title: Pressure- and thermally-induced reversible changes in the secondary structure of ribonuclease A studied by FT-IR spectroscopy.
abstract: Fourier transform infrared (FT-IR) spectroscopy combined with a resolution enhancement technique has been used to characterize pressure and thermal effects on the secondary structure of ribonuclease A. The experiments were performed at pD 7.0 with 50 mg/mL protein solution in D2O buffer. According to the observed changes in the amide I' band, secondary structure elements such as alpha-helices, beta-sheets, and turns are cooperatively disrupted by application of either pressures above 570 MPa at 30 degrees C or temperatures above 60 degrees C at 0.1 MPa. Pressure- and thermally-denatured ribonuclease A are fully unfolded and do not contain any residual secondary structures. Both the structural changes are intrinsically reversible, although the pressure-induced transition shows a hysteresis. It is found that nonnative turn structures are formed prior to the appearance of the native secondary structure in the folding from the pressure-unfolded state. The structural features upon the pressure-induced unfolding are additionally characterized by the interesting behavior of hydrogen-deuterium exchange at high pressure. Most of the backbone amide protons protected at atmospheric pressure, which are involved in the alpha-helices and beta-sheet, are exchanged with solvent deuterons in the pressure range where the two secondary structural elements are virtually identified as intact. There is a possibility that, for ribonuclease A, application of high pressure up to 570 MPa induces such a partially unfolded state as has native-like secondary structure but permits solvent to be highly accessible to the internal regions.
authors.affiliation: Department of Chemistry, Faculty of Science and Engineering, Ritsumeikan University, Shiga, Japan.
authors.forename: N
authors.initials: N
authors.lastname: Takeda
authors.forename: M
authors.initials: M
authors.lastname: Kato
authors.forename: Y
authors.initials: Y
authors.lastname: Taniguchi
mesh_terms: D000818:Animals; D002417:Cattle; D002942:Circular Dichroism; D003903:Deuterium; D006358:Hot Temperature; D006859:Hydrogen / Q000378:metabolism; D011312:Pressure; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D012259:Ribonuclease, Pancreatic / Q000737:chemistry*; D017550:Spectroscopy, Fourier Transform Infrared
chemical_list: D006859:Hydrogen; D003903:Deuterium; D012259:Ribonuclease, Pancreatic