title: Native state dynamics affects the folding transition of porcine pancreatic phospholipase A2.
abstract: Porcine pancreatic phospholipase A2, a small and disulfide rich protein, is extremely resistant against chemically or thermally induced unfolding. Despite this marked resistance, the protein displays broad unfolding transitions resulting in comparatively low apparent thermodynamic stability. Broad unfolding transitions may result from undetected folding intermediates, residual structures in the unfolded state or an inhomogeneity of the native state. Using circular dichroism, fluorescence, and NMR spectroscopy, we ruled out the existence of stably populated folding intermediates, whereas UV absorbance measurements hinted at stable residual structures in the unfolded state. These residual structures proved, however, to have no impact on the folding parameters. Studies by limited proteolysis, CD, and NMR spectroscopy under non-denaturing conditions suggested pronounced dynamics of the protein in the native state, which as long as unrestrained by acidic pH or bound Ca(2+) ions exert considerable influence on the unfolding transition.
authors.affiliation: Biomolecular & Analytical Mass Spectrometry, Chemistry Department, University of Antwerp, Groenenborgerlaan 171, 2020 Antwerp, Belgium. Electronic address: Knut.Koelbel@uantwerpen.be.
authors.forename: Knut
authors.initials: K
authors.lastname: Kölbel
authors.affiliation: Department of Biophysical Chemistry, Lund University, PO Box 124, SE-22100 Lund, Sweden.
authors.forename: Ulrich
authors.initials: U
authors.lastname: Weininger
authors.affiliation: Institute of Pharmacy, Martin-Luther-University Halle-Wittenberg, Wolfgang-Langenbeck-Strasse 4, D-06120 Halle, Germany.
authors.forename: Christian
authors.initials: C
authors.lastname: Ihling
authors.affiliation: Institute of Biochemistry and Biotechnology, Martin-Luther-University Halle-Wittenberg, Kurt-Mothes-Strasse 3, D-06120 Halle, Germany.
authors.forename: Carmen
authors.initials: C
authors.lastname: Mrestani-Klaus
authors.affiliation: Institute of Biochemistry and Biotechnology, Martin-Luther-University Halle-Wittenberg, Kurt-Mothes-Strasse 3, D-06120 Halle, Germany.
authors.forename: Renate
authors.initials: R
authors.lastname: Ulbrich-Hofmann
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002942:Circular Dichroism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010179:Pancreas / Q000737:chemistry / Q000201:enzymology*; D054467:Phospholipases A2 / Q000737:chemistry*; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D058767:Protein Unfolding; D059748:Proteolysis; D016415:Sequence Alignment; D013552:Swine; D013816:Thermodynamics
chemical_list: D054467:Phospholipases A2
keywords: Equilibrium; Folding intermediate; Limited proteolysis; Protein unfolding; Residual structure; Spectroscopy; Thermodynamic parameter; Unfolded state