title: Identifying the site of initial tertiary structure disruption during apomyoglobin unfolding.
abstract: Structural characterization of protein unfolding intermediates [Kiefhaber et al. (1995) Nature 375, 513; Hoeltzli et al.(1995) Proc. Natl. Acad. Sci. U.S.A. 92, 9318], which until recently were thought to be nonexistent, is beginning to give information on the mechanism of unfolding. To test for apomyoglobin unfolding intermediates, we monitored kinetics of urea-induced denaturation by stop-flow tryptophan fluorescence and quench-flow amide hydrogen exchange. Both measurements yield a single, measurable kinetic phase of identical rate, indicating that the reaction is highly cooperative. A burst phase in fluorescence, however, suggests that an intermediate is rapidly formed. To structurally characterize it, we carried out stop-flow thiol-disulfide exchange studies of 10 single cysteine-containing mutants. Cysteine probes buried at major sites of helix-helix pairing revealed that side chains throughout the protein unpack and become accessible to the labeling reagent [5, 5'-dithiobis (2-nitrobenzoic acid)] with one of two rates. Probes located at all helical-packing interfaces-except for one-become exposed at the rate of global unfolding as determined by fluorescence and hydrogen exchange measurements. In contrast, probes located at the A-E helical interface undergo complete thiol-disulfide exchange within the mixing dead time of 6 ms. These results point to the existence of a burst-phase unfolding intermediate that contains globally intact hydrogen bonds but locally disrupted side-chain packing interactions. Dissolution of secondary and tertiary structure are therefore not tightly coupled processes. We suggest that disruption of tertiary structure may be a stepwise process that begins at the weakest point of the native fold, as determined by native-state hydrogen-exchange parameters.
authors.affiliation: SUNY Health Science Center at Syracuse, Department of Biochemistry and Molecular Biology, 750 East Adams Street, Syracuse New York 13210, USA.
authors.forename: Z
authors.initials: Z
authors.lastname: Feng
authors.forename: J H
authors.initials: JH
authors.lastname: Ha
authors.forename: S N
authors.initials: SN
authors.lastname: Loh
mesh_terms: D000818:Animals; D001059:Apoproteins / Q000737:chemistry* / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D003545:Cysteine / Q000235:genetics; D066298:In Vitro Techniques; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009211:Myoglobin / Q000737:chemistry* / Q000235:genetics; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D014508:Urea; D014907:Whales
chemical_list: D001059:Apoproteins; D009211:Myoglobin; D011994:Recombinant Proteins; C010857:apomyoglobin; D014508:Urea; D003545:Cysteine