathway of refolding of reduced bovine pancreatic trypsin inhibitor that accompanies disulphide bond formation is explained in the light of a recent suggestion that the inability of certain Cys residues to form disulphide bonds could be explained simply by their thiol groups being inaccessible to disulphide reagents. This explanation is not valid, because part of the experimental evidence for inability to form disulphides is that the Cys residues accumulate as mixed-disulphides with the reagent. That these thiol groups are observed to react normally with the reagent, and with iodoacetic acid, is direct positive proof that they were not inaccessible or otherwise unreactive. The experimentally determined refolding pathway accurately reflects the energetics of the protein folding transitions and is consistent with all general observations of the folding transitions of other small proteins, whether or not disulphide bond formation is involved.     

SP-40,40, a protein involved in the control of the complement pathway, possesses a unique array of disulphide bridges.

SP-40,40 is a two-chain serum protein which acts in vitro as a potent inhibitor of the assembly of the membrane attack complex of human complement. It contains 10 cysteine residues, the numbers and locations of which are conserved in several mammalian species. Evidence is presented that all the cysteine residues are involved in interchain (alpha-beta) disulphide bonds. There are no free cysteine residues. The disulphide bond motif established in this study for SP-40,40 is unique and bears no obvious homology to those complement components whose disulphide bonds have been assigned, nor is there any homology apparent between SP-40,40 and other multi-chain proteins containing disulphide bonds.     

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.     

Renaturation of the reduced bovine pancreatic trypsin inhibitor

Refolding of the reduced pancreatic trypsin inhibitor has been investigated using thiol-disulphide exchange with various disulphide reagents to regenerate the three disulphide bonds. Essentially quantitative renaturation was routinely achieved. The refolded inhibitor was indistinguishable from the original protein in interaction with trypsin and chymotrypsin, electrophoretic mobility, and nature of disulphide bonds.The kinetics of refolding using oxidized dithiothreitol to form the disulphide bonds have been studied in some detail. The renaturation reaction is usually of second-order, being first-order in both inhibitor and disulphide reagent concentrations. A short lag period in the appearance of inhibitor activity and the inhibition of the rate, but not the extent, of renaturation by low levels of reduced dithiothreitol suggest the accumulation of metastable intermediates. In addition, heterogeneity of the refolding reaction is apparent at high concentrations of disulphide reagent, with a fraction of the material being only slowly renatured.     

Both the stroma and thylakoid lumen of tobacco chloroplasts are competent for the formation of disulphide bonds in recombinant proteins

Plant chloroplasts are promising vehicles for recombinant protein production, but the process of protein folding in these organelles is not well understood in comparison with that in prokaryotic systems, such as Escherichia coli . This is particularly true for disulphide bond formation which is crucial for the biological activity of many therapeutic proteins. We have investigated the capacity of tobacco ( Nicotiana tabacum ) chloroplasts to efficiently form disulphide bonds in proteins by expressing in this plant cell organelle a well-known bacterial enzyme, alkaline phosphatase, whose activity and stability strictly depend on the correct formation of two intramolecular disulphide bonds. Plastid transformants have been generated that express either the mature enzyme, localized in the stroma, or the full-length coding region, including its signal peptide. The latter has the potential to direct the recombinant alkaline phosphatase into the lumen of thylakoids, giving access to this even less well-characterized organellar compartment. We show that the chloroplast stroma supports the formation of an active enzyme, unlike a normal bacterial cytosol. Sorting of alkaline phosphatase to the thylakoid lumen occurs in the plastid transformants translating the full-length coding region, and leads to larger amounts and more active enzyme. These results are compared with those obtained in bacteria. The implications of these findings on protein folding properties and competency of chloroplasts for disulphide bond formation are discussed.     

HPr as a model protein in structure, interaction, folding and stability studies

The small size and lack of disulphide bonds or cofactors in the Histidine-containing phosphocarrier protein (HPr) makes it an attractive system with which to study structure, interaction to its enzymatic partners, and its stability and folding. Here we give an overview on the immense work that has been performed on this protein and we will show that HPr has been widely used as a model protein to study important aspects in modern Structural Biology.