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C Woodward

Publications and source records attributed to C Woodward.

At least 55 records · Page 3Linked to original sources
PubMed1995

Partially folded, molten globule and molten coil states of bovine pancreatic trypsin inhibitor.

Three denatured states of bovine pancreatic trypsin inhibitor have been characterized, using two chemically synthesized analogues designed for study of folding intermediates. One analogue, [14-38]Abu, retains only the 14-38 disulphide. At pH 4.5-6 and 1-7 degrees C, [14-38]Abu is a highly ordered beta-sheet molten globule; it has the circular dichroism (CD), ANS-binding and folding kinetics of a molten globule; is partially folded by NMR analysis; and undergoes cooperative thermal denaturation. At low temperature [14-38]Abu also forms an acid state at pH 1.5, as well as a denatured state at pH 2.5. A second BPTI analogue with all three disulphide bridges eliminated, [R]Abu, lacks detectable secondary and tertiary structure but has stable hydrophobic surfaces and is collapsed. We term this species a 'molten coil'.

Anilino Naphthalenesulfonates↗
PubMed1995

Unfolded BPTI variants with a single disulfide bond have diminished non-native structure distant from the crosslink.

Backgound. NMR studies of denatured states, both fully unfolded and partially folded, give insight into the conformations and interactions favored in initial stages of folding, and in early intermediates formed during folding. We have characterized non-random structures favored in unfolded, reduced BPTI [1], and in partially folded BPTI [2]. Here, we report NMR-detected structure of two analogs of unfolded BPTI with one native 14-38 disulfide bond. Results. Analogs Y21A[14-38]Abu and Y23A[14-38]Abu, obtained by chemical synthesis of [14-38]Abu with Y21 or Y23 replaced by alanine, are models for unfolded BPTI with 14-38 the only disulfide. Compared to unfolded BPTI with all three disulfides broken, the unfolded 14-38 BPTI analogs have numerous differences, including loss of non-native, turn-like conformations for beta2 residues, diminished non-native aromatic-aliphatic NOEs, and increased intermediate chemical exchange of residues that have native-like conformations in partially folded BPTI. Although the Y21A and Y23A analogs have similar CD and NMR properties, specific differences in NOE patterns and in exchange broadening are observed. Conclusion. Changes in unfolded BPTI associated with formation of the 14-38 disulfide bond are consistent with less non-native structure, and more native-like structure, in residues composing the stable core of antiparallel beta-sheet in partially folded BPTI. Specific differences between Y21A[14-38]Abu and Y23A[14-38]Abu indicate that replacement of Y23 results in less ordered structure than replacement of Y21.

Journal Article↗
PubMed1994

Patient-physician sexual involvement: a Canadian survey of obstetrician-gynecologists.

OBJECTIVE: To determine obstetrician-gynecologists' (ob-gyns') awareness of and experience with sexual abuse of patients and former patients and their opinions about appropriate consequences. DESIGN: Mailed survey. SETTING: Canada. PARTICIPANTS: All 792 members of the Society of Obstetricians and Gynaecologists of Canada (SOGC); 618 (78%) responded. Approximately half of all ob-gyns in Canada belong to the SOGC. MAIN OUTCOME MEASURES: Knowledge of sexual involvement by an ob-gyn colleague with a patient or former patient (as defined by the respondents and by the College of Physicians and Surgeons of Ontario [CPSO]), self-report of such involvement, attitudes toward physician sexual abuse, desirable length of time a physician should wait before seeing a former patient in a situation that could lead to a sexual encounter, suggested consequences of sexual abuse. RESULTS: Overall, 10% of the respondents indicated that they knew about another ob-gyn who at some time had been sexually involved with a patient. In all, 3% of the male respondents and 1% of the female respondents reported sexual involvement with a patient; the corresponding proportions of those who reported having been accused of sexual abuse by a patient were 4% and 2%. Significantly more of the female ob-gyns than of their male counterparts (37% v. 19%) reported awareness of a colleague's sexual involvement with a patient that would meet the CPSO's definition of sexual impropriety, transgression or violation. Most of the respondents felt that the consequence of proven sexual impropriety should be reprimand and fine (chosen by 33%) or rehabilitation without loss of licence (28%). Most of the physicians supported loss of licence for proven sexual transgression (57%) or proven sexual violation (74%), but fewer felt that loss of licence should be permanent for these types of abuse (4% and 24% respectively). The female ob-gyns supported stronger sanctions against sexual transgression and sexual violation than the male ob-gyns. A wide range of opinion was seen regarding the propriety of sexual relationships with former patients. CONCLUSIONS: Ob-gyns have varied opinions about how sexual abuse of patients should be defined and how it should be sanctioned. There is a discrepancy between proposed public policy and the beliefs of physicians to whom the policy is to be applied.

Canada↗
PubMed1993

Protein internal flexibility and global stability: effect of urea on hydrogen exchange rates of bovine pancreatic trypsin inhibitor.

The hydrogen isotope exchange kinetics of buried NH protons in bovine pancreatic trypsin inhibitor (BPTI) was measured in 8 M urea at 30 degrees C and pH 3.5. The data were analyzed by the two-process model in which slower exchanging protons utilize an unfolding mechanism and more rapidly exchanging protons exchange from the folded state. Urea accelerates the set of protons exchanging by the unfolding mechanism, all of which have approximately the same exchange rate constants in urea. For protons in this set, the ratio of exchange rate constants in the presence and absence of urea is used to estimate delta delta G(0-->8M urea) = 6.6 kcal/mol. For the set of protons exchanging from the folded state, 8 M urea either has no effect or slows exchange. Slowing of exchange by urea implies binding of urea to sites at or near the exchanging proton. Some buried protons exchanging from the folded state have diminished rates in 8 M urea, meaning that urea is accessible to these buried sites. Several unassigned side-chain NH's of arginine or lysine are highly protected from exchange by urea, suggesting that they are the location of urea binding sites on the surface of the molecule.

Animals↗
PubMed1993

Determination of the pKa values of active-center cysteines, cysteines-32 and -35, in Escherichia coli thioredoxin by Raman spectroscopy.

We have determined the pKa values for thiol-thiolate equilibria of Escherichia coli thioredoxin and compared structural properties of reduced and oxidized forms of the protein in solution by Raman spectroscopy. Ionization and hydrogen-bonding states of the two cysteine sulfhydryls (Cys32 and Cys35) in reduced thioredoxin were determined by monitoring the complex Raman SH stretching band (Li & Thomas, 1991) as a function of pH in the range 4.0 < pH < 12.2. The Raman SH markers indicate the following: (i) Both sulfhydryls of the native protein are relatively robust hydrogen-bond donors, but one is a stronger hydrogen-bond donor than the other. (ii) The sulfhydryl which donates the weaker hydrogen bond, assigned tentatively to Cys32, is preferentially titrated to the thiolate ion (S-) as the solution pH is increased from 4 to 7. Water or a hydroxyl oxygen is the probable acceptor for the Cys32 S-H donor. (iii) The sulfhydryl which donates the stronger hydrogen bond, Cys35, resists substantial ionization until roughly 50% of the more acidic sulfhydryl has been titrated. A carbonyl oxygen is proposed as the likely acceptor of the Cys35 S-H donor. (iv) The Raman titration data indicate pK1 = 7.1 +/- 0.2 and pK2 = 7.9 +/- 0.2 for the two thiol-thiolate equilibria. The lower pKa, which is the more strongly perturbed, is assigned tentatively to Cys32.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Crevice-forming mutants in the rigid core of bovine pancreatic trypsin inhibitor: crystal structures of F22A, Y23A, N43G, and F45A.

Crystal structures of four mutants of bovine pancreatic trypsin inhibitor (F22A, Y23A, N43G, and F45A), engineered to alter their stability properties, have been determined. The mutated residues, which are highly conserved among Kunitz-type inhibitors, are located in the rigid core of the molecule. Replacement of the partially buried bulky residues of the wild-type protein with smaller residues resulted in crevices open to the exterior of the molecule. The overall three-dimensional structure of these mutants is very similar to that of the wild-type protein and only small rearrangements are observed among the atoms lining the crevices.

Animals↗
PubMed1993

Crevice-forming mutants of bovine pancreatic trypsin inhibitor: stability changes and new hydrophobic surface.

Four mutants of bovine pancreatic trypsin inhibitor (BPTI) with replacements in the rigid core result in the creation of deep crevices on the surface of the protein. Other than crevices at the site of the mutation, few other differences are observed in the crystal structures of wild-type BPTI and the mutants F22A, Y23A, N43G, and F45A. These mutants are highly destabilized relative to wild type (WT). The differences between WT and mutants in the free energy change associated with cooperative folding/unfolding, delta delta G0 (WT-->mut), have been measured by calorimetry, and they are in good agreement with delta delta G0(WT-->mut) values from hydrogen exchange rates. For F22A the change in free energy difference is about 1.7 kcal/mol at 25 degrees C; for the other three mutants it is in the range of 5-7 kcal/mol at 25 degrees C. The experimental delta delta G0(WT-->mut) values of F22A, Y23A, and F45A are reasonably well accounted for as the sum of two terms: the difference in transfer free energy change, and a contribution from exposure to solvent of new surface (Eriksson, A.E., et al., 1992, Science 255, 178-183), if the recently corrected transfer free energies and surface hydrophobicities (De Young, L. & Dill, K., 1990, J. Phys. Chem. 94, 801-809; Sharp, K.A., et al., 1991a, Science 252, 106-109) are used and only nonpolar surface is taken into account. In N43G, three protein-protein hydrogen bonds are replaced by protein-water hydrogen bonds.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1993

Thermodynamics of BPTI folding.

A calorimetric study of the basic pancreatic trypsin inhibitor (BPTI) has been performed using the new generation of the adiabatic scanning microcalorimeters, operating in an extended temperature range of 5-130 degrees C. Precise measurements of the heat capacities of the native and unfolded states of BPTI show that the heat capacity change upon unfolding strongly depends on temperature; its value is maximal at about 50 degrees C and diminishes as the temperature is increased. The temperature dependencies of the enthalpy and entropy changes upon BPTI unfolding were found to be similar to those normally observed for other small globular proteins. The stability of BPTI has been correlated with its structure.

Aprotinin↗
PubMed1992

Identification of the three-dimensional thioredoxin motif: related structure in the ORF3 protein of the Staphylococcus aureus mer operon.

We have developed a computerized search pattern for recognition of the three-dimensional redox site of thioredoxins based on primary and predicted secondary structure. This pattern, developed in the ARIADNE protein expert system, is used to search for thioredoxin-like tertiary structural motif among proteins for which the only structural information is the primary sequence. The pattern was trained on 102 protein sequences (25 functionals and 77 controls); it matches all 25 members of the functional set under cutoff conditions that include only 2 members of the control set, for a sensitivity of 1.0 and a specificity of 0.97. The pattern matches only one of the two thioredoxin-like domains in protein disulfide isomerases (PDIs) and their analogues, suggesting that the C-terminal domain is more structurally similar to thioredoxin than the N-terminal domain. The Escherichia coli DsbA protein, a possible PDI analogue, appears to be more structurally similar to the N-terminal thioredoxin-like domain of PDIs. Thioredoxin-like redox functionality has been proposed for lutropin and follitropin, in part on the basis of their having -Cys-X-Pro-Cys- sequences. None match our pattern; all lack a predicted alpha-helix pattern element immediately after the active site. Hypothetical proteins in the National Biomedical Research Foundation Protein Identification Resource database were searched for matches to the pattern. The most interesting match was a hypothetical protein (161 residues) from the third open reading frame in the Staphylococcus aureus mer operon, which is involved in mercury detoxification. The match to our pattern and the hydrophobicity distribution in aligned elements of secondary structure not in our pattern strongly suggest that it has thioredoxin-like structure.

Amino Acid Sequence↗
PubMed1992

Comparison of hydrogen exchange rates for bovine pancreatic trypsin inhibitor in crystals and in solution.

Hydrogen exchange rate constants for the 17 slowest exchanging amide NH groups in bovine pancreatic trypsin inhibitor (BPTI) were measured in solution and in form II and form III crystals. All 17 amide hydrogens are buried and intramolecularly hydrogen bonded in the crystal structure, except Lys 41 which is buried and hydrogen bonded to a buried water. Large-scale crystallization procedures were developed for these experiments, and rate constants for both crystal and solution exchange were measured by 1H NMR spectroscopy of exchange-quenched samples in solution. Two conditions of pH and temperature, pH 9.8 and 35 degrees C, and pH 9.4 and 25 degrees C, bring two groups of hydrogens into the experimental time window (minutes to weeks). One consists of the 10 slowest exchanging hydrogens, all of which are associated with the central beta-sheet of BPTI. The second group consists of seven more rapidly exchanging hydrogens, which are distributed throughout the molecule, primarily in a loop or turn. In both groups, most hydrogens exchange more slowly in crystals, but there is considerable variation in the degree to which the exchange is depressed in crystals. Many differences observed for the more rapidly exchanging hydrogens can be attributed to local surface effects arising from intermolecular contacts in the crystal lattice. Within the slower group, however, a very large effect on exchange of Ile 18 and Tyr 35 appears to be selectively transmitted through the matrix of the molecule.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1991

Crystal structure of a Y35G mutant of bovine pancreatic trypsin inhibitor.

The structure of a Y35G mutant of bovine pancreatic trypsin inhibitor (BPTI) was solved by molecular replacement and was refined by both simulated annealing and restrained least-squares at 1.8 A resolution. The crystals belong to the space group P42212, with unit cell dimensions a = b = 46.75 A, c = 50.61 A. The final R-factor is 0.159 and the deviation from ideality for bond distances is 0.02 A. The structure of the mutant differs from that of the native protein, showing an overall root-mean-square (r.m.s.) difference of 1.86 A for main-chain atoms. However, the change is mostly localized in the two loops (respective r.m.s. values of 2.04 A and 3.93 A) and the C terminus (r.m.s. 6.79 A), while the core of the protein is well conserved (r.m.s. 0.45 A). The change in the loop regions can be clearly attributed to the mutation while the difference in the C terminus might be only due to a different crystal packing. Seventy water molecules were included in the model but only seven of them are shared with the native structure. Thermal parameters are showing a good correlation with those for the wild-type of BPTI.

Amino Acid Sequence↗
PubMed1991

The conserved, buried aspartic acid in oxidized Escherichia coli thioredoxin has a pKa of 7.5. Its titration produces a related shift in global stability.

Aspartic acid 26 in Escherichia coli thioredoxin is located at the bottom of a hydrophobic cavity, near the redox-active disulfide of the active site. Asp 26 is embedded in the protein except for part of the surface of one carboxyl oxygen. The high degree of evolutionary conversion of Asp 26 suggests that it plays a critical role in thioredoxin function. We have determined the pKa of Asp 26 by a novel electrophoretic method based on the relative electrophoretic mobilities of wild-type thioredoxin and of D26A thioredoxin (with Asp 26 replaced by alanine). The pKa of Asp 26 determined by this technique is 7.5, more than 3 units above the pKa of a solvated carboxyl side chain. The titration of Asp 26 is thermodynamically linked to the stability of thioredoxin. As expected if thioredoxin stability depends on the ionization state of Asp 26, delta Go WT, the free energy of the cooperative denaturation reaction of wild-type thioredoxin by guanidine hydrochloride, varies with pH in a sigmoidal fashion in the vicinity of pH 7.5. Over the same pH range, the free energy for D26A folding, delta Go D26A, is pH independent and D26A is highly stabilized compared to wild type. From the thermodynamic cycle describing the linkage of Asp 26 titration to thioredoxin stability, the difference in free energy between wild-type thioredoxin with protonated Asp 26 and wild-type thioredoxin with deprotonated Asp 26, delta delta Go (COOH----COO-), is calculated to be 4.9 kcal/mol.(ABSTRACT TRUNCATED AT 250 WORDS)

Alanine↗
PubMed1991

Linkage of thioredoxin stability to titration of ionizable groups with perturbed pKa.

The highly conserved, buried, Asp 26 in Escherichia coli thioredoxin has a pKa = 7.5, and its titration is associated with a sizable destabilization of the protein [Langsetmo, K., Fuchs, J., & Woodward, C. (1991) Biochemistry (preceding paper in this issue)]. A fit of the experimental pH dependence of thioredoxin stability to a theoretical expression for the pH/stability relation in proteins agrees closely with a pKa value of 7.5 for Asp 26. The agreement between the experimental and theoretical changes in protein stability due to substitution of Asp 26 by alanine is also good. The local structure in the vicinity of Asp 26 in the low-pH crystal structure (with uncharged Asp 26) is hydrophobic, indicating that the aspartate would be highly destabilized. In theoretical calculations, the desolvation penalty for deprotonating Asp 26 in this environment is similar to the total protein folding energy. As a consequence, the Asp 26 pKa would be much greater than 7.5, and/or the protein might not fold. This suggests that a compensating process partially stabilizes the Asp 26 carboxyl group when it is charged. A simple model for this proposed, whereby the Lys 57 side chain rotates to form a salt bridge with Asp 26 when it is deprotonated.

Aspartic Acid↗