title: Folding analysis of the most complex Stevedore's protein knot.
abstract: DehI is a homodimeric haloacid dehalogenase from Pseudomonas putida that contains the most complex 61 Stevedore's protein knot within its folding topology. To examine how DehI attains such an intricate knotted topology we combined far-UV circular dichroism (CD), intrinsic fluorescence spectroscopy and small angle X-ray scattering (SAXS) to investigate its folding mechanism. Equilibrium unfolding of DehI by chemical denaturation indicated the presence of two highly populated folding intermediates, I and I'. While the two intermediates vary in secondary structure contents and tertiary packing according to CD and intrinsic fluorescence, respectively, their overall dimension and compactness are similar according to SAXS. Three single-tryptophan variants (W34, W53, and W196) were generated to probe non-cooperative unfolding events localized around the three fluorophores. Kinetic fluorescence measurements indicated that the transition from the intermediate I' to the unfolded state is rate limiting. Our multiparametric folding analyses suggest that DehI unfolds through a linear folding pathway with two distinct folding intermediates by initial hydrophobic collapse followed by nucleation condensation, and that knotting precedes the formation of secondary structures.
authors.affiliation: Institute of Biological Chemistry, Academia Sinica, 128, Section 2, Academia Road, Taipei 11529, Taiwan.
authors.forename: Iren
authors.initials: I
authors.lastname: Wang
authors.affiliation: Institute of Biological Chemistry, Academia Sinica, 128, Section 2, Academia Road, Taipei 11529, Taiwan.
authors.forename: Szu-Yu
authors.initials: SY
authors.lastname: Chen
authors.affiliation: Institute of Biological Chemistry, Academia Sinica, 128, Section 2, Academia Road, Taipei 11529, Taiwan.
authors.forename: Shang-Te Danny
authors.initials: ST
authors.lastname: Hsu
mesh_terms: D002942:Circular Dichroism; D006867:Hydrolases / Q000737:chemistry*; D007700:Kinetics; D008958:Models, Molecular; D011487:Protein Conformation; D017510:Protein Folding; D016958:Pseudomonas putida / Q000201:enzymology; D053838:Scattering, Small Angle; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014961:X-Ray Diffraction
chemical_list: D006867:Hydrolases; C034507:2-haloacid dehalogenase