title: Stability of alpha-helices in a molten globule state of cytochrome c by hydrogen-deuterium exchange and two-dimensional NMR spectroscopy.
abstract: To distinguish between intrinsically stable helices and those stabilized by the protein three-dimensional structure, we report the hydrogen-deuterium exchange (H2H) rates of 29 amide protons of ferricytochrome c in a molten globule state (at 35 degrees C, pH 2.0 with 0.5 M KCl), monitored by 2D-NMR. The results of the H2H-exchange experiments have been analyzed to calculate the protection factors. The helices were not uniformly stable and amide protons of residues belonging to the N and C-terminal helices had high protection factors. The protection factors of amide protons involved in the 50's helix were low, and could not be determined quantitatively. In the 60's helix we found two amide protons with protection factors comparable to those of the N and C-terminal helices. These results, compared with previously reported intrinsic helicities of peptide fragments, indicate that the relative helicities of isolated fragments are not directly reflected in the stability of the helices in the molten globule state, even though this state has no rigid tertiary structure. This suggests that loose interactions between helices are present in the molten globule state of cytochrome c, and that they are essential for keeping the helicity of the helical segments. The loose tertiary interactions discussed here differ from the usual tertiary interaction found in the native state in that the specific interdigitization between side-chains is absent.
authors.affiliation: Protein Engineering Research Institute, Osaka, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Kuroda
authors.forename: S
authors.initials: S
authors.lastname: Endo
authors.forename: K
authors.initials: K
authors.lastname: Nagayama
authors.forename: A
authors.initials: A
authors.lastname: Wada
mesh_terms: D000595:Amino Acid Sequence; D003574:Cytochrome c Group / Q000737:chemistry*; D003903:Deuterium / Q000737:chemistry; D006859:Hydrogen / Q000737:chemistry; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D017434:Protein Structure, Tertiary; D013057:Spectrum Analysis
chemical_list: D003574:Cytochrome c Group; D006859:Hydrogen; D003903:Deuterium