The importance of phosphate ester backbone flexibility in protein-DNA recognition

Maria Victoria Esperon Botuyan, Purdue University

Abstract

The N-terminal 56 amino acid headpieces of the wild-type and mutant Y7I lac repressors and the 14-22 base pair DNAs containing the symmetric lac operator sequences can be used to study protein-DNA interaction. Phosphorus-31 NMR gives direct information as to the type of phosphate conformations present in the DNA. Thus, it can potentially provide information about protein-DNA phosphate backbone interaction. The alkaline phosphatase assay can be used to measure the dissociation constants of the headpiece:operator complexes. The wild-type operator forms the strongest complex with the wild-type headpiece while the mutant operators form weaker complexes. Relative to the wild-type headpiece, the Y7I headpiece forms weaker complexes with the operators. Although the recognition helix of this mutant headpiece is partially destroyed, its overall folding is still comparable to that of the wild-type headpiece. Most of the tertiary structure crosspeaks in the wild-type headpiece are still found in the Y7I headpiece. Results from the phosphorus-31 titrations and binding studies suggest that specific, strongly bound complexes retain the inherent conformational flexibility of the operator itself, whereas more weakly bound, but still specific, operator:protein complexes restrict the phosphate ester conformational freedom in the complex relative to the free DNA. NMR and molecular dynamics methodologies can be successfully applied to structure refinement of macromolecules. Using these methods, the 14-mer 7G 434 operator has been found to have both A- and B-like DNA conformations.

Degree

Ph.D.

Advisors

Gorestein, Purdue University.

Subject Area

Biochemistry

COinS