Formation of native insulin from the scrambled molecule by protein disulphide-isomerase.

The formation of native insulin either from scrambled insulin or from the separated A chain and B chain S-sulphonates by protein disulphide-isomerase was demonstrated with yields of 20-30% as measured by h.p.l.c. analysis, receptor binding and stimulation of lipogenesis. The h.p.l.c. profile of the reaction products shows that, among all the possible isomers containing both chains, the native hormone is by far the predominating product and consequently the most stable under certain conditions.     

Reconstitution of the thermostable trimeric phage P22 tailspike protein from denatured chains in vitro

Intermediates in the intracellular chain folding and association pathway of the P22 tailspike endorhamnosidase have been identified previously by physiological and genetic methods. Conditions have now been found for the in vitro refolding of this large (Mr = 215,000) oligomeric protein. Purified Salmonella phage P22 tailspikes, while very stable to urea in neutral solution, were dissociated by moderate concentrations of urea at acidic pH. The tailspike protein was denatured to unfolded polypeptide chains in 6 M urea, pH 3, as disclosed by analytical ultracentrifugation, fluorescence, and circular dichroism. Upon dilution into neutral buffer at 10 degrees C, the polypeptides fold spontaneously and associate to form trimeric tailspikes with high yield. Like native phage P22 tailspikes, the reconstitution product is resistant to denaturation by dodecyl sulfate in the cold and displays endorhamnosidase activity. Sedimentation coefficients, electrophoretic mobility, and fluorescence emission maxima of native and reconstituted tailspikes are identical within experimental error. By characterization of intermediates, localization of temperature-sensitive steps, and analysis of the effect of previously identified folding mutations, the reconstitution system described should allow comparison of in vivo and in vitro folding pathways of this large protein oligomer.     

In vivo assembly of aspartate transcarbamoylase from fragmented and circularly permuted catalytic polypeptide chains

Previous studies on Escherichia coli aspartate transcarbamoylase (ATCase) demonstrated that active, stable enzyme was formed in vivo from complementing polypeptides of the catalytic (c) chain encoded by gene fragments derived from the pyrBI operon. However, the enzyme lacked the allosteric properties characteristic of wild-type ATCase. In order to determine whether the loss of homotropic and heterotropic properties was attributable to the location of the interruption in the polypeptide chain rather than to the lack of continuity, we constructed a series of fragmented genes so that the breaks in the polypeptide chains would be dispersed in different domains and diverse regions of the structure. Also, analogous molecules containing circularly permuted c chains with altered termini were constructed for comparison with the ATCase molecules containing fragmented c chains. Studies were performed on four sets of ATCase molecules containing cleaved c chains at positions between residues 98 and 99, 121 and 122, 180 and 181, and 221 and 222; the corresponding circularly permuted chains had N termini at positions 99, 122, 181, and 222. All of the ATCase molecules containing fragmented or circularly permuted c chains exhibited the homotropic and heterotropic properties characteristic of the wild-type enzyme. Hill coefficients (n(H:)) and changes in them upon the addition of ATP and CTP were similar to those observed with wild-type ATCase. In addition, the conformational changes revealed by the decrease in sedimentation coefficient upon the addition of a bisubstrate analog were virtually identical to that for the wild-type enzyme. Differential scanning calorimetry showed that neither the breakage of the polypeptide chains nor the newly formed covalent bond between the termini in the wild-type enzyme had a significant impact on the thermal stability of the assembled dodecamers. The studies demonstrate that continuity of the polypeptide chain within structural domains is not essential for the assembly, activity, and allosteric properties of ATCase.     

Protein disulphide-isomerase activity in chick-embryo tissues. Correlation with the biosynthesis of procollagen.

Protein disulphide-isomerase activity was determined in homogenates of chick-embryo tissues. Activities were highest in tissues active in procollagen synthesis and were maximal at the developmental stage of maximal procollagen synthesis. These variations in protein disulphide-isomerase activity correlate closely with those previously observed for protocollagen hydroxylase activities.     

Hydroxylation of peptide-bound proline and lysine before and after chain completion of the polypeptide chains of procollagen

The synthesis of procollagen hydroxyproline and hydroxylysine was examined in matrix-free cells which were isolated from embryonic tendon by controlled enzymic digestion and then incubated in suspension. After the cells were labeled with [¹⁴C]proline for 2 min, or about one-third the synthesis time for a Pro-α chain, [¹⁴C]hydroxyproline was found in short peptides considerably smaller than the Pro-α chains of procollagen. The results, therefore, confirmed previous reports indicating that the hydroxylation of proline can begin on nascent chains. In similar experiments in which the cells were labeled with [¹⁴C]lysine, [¹⁴C]hydroxylysine was found in short, newly synthesized peptides, providing the first evidence that the hydroxylation of lysine can also begin on nascent peptides. However, further experiments demonstrated that the synthesis of hydroxyproline and hydroxylysine continues until some time after assembly of the polypeptide chains is completed.     

Functional protein domains from the thermally driven motion of polypeptide chains: A proposal

It is proposed that the thermally driven motion of certain polypeptide chains, including those that are part of an otherwise stable folded protein, produces time-averaged three-dimensional domains that confer unique functions to a protein. These domains may be controlled by collapsing the polypeptide into an enthalpically favored structure, or extending it into an entropically dominated form. In the extended form, these domains occupy a relatively large space, which may be used to regulate protein–protein interactions and confer mechanical properties to proteins. This “entropic bristle” model makes several predictions about the structure and properties of these domains, and the predictions are used to reevaluate a range of biophysical studies on proteins. The outcome of the analysis suggests that the entropic bristle can be used to explain a wide range of disparate and apparently unrelated experimental observations. Proteins 32:223–228, 1998. © 1998 Wiley-Liss, Inc.     

Resolution of protein disulphide-isomerase and glutathione-insulin transhydrogenase activities by covalent chromatography.

1. Protein disulphide-isomerase (EC 5.3.4.1) and glutathione-insulin transhydrogenase (EC 1.8.4.2) were resolved by covalent chromatography. Both activities, in a partially purified preparation from bovine liver, bind covalently as mixed disulphides to activated thiopropyl-Sepharose 6B, in a new stepwise elution procedure protein disulphide-isomerase is displaced in mildly reducing conditions whereas glutathione-insulin transhydrogenase is only displaced by more extreme reducing conditions. 2. This together with evidence for partial resolution of the two activities by ion-exchange chromatography, conclusively establishes that the two activities are not alternative activities of a single bovine liver enzyme. 3. Protein disulphide-isomerase, partially purified by a published procedure, has now been further purified by covalent chromatography and ion-exchange chromatography. The final material is 560-fold purified relative to a bovine liver homogenate; it has barely detectable glutathione-insulin transhydrogenase activity. 4. The purified protein disulphide-isomerase shows a single major band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis corresponding to a mol.wt. of 57000. 5. The purified protein disulphide-isomerase has Km values for 'scrambled' ribonuclease and dithiothreitol of 23 microgram/ml and 5.4 microM respectively and has a sharp pH optimum at 7.5. The enzyme has a broad thiol-specificity, and several monothiols, at 1mM, can replace dithiothreitol. 6. The purified protein disulphide-isomerase is completely inactivated after incubation with a 2-3 fold molar excess of iodoacetate. The enzyme is also significantly inhibited by low concentrations of Cd2+ ions. These findings strongly suggest the existence of a vicinal dithiol group essential for enzyme activity. 7. When a range of thiols were used as co-substrates for protein disulphide-isomerase activity, the activities were found to co-purify quantitatively, implying the presence of a single protein disulphide-isomerase of broad thiol-specificity. Glutathione-disulphide transhydrogenase activities, assayed with a range of disulphide compounds, did not co-purify quantitatively.     

The insulin A and B chains contain structural information for the formation of the native molecule. Studies with protein disulphide-isomerase.

It has been shown previously [Tang, Wang & Tsou (1988) Biochem. J. 255, 451-455] that, under appropriate conditions, native insulin can be obtained from scrambled insulin or the S-sulphonates of the chains with a yield of 25-30%, together with reaction products containing the separated A and B chains. The native hormone is by far the predominant product among the isomers containing both chains. It is now shown that the presence of added C peptide has no appreciable effect on the yield of native insulin. At higher temperatures the content of the native hormone decreases whereas those of the separated chains increase, and in no case was scrambled insulin containing both chains the predominant product in the absence of denaturants. Both the scrambling and the unscrambling reactions give similar h.p.l.c. profiles for the products. Under similar conditions cross-linked insulin with native disulphide linkages can be obtained from the scrambled molecule or from the S-sulphonate derivative with yields of 50% and 75% respectively at 4 degrees C, and with a dilute solution of the hexa-S-sulphonate yields better than 90% can be obtained. The regenerated product is shown to have the native disulphide bridges by treatment with CNBr to give insulin and by the identity of the h.p.l.c. profile of its peptic hydrolysate with that for cross-linked insulin. It appears that the insulin A and B chains contain sufficient information for the formation of the native molecule and that the role of the connecting C peptide is to bring and to keep the two chains together.     

Induction of expression of protein disulphide-isomerase during lymphocyte maturation stimulated by bacterial lipopolysaccharide

Protein disulphide-isomerase (PDI) activity, and the level of immunodetectable PDI protein, were monitored in splenic lymphocytes and in BCL1 cells during culture in the presence of various activating factors. Bacterial lipopolysaccharide stimulated induction of PDI in splenic B cells and BCL1 cells. The time-course and specificity of induction indicated that the increase in expression of PDI is closely coupled to the final stages of B cell differentiation into antibody-producing plasma cells. The system will prove valuable in studies on the control of expression of PDI.     

Describing the structure and assembly of protein filaments by EPR spectroscopy of spin-labeled side chains

In this review we summarize our approach to the study of Intermediate Filament (IF) structure and assembly by electron paramagnetic resonance (EPR) spectroscopy of site-directed spin labels. Using vimentin, a homopolymeric type III IF protein, we demonstrate that this approach serves as a general paradigm for studying protein filament structure and assembly. These strategies will be useful in exploring the structure and assembly properties of other filamentous or aggregation-prone systems.     

Structural properties of homogeneous protein disulphide-isomerase from bovine liver purified by a rapid high-yielding procedure.

Protein disulphide-isomerase from bovine liver was purified to homogeneity as judged by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, two-dimensional electrophoresis and N-terminal amino acid analysis. The preparative procedure, a modification of that of Carmichael, Morin & Dixon [(1977) J. Biol. Chem. 252, 7163-7167], is much faster and higher-yielding than previous procedures, and the final purified material is of higher specific activity. The enzyme has Mr 57 000 as determined by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, both in the presence and in the absence of thiol compounds. Gel-filtration studies on Sephadex G-200 indicate an Mr of 107 000, suggesting that the native enzyme is a homodimer with no interchain disulphide bonds. Ultracentrifugation studies give a sedimentation coefficient of 3.5S, implying that the enzyme sediments as the monomer. The isoelectric point, in the presence of 8 M-urea, is 4.2, and some microheterogeneity is detectable. The amino acid composition is comparable with previous analyses of this enzyme from bovine liver and of other preparations of thiol:protein disulphide oxidoreductases whose relation to protein disulphide-isomerase has been controversial. The enzyme contains a very high proportion of Glx + Asx residues (27%). The N-terminal residue is His. The pure enzyme has a very small carbohydrate content, determined as 0.5-1.0% by the phenol/H2SO4 assay. Unless specific steps are taken to remove it, the purified enzyme contains a small amount (5 mol/mol of enzyme) of Triton X-100 carried through the purification.     

Collagenous sequence governs the trimeric assembly of collagen XII

A minicollagen containing the COL1 and NC1 domains of chicken collagen XII has been produced in insect cells. Significant amounts of trimers contain a triple-helical domain in which the cysteines are not involved in inter- but in intrachain bonds. In reducing conditions, providing that the triple-helix is maintained, disulfide exchange between intra- and interchain bonding is observed, suggesting that the triple-helix forms first and that in favorable redox conditions interchain bonding occurs to stabilize the molecule. This hypothesis is verified by in vitro reassociation studies performed in the presence of reducing agents, demonstrating that the formation of interchain disulfide bonds is not a prerequisite to the trimeric association and triple-helical folding of the collagen XII molecule. Shortening the COL1 domain of minicollagen XII to its five C-terminal GXY triplets results in an absence of trimers. This can be explained by the presence of a collagenous domain that is too short to form a stable triple-helix. In contrast, the presence of five additional C-terminal triplets in COL1 allows the formation of triple-helical disulfide-bonded trimers, suggesting that the presence of a triple-helix is essential for the assembly of collagen XII.     

In vivo formation of allosteric aspartate transcarbamoylase containing circularly permuted catalytic polypeptide chains: implications for protein folding and assembly.

Because the N- and C-terminal amino acids of the catalytic (c) polypeptide chains of Escherichia coli aspartate transcarbamoylase (ATCase) are in close proximity to each other, it has been possible to form in vivo five different active ATCase variants in which the terminal regions of the wild-type c chains are linked in a continuous polypeptide chain and new termini are introduced elsewhere in either of the two structural domains of the c chain. These circularly permuted (cp) chains were produced by constructing tandem pyrB genes, which encode the c chain of ATCase, followed by application of PCR. Chains expressed in this way assemble efficiently in vivo to form active, stable ATCase variants. Three such variants have been purified and shown to have the kinetic and physical properties characteristic of wild-type ATCase composed of two catalytic (C) trimers and three regulatory (R) dimers. The values of Vmax for cpATCase122, cpATCase222, and cpATCase281 ranged from 16-21 mumol carbamoylaspartate per microgram per h, compared with 15 for wild-type ATCase, and the values for K0.5 for the variants were 4-17 mM aspartate, whereas wild-type ATCase exhibited a value of 6 mM. Hill coefficients for the three variants varied from 1.8 to 2.1, compared with 1.4 for the wild-type enzyme. As observed with wild-type ATCase, ATP activated the variants containing the circularly permuted chains, as shown by the lowering of K0.5 for aspartate and a decrease in the Hill coefficient (nH). In contrast, CTP caused both an increase in K0.5 and nH for the variants, just as observed with wild-type ATCase. Thus, the enzyme containing the permuted chains with widely diverse N- and C-termini exhibited the homotropic and heterotropic effects characteristic of wild-type ATCase. The decrease in the sedimentation coefficient of the variants caused by the binding of the bisubstrate ligand N-(phosphonacetyl)-L-aspartate (PALA) was also virtually identical to that obtained with wild-type ATCase, thereby indicating that these altered ATCase molecules undergo the analogous ligand-promoted allosteric transition from the taut (T) state to the relaxed (R) conformation. These ATCase molecules with new N- and C-termini widely dispersed throughout the c chains are valuable models for studying in vivo and in vitro folding of polypeptide chains.     

Refolding and assembly of penicillin acylase, an enzyme composed of two polypeptide chains that result from proteolytic activation

The in vitro folding and assembly of penicillin acylase (EC 3.5.1.11) (PA) to active enzyme has been studied. PA is a large bacterial protein (Mr = 86,000) comprising two peptides, alpha and beta, produced by proteolytic processing and activation of a 92-kDa precursor. Proteins that result from proteolytic processing are characteristically difficult if not impossible to refold. Different factors that affect folding and assembly of PA, including pH, ionic strength, and temperature, have been studied. Yields of 60% can be obtained, based on recovery of enzyme activity, together with another 20% of folded and associated monomer with conformation closely similar to that of the active enzyme but with the active site not formed. Evidence is presented for in vitro assembly proceeding via initial folding of the N-terminal alpha-peptide with subsequent collapse of the transiently folded beta-chain on to the surface of the former. A slow process of rearrangement follows association in vitro. Competition experiments support the proposal that the linker endopeptide in the precursor serves to increase the probability of productive collision between folded alpha- and beta-peptides. The effect of raised temperature is to interfere with the folding of the alpha-peptide, thus preventing proper folding of the precursor. This finding accounts for the basis of the temperature regulation of PA production in vivo.     

Folding of polypeptide chains induced by the amino acid side-chains

Conformational calculations with the use of semi-empirical potential functions have been applied to the analysis of the folding of peptide chains. In particular, the part played by the amino acid side-chains in the adoption of folded conformations has been investigated.The results show that the preferred conformations of short peptides are mostly extended ones. However, from a given peptide chain-length, the side-chain to backbone and side-chain to side-chain interactions become strong enough so that definite sequences of amino acids can induce a transition from extended to folded conformations. We propose to call these folded structures “conformational nuclei”. The type of “nucleus” formed is dependent on both the amino acid composition and the sequence.Our results strongly support the hypothesis that folding of polypeptide chains can occur through a nucleation process that could be induced by the side-chains.     

Optical anisotropy of polypeptide chains

Optical anisotropies γ² of N-t-butylacetamide (tBA), N-Methylacetamide (MA), and N, N-dimethylacetamide (DMA) have been determined from the Rayleigh ratios for depolarzed scattering by dilute solutions of the amides in p-dioxane. Traceless optical polarizability tensors \documentclass{article}\pagestyle{empty}\begin{document}$ \widehat{\rm \alpha } $\end{document} for the amides are derived from these results in conjunction with the Kerr constant for tBA determined by LeGèvre and co-workers. It is shown that the tensor \documentclass{article}\pagestyle{empty}\begin{document}$ \widehat{\rm \alpha } $\end{document}_i for the glycyle unit in a polypeptide chain may be identified with \documentclass{article}\pagestyle{empty}\begin{document}$ \widehat{\rm \alpha } $\end{document}MA . Methods for deriving corresponding tensors for other peptide units are indicated and the traceless polarizability tensor \documentclass{article}\pagestyle{empty}\begin{document}$ \widehat{\rm \alpha } $\end{document} for a polypeptide chain in any specified configuration is formulated.     

Comparisons of the polypeptide chains of globins

Summary Vertebrate myoglobins and hemoglobins each consist of units containing a heme group and an associated polypeptide chain. The polypeptide chains form an homologous series and can be compared with each other to measure amino acid differences and minimum base differences per codon. These differences are the result of mutations which have been incorporated during divergent evolution from a common ancestral gene. Each such base replacement is termed an evolutionary event. Each amino acid replacement is the result of one or more evolutionary events. However there can be only one amino acid difference between two sites. The minimum mutational difference between two sites is perceptible only as one, two or three base differences per codon, but there may be more evolutionary events than base differences, because of revertants and multiplicity of base replacements at the same site. When all three bases in a codon are changed, the result is recognizable in only about five per cent of cases. Therefore such recognizable three-base changes indicate a large number of evolutionary events.     

Detection of protein disulphide-isomerase in sheep pancreas fractions

Conclusions The use of diethylpyrocarbonate to inhibit endogenous ribonuclease in sheep pancreas allows the detection of protein-disulphide-isomerase activity in homogenates, at specific activities of up to 4 units/g. This is higher than the specific activity in sheep liver homogenates (about 2 units/g) or in homogenates of other sheep tissues (16). It is thus evident that high levels of protein-disulphide-isomerase activity are present in sheep pancreas. This is consistent both with the postulated general role of protein disulphide-isomerase in protein biosynthesis (10,11) and with the in vitro action of the enzyme on its conventional substrate scrambled ribonuclease, since pancreas is the major site of ribonuclease synthesis.