Oxidative folding of Amaranthus alpha-amylase inhibitor: disulfide bond formation and conformational folding.
Oxidative folding is the fusion of native disulfide bond formation with conformational folding. This complex process is guided by two types of interactions: first, covalent interactions between cysteine residues, which transform into native disulfide bridges, and second, non-covalent interactions giving rise to secondary and tertiary protein structure. The aim of this work is to understand both types of interactions in the oxidative folding of Amaranthus alpha-amylase inhibitor (AAI) by providing information both at the level of individual disulfide species and at the level of amino acid residue conformation. The cystine-knot disulfides of AAI protein are stabilized in an interdependent manner, and the oxidative folding is characterized by a high heterogeneity of one-, two-, and three-disulfide intermediates. The formation of the most abundant species, the main folding intermediate, is favored over other species even in the absence of non-covalent sequential preferences. Time-resolved NMR and photochemically induced dynamic nuclear polarization spectroscopies were used to follow the oxidative folding at the level of amino acid residue conformation. Because this is the first time that a complete oxidative folding process has been monitored with these two techniques, their results were compared with those obtained at the level of an individual disulfide species. The techniques proved to be valuable for the study of conformational developments and aromatic accessibility changes along oxidative folding pathways. A detailed picture of the oxidative folding of AAI provides a model study that combines different biochemical and biophysical techniques for a fuller understanding of a complex process.
Cemazar
Masa
M
International Centre for Genetic Engineering and Biotechnology, Padriciano 99, 34012 Trieste, Italy. cemazar@icgeb.org
Zahariev
Sotir
S
Pongor
Sándor
S
Hore
Peter J
PJ
D027721:Amaranthus / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D007700:Kinetics; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010940:Plant Proteins / Q000737:chemistry*; D017510:Protein Folding; D014361:Trypsin Inhibitors; D000516:alpha-Amylases / Q000037:antagonists & inhibitors
D010940:Plant Proteins; D014361:Trypsin Inhibitors; D000516:alpha-Amylases