CRITICAL AMINO-ACIDS IN THE TRANSCRIPTIONAL ACTIVATION DOMAIN OF THE HERPESVIRUS PROTEIN VP16 ARE SOLVENT-EXPOSED IN HIGHLY MOBILE PROTEIN SEGMENTS - AN INTRINSIC FLUORESCENCE STUDY

Eukaryotic transcriptional regulatory proteins typically comprise both a DNA binding domain and a regulatory domain. Although the structures of many DNA binding domains have been elucidated, no detailed structu res are yet available for transcriptional activation domains. The acti vation domain of the herpesvirus protein VP16 has been an important mo del in mutational and biochemical studies. Here, we characterize the V P16 activation domain using time-resolved and steady-state fluorescenc e. Unique intrinsic fluorescent probes were obtained by replacing phen ylalanine residues with tryptophan at position 442 or 473 of the activ ation domain of VP16 (residues 413-490, or subdomains thereof), linked to the DNA binding domain of the yeast protein GAL4. Emission spectra and quenching properties of Trp at either position were characteristi c of fully exposed Trp. Time-resolved anisotropy decay measurements su ggested that both Trp residues were associated with substantial segmen tal motion. The Trp residues at either position showed nearly identica l fluorescence properties in either the full-length activation domain or relevant subdomains, suggesting that the two subdomains are similar ly unstructured and have little effect on each other. As this domain m ay directly interact with several target proteins, it is likely that a significant structural transition accompanies these interactions.