title: Protein Folding Mechanism of the Dimeric AmphiphysinII/Bin1 N-BAR Domain.
abstract: The human AmphyphisinII/Bin1 N-BAR domain belongs to the BAR domain superfamily, whose members sense and generate membrane curvatures. The N-BAR domain is a 57 kDa homodimeric protein comprising a six helix bundle. Here we report the protein folding mechanism of this protein as a representative of this protein superfamily. The concentration dependent thermodynamic stability was studied by urea equilibrium transition curves followed by fluorescence and far-UV CD spectroscopy. Kinetic unfolding and refolding experiments, including rapid double and triple mixing techniques, allowed to unravel the complex folding behavior of N-BAR. The equilibrium unfolding transition curve can be described by a two-state process, while the folding kinetics show four refolding phases, an additional burst reaction and two unfolding phases. All fast refolding phases show a rollover in the chevron plot but only one of these phases depends on the protein concentration reporting the dimerization step. Secondary structure formation occurs during the three fast refolding phases. The slowest phase can be assigned to a proline isomerization. All kinetic experiments were also followed by fluorescence anisotropy detection to verify the assignment of the dimerization step to the respective folding phase. Based on these experiments we propose for N-BAR two parallel folding pathways towards the homodimeric native state depending on the proline conformation in the unfolded state.
authors.affiliation: Martin-Luther University Halle-Wittenberg, Institute of Physics, Betty-Heimann Str. 7, 06120, Halle, Germany.
authors.forename: Tobias
authors.initials: T
authors.lastname: Gruber
authors.affiliation: Martin-Luther University Halle-Wittenberg, Institute of Physics, Betty-Heimann Str. 7, 06120, Halle, Germany.
authors.forename: Jochen
authors.initials: J
authors.lastname: Balbach
mesh_terms: D000595:Amino Acid Sequence; D006801:Humans; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000737:chemistry*; D017510:Protein Folding; D055503:Protein Multimerization; D017434:Protein Structure, Tertiary
chemical_list: D009419:Nerve Tissue Proteins; C075637:amphiphysin