THE ENERGETICS OF HMG BOX INTERACTIONS WITH DNA - THERMODYNAMIC DESCRIPTION OF THE BOX FROM MOUSE SOX-5

The structural energetics of the HMG box from the DNA-binding protein mouse Sox-5 were examined calorimetrically. It was found that this box , notwithstanding its small size (molecular mass about 10 kDa), does n ot behave as a single cooperative unit and, on heating, the box revers ibly unfolds In two separate stages. The first transition (t(t) simila r to 34 degrees C) involves about 40% of the total enthalpy and the se cond (t(t) similar to 46 degrees C) the remainder. Both transitions pr oceed with significant heat capacity increment, showing that they are associated with the unfolding, of two sub-domains having non-polar cor es. According to heat capacity, ellipticity, fluorescence and NMR crit eria, this HMG box is in a fully compact native state only below 5 deg rees C. HMG boxes consist of two approximately orthogonal wings: the m inor wing comprises helix 3 and its associated antiparallel N-terminal strand, whilst the major wing is composed of helices I and II. Analys is of the fluorescence and NMR spectra for this;box obtained at differ ent temperatures shows that the lower melting transition can be assign ed to the minor wing and the upper transition to the major wing. Under physiological conditions (37 degrees C), the minor wing is considerab ly unfolded, whilst the major wing is essentially fully folded. DNA bi nding in vivo therefore involves refolding of the minor wing. (C) 1998 Academic Press.