Unfolded Bovine Pancreatic Trypsin Inhibitor Research Articles

Chaperone properties of Escherichia coli thioredoxin and thioredoxin reductase.

Thioredoxin, thioredoxin reductase and NADPH form the thioredoxin system and are the major cellular protein disulphide reductase. We report here that Escherichia coli thioredoxin and thioredoxin reductase interact with unfolded and denatured proteins, in a manner similar to that of molecular chaperones that are involved in protein folding and protein renaturation after stress. Thioredoxin and/or thioredoxin reductase promote the functional folding of citrate synthase and alpha-glucosidase after urea denaturation. They also promote the functional folding of the bacterial galactose receptor, a protein without any cysteines. Furthermore, redox cycling of thioredoxin/thioredoxin reductase in the presence of NADPH and cystine stimulates the renaturation of the galactose receptor, suggesting that the thioredoxin system functions like a redox-powered chaperone machine. Thioredoxin reductase prevents the aggregation of citrate synthase under heat-shock conditions. It forms complexes that are more stable than those formed by thioredoxin with several unfolded proteins such as reduced carboxymethyl alpha-lactalbumin and unfolded bovine pancreatic trypsin inhibitor. These results suggest that the thioredoxin system, in addition to its protein disulphide isomerase activity possesses chaperone-like properties, and that its thioredoxin reductase component plays a major role in this function.

Native-like interactions favored in the unfolded bovine pancreatic trypsin inhibitor have different roles in folding.

Folding kinetics of a series of bovine pancreatic trypsin inhibitor (BPTI) variants with similar stabilities and structures have been measured. All are strongly destabilized relative to WT. In Y21A, F22A, Y23A, G37A, and F45A, the three native disulfide bonds are retained. In RM(14-38), Cys14 and Cys38 thiols are methylated while C30-C51 and C5-C55 disulfides remain intact. At pH 2 and 20 degrees C, relaxation rate constants of the major kinetic phase range from approximately 10 ms to 0.71 s in the absence of denaturant. All mutants except G37A exhibit standard two-state behavior. Y21A, F22A, and Y23A fold much more slowly than other mutants. The experiments were designed to test the hypothesis that native-like structure detected in the unfolded BPTI is important in folding. Two native-like contacts are implied by NOEs in reduced and unfolded BPTI, between residues Tyr23 and Ala25, and between Gly37 NH and the Tyr35 ring. The results support an earlier hypothesis that formation of the central beta-hairpin, monitored by a local native interaction between Tyr23 and Ala25, is crucial to initiation of BPTI folding. The second native-like contact is important, not in folding initiation, but in preventing a kinetic trap later in the process. Evidence for this comes from mutant G37A, which behaves very differently from the others in displaying a phenomenon called rollover. G37A is, to our knowledge, the first reported case in which a single-site replacement causes rollover, while the wild type and all other known mutants of the same protein show typical two-state chevron plots. The best explanation is that the G37A mutation introduces a kinetic trap of the type described by Chan and Dill [(1998) Proteins 30, 2-33]. In native BPTI, there is an unusual polar interaction between the ring of Tyr35 and the backbone NH of Gly37. Our results suggest that the NH-aromatic interaction between residues 37 and 35 is important throughout folding in stabilizing native-like loop conformations and in preventing the flexible loops from being trapped in nonfunctional conformations during later stages of folding.

Electron Spin Resonance and Fluorescence Studies of the Bound-state Conformation of a Model Protein Substrate to the Chaperone SecB

SecB is a homotetrameric, cytosolic chaperone that forms part of the protein translocation machinery in Escherichia coli. We have investigated the bound-state conformation of a model protein substrate of SecB, bovine pancreatic trypsin inhibitor (BPTI) as well as the conformation of SecB itself by using proximity relationships based on site-directed spin-labeling and pyrene fluorescence methods. BPTI is a 58-residue protein and contains three disulfide groups between residues 5 and 55, 14 and 38, as well as 30 and 51. Mutants of BPTI that contained only a single disulfide were reduced, and the free cysteines were labeled with either thiol-specific spin labels or pyrene maleimide. The relative proximity of the labeled residues was studied using either electron spin resonance spectroscopy or fluorescence spectroscopy. The data suggest that SecB binds a collapsed coil of reduced unfolded BPTI, which then undergoes a structural rearrangement to a more extended state upon binding to SecB. Binding occurs at multiple sites on the substrate, and the binding site on each SecB monomer accommodates less than 21 substrate residues. In addition, we have labeled four solvent-accessible cysteine residues in the SecB tetramer and have investigated their relative spatial arrangement in the presence and absence of the substrate protein. The electron spin resonance data suggest that these cysteine residues are in close proximity (15 A) when no substrate protein is bound but move away to a distance of greater than 20 A when SecB binds substrate. This is the first direct evidence of a conformational change in SecB upon binding of a substrate protein.

Stability of the residual structure in unfolded BPTI in different conditions of temperature and solvent composition measured by disulphide kinetics and double mutant cycle analysis

The folding funnel model proposes a clear description of the protein folding process. To test this model, additional data on the structures populated in different stages of folding and their influence on further folding are required. Here, we use the double mutant strategy and disulphide formation kinetics measurements to study the impact on folding of the residual structure in unfolded bovine pancreatic trypsin inhibitor (BPTI). We show how five amino acid residues stabilise a folding initiation site, possibly a β-hairpin, and influence the shape of the upper region of the folding funnel in BPTI in different conditions of temperature and solvent composition. Our data provide experimental evidence for the mechanism by which a fast search for a proper chain topology is made possible early in the folding of proteins. The results apply to proteins in general, not necessarily just to disulphide bonded proteins, since cysteine residues are used here merely as reporter groups.

Chaperone Properties of Bacterial Elongation Factor EF-Tu

Elongation factor Tu (EF-Tu) is involved in the binding and transport of the appropriate codon-specified aminoacyl-tRNA to the aminoacyl site of the ribosome. We report herewith that the Escherichia coli EF-Tu interacts with unfolded and denatured proteins as do molecular chaperones that are involved in protein folding and protein renaturation after stress. EF-Tu promotes the functional folding of citrate synthase and alpha-glucosidase after urea denaturation. It prevents the aggregation of citrate synthase under heat shock conditions, and it forms stable complexes with several unfolded proteins such as reduced carboxymethyl alpha-lactalbumin and unfolded bovine pancreatic trypsin inhibitor. The EF-Tu.GDP complex is much more active than EF-Tu.GTP in stimulating protein renaturation. These chaperone-like functions of EF-Tu occur at concentrations that are at least 20-fold lower than the cellular concentration of this factor. These results suggest that EF-Tu, in addition to its function in translation elongation, might be implicated in protein folding and protection from stress.

Reduced BPTI is collapsed. A pulsed field gradient NMR study of unfolded and partially folded bovine pancreatic trypsin inhibitor.

Pulsed field gradient NMR was used to measure the hydrodynamic behavior of unfolded variants of bovine pancreatic trypsin inhibitor (BPTI). The unfolded BPTI species studied were [R]Abu, at pH 4.5 and pH 2.5, and unfolded [14-38]Abu, at pH 2.5. These were prepared by chemical synthesis. [R]Abu is a model for reduced BPTI; all cysteine residues are replaced by alpha-amino-n-butyric acid (Abu). [14-38]Abu retains cysteines 14 and 38, which form a disulfide bond, while the other cysteine residues are replaced by Abu. In the PFG experiments, the diffusion coefficient is measured as a function of protein concentration, and the value of D degree -the diffusion coefficient extrapolated to infinite dilution-is determined. From D degree, a value of the hydrodynamic radius. Rh, is computed from the Stokes-Einstein relationship. At pH 4.5, [R]Abu has an Rh value significantly less than the value calculated for a random coil, while at pH 2.5 the experimental Rh value is the same as for a random coil. In view of the changes in NMR detected structure of [R]Abu at pH 4.5 versus pH 2.5 (Pan H, Barbar E, Barany G, Woodward C. 1995. Extensive non-random structure in reduced and unfolded bovine pancreatic trypsin inhibitor. Biochemistry 34:13974-13981), the collapse of reduced BPTI at pH 4.5 may be associated with the formation of non-native hydrophobic clusters of pairs of side chains one to three amino acids apart in sequence. The diffusion constant of [14-38]Abu was also measured at pH 4.5, where the protein is partially folded. An increase in hydrodynamic radius of partially folded [14-38]Abu, relative to native BPTI, is similar to the increase in radius of gyration measured for other proteins under "molten globule" conditions.

Chaperone properties of the bacterial periplasmic substrate-binding proteins.

Bacterial periplasmic substrate-binding proteins are initial receptors in the process of active transport across cell membranes and/or chemotaxis. Each of them binds a specific substrate (e.g. sugar, amino acid, or ion) with high affinity. For transport, each binding protein interacts with a cognate membrane complex consisting of two hydrophobic proteins and two subunits of a hydrophilic ATPase. For chemotaxis, binding proteins interact with specific membrane chemotaxis receptors. We report, herewith, that the oligopeptide-binding protein OppA of Escherichia coli, the maltose-binding protein MalE of E. coli, and the galactose-binding protein MglB of Salmonella typhimurium interact with unfolded and denatured proteins, such as the molecular chaperones that are involved in protein folding and protein renaturation after stress. These periplasmic substrate-binding proteins promote the functional folding of citrate synthase and alpha-glucosidase after urea denaturation. They prevent the aggregation of citrate synthase under heat shock conditions, and they form stable complexes with several unfolded proteins, such as reduced carboxymethyl alpha-lactalbumin and unfolded bovine pancreatic trypsin inhibitor. These chaperone-like functions are displayed by both the liganded and ligand-free forms of binding proteins, and they occur at binding protein concentrations that are 10-100-fold lower than their periplasmic concentration. These results suggest that bacterial periplasmic substrate-binding proteins, in addition to their function in transport and chemotaxis, might be implicated in protein folding and protection from stress in the periplasm.

Extensive nonrandom structure in reduced and unfolded bovine pancreatic trypsin inhibitor.

Two-dimensional 1H NMR spectra of an analog of reduced BPTI at pH 4.5, 1 degrees C, have been assigned. Spectra indicate considerable conformational averaging, as expected for a flexible, unfolded protein. The presence of extensive nonrandom structure is detected by the presence of NHi-NHi + 1 and aromatic-aliphatic NOEs. Sequential amide-amide NOEs indicate that turn-like conformations are significantly populated at 18 pairs of residues along the chain. Many of these are located in a turn, loop, or helix in native BPTI, but six are observed for contiguous pairs in the segment composed of residues 29-35, which in native BPTI constitute a strand of extended sheet. A novel finding for unfolded proteins is our observation of NOEs implying non-native hydrophobic interactions. Multiple aromatic-aliphatic NOEs are observed for pairs of residues that are within 1-3 residues of each other. Most are non-native and involve residues in both strands of the central antiparallel strand-turn-strand of native BPTI comprised of residues 18-35. All NOEs reported for oligopeptides spanning the BPTI sequence [Kemmink, J., & Creighton, T. (1993) J. Mol. Biol. 234, 861-878] are observed in reduced BPTI, but many others are present as well. Similar spectra are obtained for naturally occurring BPTI reduced by dithiothreitol, BPTI with cysteines replaced by alpha-amino-n-butyric acid, and BPTI mutant F45A reduced by dithiothreitol.(ABSTRACT TRUNCATED AT 250 WORDS)

Autostimulation of the DnaK (HSP 70) ATPase of Escherichia coli

The ATPase activity of DnaK, the 70-kDa chaperone of Escherichia coli, is stimulated by an unfolded protein. However, the stimulation of the DnaK ATPase by unfolded bovine pancreatic trypsin inhibitor can only be observed at low DnaK protein concentrations. At higher DnaK concentrations, the ATPase activity of DnaK cannot be stimulated by the addition of unfolded bovine pancreatic trypsin inhibitor. This is a consequence of the autostimulation of the DnaK ATPase at higher DnaK concentrations. The autostimulation of DnaK is reflected by a non-linear dependence of ATP hydrolysis on DnaK concentration. Furthermore, DnaK exists as a mixture of monomers and dimers in equilibrium, and the dimers dissociate into monomers in the presence of ATP.

Small effects of amino acid replacements on the reduced and unfolded state of pancreatic trypsin inhibitor.

The effects of amino acid replacements on the hydrodynamic volume of reduced and unfolded bovine pancreatic trypsin inhibitor (BPTI) have been examined by gel electrophoresis. The electrophoretic mobilities of the reduced forms of 46 BPTI variants were compared at room temperature in the absence of denaturants. The single substitutions examined include many different types of replacements at sites throughout the polypeptide, and, collectively, alter 22 of the 58 residues of the wild-type protein. The only substitutions found to alter the electrophoretic mobility of the reduced protein by more than approximately 3% are those that change the net charge of the protein. For nine mutants, the rates of disulfide formation in the reduced protein were also examined and found to be very similar to that of the wild-type protein. These results suggest that any structure that may be present in the reduced protein is either relatively insensitive to amino acid replacements or does not greatly influence the averaged properties of the polypeptide chain.

Nonlocal interactions stabilize compact folding intermediates in reduced unfolded bovine pancreatic trypsin inhibitor.

To further our understanding of the protein folding process, it is desirable to examine the structural intermediates (equilibrium and kinetic) that are populated between the statistical coil state and the folded molecule. X-ray crystallography and NMR structural studies are unable to determine long-range distances in proteins under denaturing solution conditions. Nonradiative (Förster) energy transfer, however, has been shown to be a spectroscopic ruler for the measurement of distance distributions and diffusion between selected sites in proteins under a range of different solution conditions. The distributions of distances between a donor probe at the N-terminal residue and an acceptor attached to one of the four lysine residues (15, 26, 41, 46) of reduced and unfolded (in 6 M guanidine hydrochloride and 20 mM dithiothreitol) bovine pancreatic trypsin inhibitor (BPTI) were measured as a function of temperature. Even in strong denaturant and reducing agent, BPTI does not exist as a statistical coil polypeptide. It appears that nonlocal (long-range) interactions are already beginning to "fold" the protein toward a more compact, native conformation. As the temperature is increased under these conditions, hydrophobic interactions lead to an even more compact structure consistent with the predictions of phase diagrams for globular proteins.

Detection of local structures in reduced unfolded bovine pancreatic trypsin inhibitor.

The structure of BPTI and reduced BPTI in concentrated guanidinium HCl (GUHCl) in the presence of glycerol has been probed by measurements of dynamic nonradiative excitation energy transfer between probes attached to its amino groups. Interprobe distance distributions were obtained from analysis of donor fluorescence decay curves and used to characterize local structures in unordered states of the protein. Site specifically fluorescently labeled BPTI derivatives (1-n)BPTI (n = 15, 20, 41, 46) were used, each carrying a 2-methoxy-naphthyl-1-methylenyl group (MNA) at the N-terminal amino group of arg1 and 7-(dimethylamino)-coumarin-4-yl-acetyl residue (DA-coum) at one of its epsilon-NH2 groups of the lysine side chains. Analysis of donor fluorescence decay kinetics gave the interprobe distance distributions in the native and denatured states. The N-terminal-segment, residues 1-15, is in an extended conformation (with an average interprobe distance of 34 +/- 2 A) in the native state. Upon unfolding by reduction with DTT or beta-mercapto ethanol in 6 M GUHCl/glycerol mixture, the conformation of this segment relaxed to a state characterized by a reduced average interprobe distance and a larger width of the distances distribution. The average distance between residues 1 and 26, i.e., between the N-terminus and the turn of the twisted beta sheet element (residues 18-35), increased upon unfolding. At -30 degrees C in the above solvent, the distribution between these two sites was probably composed of two conformational subpopulations. About 45 +/- 20% of the molecules were characterized by a short interprobe distance (like the native state) representing a compact conformation, and 55 +/- 20% of the molecules showed large interprobe distances representing an expanded (unfolded) conformation. Thus local structures seem to exist in reduced denatured BPTI even under denaturing conditions in 6 M GUHCl/glycerol mixtures. Some of those structures are unstable in guanidinium isothiocyanate (GUSCN). The method introduced here is suitable for probing local structures and very long range interactions in unfolded proteins and for search for folding initiation sites (FISs) and early folding intermediates.