Folding and stability of EBNA1 DNA binding domain.

FREIRE, E., ODDO, C. AND PRAT GAY, G. DE

Fundación Instituto Leloir, 29 y 30 de marzo de 2004.

Simposio; Simposio satélite de Biología Molecular Estructural; 2004

Fundación Instituto Leloir

“Folding and stability of EBNA1 DNA binding domain” Freire, Eleonora; Oddo, Cristian and Prat Gay, Gonzalo. Instituto Leloir and FCEyN-UBA. Buenos Aires. E-mail: efreire@leloir.org.ar The Epstein Barr nuclear antigen 1 (EBNA1) is a DNA origin binding protein (OBP). It´s DNA binding domain targets the EBV replication origin OriP and plays several roles such as activation of DNA replication, recruitment of host replication factors, destabilization of origin DNA, segregation of EBV episomes, transactivation of latent viral gene products, and repression of its own expression. The crystal structure of this domain (EBNA 459-607) free or bound to DNA revealed a particular fold, the dimeric ß-barrel, only shared with the papillomavirus E2 DNA binding domain. We have investigated its folding mechanism and found it is reversible and highly resistant to chemical denaturants, temperature and pH. The guanidine chloride denaturation midpoint is at least 6.0M, and it can be only unfolded completely using guanidine isothiocyanate, with a midpoint of 2.2M. The domain is also stable to chemical denaturation at pH 2.8. Light scattering experiments confirm it is a monomer at the end of the denaturant transition. Preliminary kinetic unfolding data shows different t1/2 when following the reaction by Trp fluorescence or Molar Ellipticity at 225nm. This difference could be assigned to the presence of secondary structure while tryptophan residues are already exposed to solvent, indicating the existence of a reaction intermediate. Both decays (Fluorescence and CD) show a multiexponential fit, which are under analysis.