title: The thermal denaturation of ribonuclease A in aqueous-methanol solvents.
abstract: Circular dichroism was used to monitor the thermal unfolding of ribonuclease A in 50% aqueous methanol. The spectrum of the protein at temperatures below -10 degrees C (pH* 3.0) was essentially identical to that of native ribonuclease A in aqueous solution. The spectrum of the thermally denatured material above 70 degrees C revealed some residual secondary structure in comparison to protein unfolded by 5 M Gdn.HCl at 70 degrees C in the presence or absence of methanol. The spectra as a function of temperature were deconvoluted to determine the contributions of different types of secondary structure. The position of the thermal unfolding transition as monitored by alpha-helix, with a midpoint at 38 degrees C, was at a much higher temperature than that monitored by beta-sheet, 26 degrees C, which also corresponded to that observed by delta A286, tyrosine fluorescence and hydrodynamic radius (from light scattering measurements). Thus, the loss of beta-sheet structure is decoupled from that of alpha-helix, suggesting a step-wise unfolding of the protein. The transition observed for loss of alpha-helix coincides with the previously measured transition for His-12 by NMR from a partially folded state to the unfolded state, suggesting that the unfolding of the N-terminal helix in RNase A is lost after unfolding of the core beta-sheet during thermal denaturation. The thermally denatured protein was relatively compact, as measured by dynamic light scattering.
authors.affiliation: Department of Chemistry, University of California, Santa Cruz 95064.
authors.forename: B
authors.initials: B
authors.lastname: Lustig
authors.forename: A L
authors.initials: AL
authors.lastname: Fink
mesh_terms: D002942:Circular Dichroism; D006358:Hot Temperature; D006863:Hydrogen-Ion Concentration; D000432:Methanol; D011487:Protein Conformation; D011489:Protein Denaturation; D012259:Ribonuclease, Pancreatic / Q000737:chemistry*; D012542:Scattering, Radiation; D012997:Solvents; D014867:Water
chemical_list: D012997:Solvents; D014867:Water; D012259:Ribonuclease, Pancreatic; D000432:Methanol