title: The confirmation of the denatured structure of pyrrolidone carboxyl peptidase under nondenaturing conditions: difference in helix propensity of two synthetic peptides with single amino acid substitution.
abstract: In the denatured state (D(1) state) of cystein-free pyrrolidone carboxyl peptidase (PCP-0SH) from Pyrococcus furiosus, a hyperthermophile under nondenaturing conditions, a fairly stable alpha-helix (alpha6-helix) has been determined from H/D exchange-NMR experiments. On the other hand, the alpha6-helix region of the proline-mutant at position 199 (A199P) was unstructured in the D(1) state unlike that of the wild-type PCP-0SH, although the folded conformations of both proteins were almost identical to each other. This finding has been deduced from the information regarding the remaining amide hydrogens in the HSQC spectra after H/D exchanges in the D(1) state. To confirm this inference, we examined the helical propensities of two synthetic peptides from their NMR structural analysis in the presence of trifluoroethanol (TFE). One is an 18-residue peptide called the wild-type H6-peptide corresponding to the alpha6-helix (from Ser188 to Glu205) of the wild-type PCP-0SH, and the other is the mutant H6-peptide corresponding to the alpha6-helix region of A199P. The NOE-contact information obtained from the 2D-(1)H-NOESY spectra measured for both peptides in the presence of 30% TFE clearly demonstrated that the wild-type H6-peptide had a high helical propensity, but the mutant H6-peptide was almost totally unstructured. The TFE-induced helical propensities for these peptide fragments confirmed the conclusions deduced from the H/D exchange data measured in the D(1) states of two proteins.
authors.affiliation: School of Science and Technology, Kwansei Gakuin University, 2-1 Gakuen, Sanda, Hyogo 669-1337, Japan.
authors.forename: Taro
authors.initials: T
authors.lastname: Umezaki
authors.forename: Satoshi
authors.initials: S
authors.lastname: Iimura
authors.forename: Yasuo
authors.initials: Y
authors.lastname: Noda
authors.forename: Shin-Ichi
authors.initials: S
authors.lastname: Segawa
authors.forename: Katsuhide
authors.initials: K
authors.lastname: Yutani
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D002942:Circular Dichroism; D041961:Deuterium Exchange Measurement; D004578:Electron Spin Resonance Spectroscopy; D019906:Nuclear Magnetic Resonance, Biomolecular; D011487:Protein Conformation; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D011751:Pyroglutamyl-Peptidase I / Q000737:chemistry* / Q000235:genetics; D014270:Trifluoroethanol
chemical_list: D014270:Trifluoroethanol; D011751:Pyroglutamyl-Peptidase I