Involvement of the N- and C-terminal fragments of bovine pancreatic deoxyribonuclease in active protein folding.
The three-dimensional structure of bovine pancreatic (bp) DNase revealed that its N- and C-termini form an antiparallel beta-sheet structure. The involvement of this beta-sheet structure in the active protein folding of bpDNase was thus investigated via a series of deletion and substitution variants. Several substitution variants of N-terminal Leu1 and C-terminal Leu259, and one variant with only the last Thr260 deleted, remained fully active. However, the other deletion variants, in which 2-10 amino acid residues were removed from the C- or N-terminus, all lost the DNase activity. The results indicated that the backbone hydrogen bonding in the antiparallel beta-sheet, rather than the side-chain interactions, is crucial for the correct protein folding. When the deletion variants were complemented with synthetic peptides of the deleted N- or C-terminal sequences, the DNase activity was generated. The highest DNase activity was generated when the C-terminal 10-residue-deleted brDNase(Delta251-260) was admixed with the C-terminal 10-residue peptide (peptide C10) in a molar ratio of 1:400. The noncovalent binding between brDNase(Delta251-260) and peptide C10 exhibited a dissociation constant of 48 microM. Circular dichroism spectra showed that the deletion variants were partially folded with mainly helical structures and that admixture with corresponding peptides facilitated their folding into the nativelike beta-sheet-rich structure. Thermal denaturation profiles also revealed that the transition temperature for brDNase(Delta251-260) was increased from 55 to 63 degrees C after incubation with peptide C10. The folding activation process for the deletion variant occurred in two stages, and Ca(2+) was required.
Institute of Biochemistry and Molecular Biology, College of Medicine, National Taiwan University, Taipei, Taiwan.
Wei-Jung
WJ
Chen
Po-Tsang
PT
Huang
Julian
J
Liu
Ta-Hsiu
TH
Liao
D019943:Amino Acid Substitution; D000818:Animals; D002417:Cattle; D003850:Deoxyribonuclease I / Q000737:chemistry*; D004795:Enzyme Stability / Q000187:drug effects; D006860:Hydrogen Bonding; D016297:Mutagenesis, Site-Directed; D010179:Pancreas / Q000201:enzymology; D010446:Peptide Fragments / Q000737:chemistry / Q000494:pharmacology*; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017384:Sequence Deletion; D013696:Temperature
D010446:Peptide Fragments; D003850:Deoxyribonuclease I