STRUCTURAL CHARACTERIZATION OF A MINIMAL FUNCTIONAL TRANSACTIVATION DOMAIN FROM THE HUMAN GLUCOCORTICOID RECEPTOR

A 58-amino acid polypeptide containing the functional core region, the tau 1 core, Of the major transactivation domain of the human glucocor ticoid receptor has been expressed in Escherichia coil and purified to homogeneity. The polypeptide retains 60-70% of the activity of the in tact domain when assayed in vivo or in vitro. This report describes a structural characterization of the tau 1 core peptide fragment, Circul ar dichroism spectroscopy shows that the tau 1 core and a larger fragm ent encompassing the intact tau 1 domain are largely unstructured in w ater solution under a variety of pH conditions, The tau 1 core, howeve r, acquires a significant alpha-helical structure when analyzed in the presence of trifluoroethanol, an agent that favors secondary structur e formation in regions that have propensity for alpha-helical conforma tion, Two- and three dimensional NMR spectroscopy of N-15-labeled tau 1 core, in the presence of trifluoroethanol, has allowed sequential as signment of H-1 and N-15 resonances and identification of three protei n segments with alpha-helical character, Potentially helix-breaking pr oline substitutions, in proposed alpha-helical regions, lead to reduce d activity, suggesting that alpha-helices are important for transactiv ation in vivo.