DsbA is a periplasmic protein of Escherichia coli that appears to be the immediate donor of disulfide bonds to proteins that are secreted. Its active site contains one accessible and one buried cysteine residue, Cys30 and Cys33, respectively, which can form a very unstable disulfide bond between them that is 10(3)-fold more reactive toward thiol groups than normal. The two cysteine residues have normal properties when in a short peptide. In DsbA, the Cys30 thiol group is shown to be reactive toward alkylating reagents down to pH 4 and to be fully ionized, on the basis of the UV absorbance of the thiolate anion at 240 nm. Its reactivity is altered by another, unknown group on the reduced protein titrating with a pKa of about 6.7. The other cysteine residue is buried and unreactive and has a high pKa value. The ionization properties of the DsbA thiol groups can explain, at least partly, the high reactivity of its disulfide bonds and thiol groups at both neutral and acidic pH values.

The effects of trifluoroethanol (TFE) on the stability of the alpha-helix formed by ribonuclease S-peptide, residues 1-19 of ribonuclease A, were studied by measuring circular dichroism as a function of TFE concentration, pH, and temperature. The S-peptide forms an unusually stable alpha-helix, which is known to be stabilized by TFE. The magnitude of the effect of charged groups on the peptide, manifested by the change in alpha-helix stability as a function of pH, was not altered significantly by either TFE concentration or temperature, indicating that the lower dielectric constant of TFE is not important in the stabilization of this alpha-helix. This suggests that the alpha-helix might be stabilized by many interactions in addition to the effects of charges. The titration curve of circular dichroism vs. TFE concentration appears to be cooperative at 0 degree C, but becomes progressively less cooperative at temperatures between 25 and 75 degrees C. The properties of the TFE stabilization indicate that TFE might be a useful probe with which to measure the stability of marginally stable peptides and small proteins.

alpha-Helix formation in the S-peptide (residues 1-19 of ribonuclease A) was studied in detail by use of two-dimensional 1H nuclear magnetic resonance to monitor the effects of 2,2,2-trifluoroethanol (TFE) at 0 degrees C and pH* 2.07. TFE stabilizes the S-peptide alpha-helix. Helix formation by a particular amino acid was monitored by the chemical shifts of the C alpha, C beta, and C gamma protons while increasing the concentration of TFE: large changes in chemical shift of a particular residue indicate that it is induced to go helical, whereas small chemical shift changes indicate little helix formation. Residues Thr-3 to Met-13 undergo chemical shift changes consistent with helix formation, whereas the other residues do not. Earlier work [Kim, P. S., & Baldwin, R. L. (1984) Nature 307, 329-334] reported that residues Thr-3 to His-12 become helical in aqueous solution. The existence of a "helix stop signal" was inferred from this behavior. We thus conclude that this helix stop signal persists in TFE solutions.

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The thermodyna~ic parameters for the double strand formation of the molecules rCAmG + rCUnG' m,n =5-7, and dC~G + dCTnG, m,n = 5,6were measured from optical melting curves. Normal helices are formed when m = n. The deoxy-oligomers are more stable than the ribo-oligomers, due to a more favorable enthalpy. Double helices with mismatched bases can be formed by mixing oligomers with m ~ n. Such helices may form several possible structures. A structure with a dangling base is favored over a structure with a bulged base. The destabilization of the double strands by the formation of a bulged base was determined to be greater than 1 .6 kcal/mol at l0°C. The extent of aggregation in the oligomer double strand rCA 7 G·rCU 7 G was determined using ultracentrifugation equilibrium. The possible effects of aggregation on the determination of the thermodynamic parameters for double strand formation are discussed.

The equilibrium constant between reduced glutathione (GSH), oxidized glutathione (GSSG), reduced dithiothreitol (DTTSH SH), and oxidized dithiothreitol (DTTS S) has been directly measured by high performance liquid chromatography analysis of equilibrium mixtures. The equilibrium constant at 25°C for the reaction GSSG + DTTSH SH ⇌ 2GSH + S S varies from approximately 200 M, below pH 8, to approximately 2800 M, above pH 11. The observed pH dependence is generally consistent with published values of acid dissociation constants of these thiols.