Complete folding of bovine pancreatic trypsin inhibitor with only a single disulfide bond.

In the oxidative folding of bovine pancreatic trypsin inhibitor (BPTI) at neutral pH, only two one-disulfide intermediates accumulate to a significant extent, namely [5-55] and [30-51]. In this paper we describe a recombinant model of [5-55], designated [5-55]Ala, which was made by replacing the cysteine residues not involved in the disulfide bond with alanine. As judged by two-dimensional NMR, [5-55]Ala folds into essentially the same conformation as native BPTI. Moreover, like native BPTI, [5-55]Ala inhibits trypsin stoichiometrically. Thus, the disulfide-bonded intermediate [5-55] corresponds not to a partially folded protein folding intermediate but rather to an essentially completely folded protein. This conclusion provides an explanation for many of the thermodynamic and kinetic properties of [5-55] in the folding pathway of BPTI.     

Kinetic role of nonnative species in the folding of bovine pancreatic trypsin inhibitor.

We have shown previously that during the oxidative folding of bovine pancreatic trypsin inhibitor only intermediates with native disulfide bonds are well populated. Nevertheless, these studies also confirmed the earlier conclusion [Creighton, T. E. (1977) J. Mol. Biol. 113, 275-293] that the rate-limiting transition in the kinetically preferred route for folding involves intramolecular disulfide bond rearrangements. Consequently, intermediates with nonnative disulfide bonds must form transiently during folding. Two specific nonnative species, denoted [30-51; 5-14] and [30-51; 5-38], in which numbers indicate residues participating in a disulfide bond, can be detected at low levels in kinetic folding experiments with bovine pancreatic trypsin inhibitor. By working with purified reversibly trapped intermediates, the role of these two nonnative species has been examined directly. These species are found to be in relatively rapid exchange with each other and with an initially formed native two-disulfide intermediate [30-51; 14-38]. Thus, the low abundance of the two nonnative species detected in kinetic folding experiments reflects primarily their low thermodynamic stability as compared to this native intermediate. To a small extent, these nonnative species form the productive native intermediate [30-51; 5-55], which is the immediate precursor to the native protein. However, an equal amount of [5-55; 14-38], a nonproductive dead-end intermediate, is also produced. Thus, the nonnative species detected during the folding of bovine pancreatic trypsin inhibitor are not committed to forming the productive native intermediate, nor do they serve to direct folding specifically toward a productive route.     

A possible folding pathway of bovine pancreatic RNase.

A theoretical pathway for the folding of RNase into its native conformation is derived from the contact map computed from crystallographic coordinates. The pathway is based on the hypothesis of Tanaka and Scheraga, according to which localized conformations stabilized by short- and medium-range interactions form before those conformational features that are stabilized primarily by long-range interactions. The pathway deduced from the contact map agrees with experimental information on intermediates detected in the thermal unfolding of RNase and in immunochemical studies on the formation of stable antigenic sites when deduced RNase is oxidized with glutathione. Ambiguities in the interpretation of the contact map are resolved by the combination of structural information contained in the contact map and experimental information.     

Isolation of pancreatic trypsin inhibitor from bovine pituitary glands.

A trypsin inhibitor has been isolated from bovine pituitary extracts. From its amino acid composition, NH2-terminal residue, mobility in paper electrophoresis, and behavior in high-performance liquid chromatography and from the tryptic pattern of the performic acid-oxidized material, it appears that the inhibitor is identical to the Kunitz and Northrop pancreatic trypsin inhibitor.     

Theoretical predictions of folding pathways by using the proximity rule, with applications to bovine pancreatic trypsin inhibitor.

We propose a phenomenological theory that accounts for entropic effects due to loop formation to predict pathways in the kinetics of protein folding. The theory, the basis of which lies in multiple folding pathways and a three-stage kinetics, qualitatively reproduces most of the kinetic measurements in the refolding of bovine pancreatic trypsin inhibitor. The resulting pathways show that nonnative kinetic transients are involved in the productive routes leading to the formation of native intermediates. Our theory emphasizes the importance of the random origin of chain folding initiation structures in directing protein folding.     

Calculation of protein conformation as an assembly of stable overlapping segments: application to bovine pancreatic trypsin inhibitor.

Conformations of bovine pancreatic trypsin inhibitor were calculated by assuming that the final structure as well as properly chosen overlapping segments thereof are simultaneously in low-energy (not necessarily the lowest-energy) conformational states. Therefore, the whole chain can be built up from building blocks whose conformations are determined primarily by short-range interactions. Our earlier buildup procedure was modified by taking account of a statistical analysis of known amino acid sequences that indicates that there is nonrandom pairing of amino acid residues in short segments along the chain, and by carrying out energy minimization on only these segments and on the whole chain [without minimizing the energies of intermediate-size segments (20-30 residues long)]. Results of this statistical analysis were used to determine the variable sizes of the overlapping oligopeptide building blocks used in the calculations; these varied from tripeptides to octapeptides, depending on the amino acid sequence. Successive stages of approximations were used to combine the low-energy conformations of these building blocks in order to keep the number of variables in the computations to a manageable size. The calculations led to a limited number of conformations of the protein (only two different groups, with very similar structure within each group), most residues of which were in the same conformational state as in the native structure.     

Genetic dissection of pancreatic trypsin inhibitor.

In a previous study, a genetic screening procedure was used to identify variants of bovine pancreatic trypsin inhibitor that can fold to an active conformation but that are inactivated much more rapidly than the wild-type protein in the presence of dithiothreitol (DTT). The mechanisms by which 30 of these DTT-sensitive variants are inactivated have now been investigated. Some of the amino acid replacements cause rapid inactivation in the presence of DTT because the three disulfides of the native protein are reduced up to 300-fold faster than for the wild-type protein, leading to complete unfolding. Other substitutions, however, do not greatly increase the rate of complete reduction and unfolding but lead to accumulation of an inactive two-disulfide species. There is a striking correlation between the locations of the DTT-sensitive amino acid replacements in the three-dimensional structure of the protein and the mechanisms by which the variants are inactivated. All of the substitutions that cause rapid unfolding are clustered at one end of the folded protein, in the vicinity of the two disulfides that are reduced most slowly during unfolding of the wild-type protein, while substitutions of the other class are all located at the other end of the protein, near the trypsin binding site. These results indicate that the kinetic stability of native bovine pancreatic trypsin inhibitor and its ability to function as a protease inhibitor are largely influenced by residues in two distinguishable regions of the folded protein.     

Sidechain torsional potentials and motion of amino acids in porteins: bovine pancreatic trypsin inhibitor.

Conformational potentials of sidechains in the bovine pancreatic trypsin inhibitor have been studied with an empirical energy function. Calculated minimumenergy positions are in excellent agreement with the x-ray structure for sidechains in the core or at the surface of the protein; as expected, angles for sidechains that are directed out into the solvent do not agree with the calculated values. The contributions to the potentials are analyzed and compared with the potentials for the free amino acid. Although there is a large restriction in the available conformational space due to nonbonded interactions, the minimum energy positions in the protein are close to those of the free amino acid; the significance of this result is discussed. To estimate the effective barriers for rotation of the aromatic rings (tyrosine and phenylalanine), calculations are done in which the protein is permitted to relax as a function of the ring orientation. Thr resulting barriers, which are much lowere than the rigid rotation barriers, are used to evaluate the rotation rates; comparison is made with the available nuclear magnetic resonance data.     

Tumour-associated trypsin inhibitor in the diagnosis of pancreatic carcinoma

The serum values of tumour-associated trypsin inhibitor (TATI) were measured in a prospective series of 97 patients with jaundice, 36 patients with unjaundiced cholestasis and 21 patients with suspicion of chronic pancreatitis or a pancreatic tumour, to assess its value in diagnosing pancreatic cancer. There were altogether 15 patients with cancer of the pancreas and 2 patients with cancer of the papilla of Vater. The highest serum TATI values were noticed in patients with choledocholithiasis, and raised values were also seen in patients with malignant disease of the liver or bile ducts. In the patients with pancreatic cancer, chronic pancreatitis or benign liver disease, the serum TATI values showed lower levels. The sensitivity of TATI in diagnosing pancreatic cancer was 41.1% with a specificity of 63.5% and an efficiency of 61.0%. In comparison to carcinoembryonic antigen (CEA), carbohydrate antigens CA 50, CA 242, tissue polypeptide antigen and tissue polypeptide-specific antigen, TATI showed a lower diagnostic value. When TATI was analysed in combination with the other markers (two tests positive), the combination of CEA with TATI reached the highest specificity (95.6%), efficiency (89.6%) and positive likelihood ratio (9.3). The results suggest that the diagnostic value of TATI is inferior to that of the established markers, but because of its different nature, it may be of help when used in combination as a complementary serum tumour marker in the diagnosis of pancreatic cancer.Abbreviations TATI tumour-associated trypsin inhibitor - TPA tissue polypeptide antigen - TPS tissue polypeptide-specific antigen - CEA carcinoembryonic antigen - CA carbohydrate antigen     

Isolation of a genomic clone for bovine pancreatic trypsin inhibitor by using a unique-sequence synthetic DNA probe.

Unique-sequence synthetic DNA probes, based on the known amino acid sequence of bovine pancreatic trypsin inhibitor, were constructed from oligodeoxynucleotides. In genomic Southern blot experiments, these probes were shown to hybridize specifically to discrete restriction fragments. A synthetic probe also was used to isolate a cloned BPTI gene from a bovine genomic library. DNA sequence analysis of this clone indicated that the BPTI coding region was neither preceded by a start codon nor immediately followed by a termination codon. This suggests that the mature form of BPTI may be produced through proteolytic processing from a larger polypeptide precursor.     

The measurement of serum immunoreactive pancreatic secretory trypsin inhibitor in gastrointestinal cancer and pancreatic disease

Summary The clinical usefulness of serum pancreatic secretory trypsin inhibitor (PSTI) in pancreatic disease and gastric and colorectal cancer has been examined. The results showed that serum PSTI in acute pancreatitis was significantly higher than in normal subjects and it was also raised in acute exacerbations of chronic pancreatitis. Although the sensitivities of serum PSTI, amylase and elastase I were similar, serum PSTI in necrotizing hemorrhagic pancreatitis was 2.7 times higher than in mild acute pancreatitis. Only a few patients with chronic pancreatitis showed increased concentrations and the mean value was near normal. The mean PSTI in patients with pancreatic and colorectal cancer was higher than normal, although that of gastric cancer was within normal limits. The sensitivity of serum PSTI measurements in patrents with these three malignant diseases was only about 30%. The results suggested that the measurement of serum PSTI could be useful in the diagnosis of acute pancreatitis, but of limited value in the diagnosis of other disease which we examined.     

The measurement of serum immunoreactive pancreatic secretory trypsin inhibitor in gastrointestinal cancer and pancreatic disease

The clinical usefulness of serum pancreatic secretory trypsin inhibitor (PSTI) in pancreatic disease and gastric and colorectal cancer has been examined. The results showed that serum PSTI in acute pancreatitis was significantly higher than in normal subjects and it was also raised in acute exacerbations of chronic pancreatitis. Although the sensitivities of serum PSTI, amylase and elastase I were similar, serum PSTI in necrotizing hemorrhagic pancreatitis was 2.7 times higher than in mild acute pancreatitis. Only a few patients with chronic pancreatitis showed increased concentrations and the mean value was near normal. The mean PSTI in patients with pancreatic and colorectal cancer was higher than normal, although that of gastric cancer was within normal limits. The sensitivity of serum PSTI measurements in patients with these three malignant diseases was only about 30%. The results suggested that the measurement of serum PSTI could be useful in the diagnosis of acute pancreatitis, but of limited value in the diagnosis of other disease which we examined.     

Structure and stability of a second molten globule intermediate in the apomyoglobin folding pathway.

Apomyoglobin folding proceeds through a molten globule intermediate (low-salt form; I1) that has been characterized by equilibrium (pH 4) and kinetic (pH 6) folding experiments. Of the eight alpha-helices in myoglobin, three (A, G, and H) are structured in I1, while the rest appear to be unfolded. Here we report on the structure and stability of a second intermediate, the trichloroacetate form of the molten globule intermediate (I2), which is induced either from the acid-unfolded protein or from I1 by > or = 5 mM sodium trichloroacetate. Circular dichroism measurements monitoring urea- and acid-induced unfolding indicate that I2 is more highly structured and more stable than I1. Although I2 exhibits properties closer to those of the native protein, one-dimensional NMR spectra show that it maintains the lack of fixed side-chain structure that is the hallmark of a molten globule. Amide proton exchange and 1H-15N two-dimensional NMR experiments are used to identify the source of the extra helicity observed in I2. The results reveal that the existing A, G, and H helices present in I1 have become more stable in I2 and that a fourth helix--the B helix--has been incorporated into the molten globule. Available evidence is consistent with I2 being an on-pathway intermediate. The data support the view that apomyoglobin folds in a sequential fashion through a single pathway populated by intermediates of increasing structure and stability.     

Crystal structure of an engineered subtilisin inhibitor complexed with bovine trypsin.

Proteinase specificity of a proteinaceous inhibitor of subtilisin (SSI; Streptomyces subtilisin inhibitor) can be altered so as to strongly inhibit trypsin simply by replacing P1 methionine with lysine (with or without concomitant change of the P4 residue) through site-directed mutagenesis. Now the crystal structure of one such engineered SSI (P1 methionine converted to lysine and P4 methionine converted to glycine) complexed with bovine trypsin has been solved at 2.6 A resolution and refined to a crystallographic R factor of 0.173. Comparing this structure with the previously established structure of the native SSI complexed with subtilisin BPN', it was found that (i) P1 lysine of the mutant SSI is accommodated in the S1 pocket of trypsin as usual, and (ii) upon complex formation, considerable conformation change occurs to the reactive site loop of the mutant SSI. Thus, in this case, flexibility of the reactive site loop seems important for successfully changing the proteinase specificity through mere replacement of the P1 residue.     

Gastric output of pancreatic secretory trypsin inhibitor is increased by misoprostol.

Pancreatic secretory trypsin inhibitor (PSTI) is a potent protease inhibitor that also has growth promoting activity. It has recently been identified in the foveolar cells of the stomach, which secrete mucus. We examined the effects of the prostaglandin E1 analogue misoprostol on gastric PSTI output. Seven normal volunteers took part. An initial period of gastric aspiration was followed by four 40 minute periods of gastric perfusion at 5 ml/minute of: 0.14 mol/l saline, 0.17 mmol/l bicarbonate, bicarbonate with misoprostol 400 micrograms, and then bicarbonate again. All perfusates contained polyethylene glycol 4000 as a marker. Misoprostol increased median gastric secretion of PSTI from 11 to 33 micrograms/hour (p less than 0.05), producing concentrations in gastric juice six times higher than those found in jejunal juice and about 1/30 of the values seen in pancreatic juice. Median mucus secretion increased to a lesser extent from 29 to 38 mg/hour during misoprostol. There was no change in intragastric concentrations of protein or of epidermal growth factor during infusion of misoprostol. Infusion of pentagastrin (6 micrograms/kg/hour) had no effect on gastric secretion of mucus, PSTI, or protein. Human gastric mucus was degraded on incubation with trypsin in vitro and this was prevented by the addition of PSTI. These results suggest that gastric PSTI may protect the gastric mucus layer against refluxed pancreatic proteases. Increased output of PSTI during microprostol may contribute to the protective effect of this drug.     

Guanidine hydrochloride stabilization of a partially unfolded intermediate during the reversible denaturation of protein disulfide isomerase.

The reversible denaturation of protein disulfide isomerase proceeds through intermediates that are stabilized by interaction with guanidine hydrochloride. At pH 7.5, the equilibrium denaturation by urea is completely reversible and the transition can be reasonably well-described by a two-state model involving only native and denatured forms. In comparison, the equilibrium denaturation by guanidine hydrochloride occurs in two distinct steps. In the presence of a low constant amount of guanidine hydrochloride (0.5-1.4 M), urea denaturation also becomes biphasic, suggesting the accumulation of an intermediate species that is stabilized by specific interaction with guanidine hydrochloride but not by high concentrations of other salts or other denaturants.