title: Stability and DNA binding ability of the DNA binding domains of interferon regulatory factors 1 and 3.
abstract: The thermodynamic properties and DNA binding ability of the N-terminal DNA binding domains of interferon regulatory factors IRF-1 (DBD1) and IRF-3 (DBD3) were studied using microcalorimetric and optical methods. DBD3 is significantly more stable than DBD1: at 20 degrees C the Gibbs energy of unfolding of DBD3 is -28.6 kJ/mol, which is 2 times larger than that of DBD1, -14.9 kJ/mol. Fluorescence anisotropy titration experiments showed that at this temperature the association constants with the PRDI binding site are 1.1 x 10(6) M(-)(1) for DBD1 and 3.6 x 10(6) M(-)(1) for DBD3, corresponding to Gibbs energies of association of -34 and -37 kJ/mol, respectively. However, the larger binding energy of DBD3 is due to its larger electrostatic component, while its nonelectrostatic component is smaller than that of DBD1. Therefore, DBD1 appears to have more sequence specificity than DBD3. Binding of DBD1 to target DNA is characterized by a substantially larger negative enthalpy than binding of DBD3, implying that the more flexible structure of DBD1 forms tighter contacts with DNA than the more rigid structure of DBD3. Thus, the strength of the DBDs' specific association with DNA is inversely related to the stability of the free DBDs.
authors.affiliation: Department of Biology, Johns Hopkins University, Baltimore, Maryland 21218, USA.
authors.forename: Victoria V
authors.initials: VV
authors.lastname: Hargreaves
authors.forename: Elena N
authors.initials: EN
authors.lastname: Makeyeva
authors.forename: Anatoly I
authors.initials: AI
authors.lastname: Dragan
authors.forename: Peter L
authors.initials: PL
authors.lastname: Privalov
mesh_terms: D004247:DNA / Q000737:chemistry / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D050856:Interferon Regulatory Factor-1 / Q000378:metabolism*; D050838:Interferon Regulatory Factor-3 / Q000378:metabolism*; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D013050:Spectrometry, Fluorescence; D055672:Static Electricity; D013816:Thermodynamics
chemical_list: D004268:DNA-Binding Proteins; D050856:Interferon Regulatory Factor-1; D050838:Interferon Regulatory Factor-3; D004247:DNA