The transcriptional activation domain of the herpesvirus protein VP16 resides in the carboxyl-terminal 78 amino acids (residues 413-490). Fluorescence analyses of this domain indicated that critical amino acids are solvent-exposed in highly mobile segments. To examine interactions between VP16 and components of the basal transcriptional machinery, we incorporated (at position 442 or 473 of VP16) tryptophan analogs that can be selectively excited in complexes with other Trp-containing proteins. TATA-box binding protein (TBP) (but not transcription factor B (TFIIB)) caused concentration-dependent changes in the steady-state anisotropy of VP16, from which equilibrium binding constants were calculated. Quenching of the fluorescence from either position (442 or 473) was significantly affected by TBP, whereas TFIIB affected quenching only at position 473. 7-aza-Trp residues at either position showed a emission spectral shift in the presence of TBP (but not TFIIB), indicating a change to a more hydrophobic environment. In anisotropy decay experiments, TBP reduced the segmental motion at either position; in contrast, TFIIB induced a slight change only at position 473. Our results support models of TBP as a target protein for transcriptional activators and suggest that ordered structure in the VP16 activation domain is induced upon interaction with target proteins.

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