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 structures are yet available for transcriptional activation domains. The activation domain of the herpesvirus protein VP16 has been an important model in mutational and biochemical studies. Here, we characterize the VP16 activation domain using time-resolved and steady-state fluorescence. Unique intrinsic fluorescent probes were obtained by replacing phenylalanine residues with tryptophan at position 442 or 473 of the activation 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 characteristic of fully exposed Trp. Time-resolved anisotropy decay measurements suggested that both Trp residues were associated with substantial segmental motion. The Trp residues at either position showed nearly identical fluorescence properties in either the full-length activation domain or relevant subdomains, suggesting that the two subdomains are similarly unstructured and have little effect on each other. As this domain may directly interact with several target proteins, it is likely that a significant structural transition accompanies these interactions.
Shen
F
F
Biochemistry Department, Michigan State University, East Lansing, Michigan 48824-1319, USA.
Triezenberg
S J
SJ
Hensley
P
P
Porter
D
D
Knutson
J R
JR
D002586:Cesium / Q000494:pharmacology; D002712:Chlorides / Q000494:pharmacology; D003001:Cloning, Molecular; D004268:DNA-Binding Proteins; D004926:Escherichia coli; D005454:Fluorescence Polarization; D005656:Fungal Proteins / Q000096:biosynthesis; D017405:Herpes Simplex Virus Protein Vmw65 / Q000096:biosynthesis / Q000737:chemistry* / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D016297:Mutagenesis, Site-Directed; D010649:Phenylalanine; D017354:Point Mutation; D011193:Potassium Iodide / Q000494:pharmacology; D011487:Protein Conformation; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D012997:Solvents; D013050:Spectrometry, Fluorescence; D014157:Transcription Factors; D014158:Transcription, Genetic; D015533:Transcriptional Activation; D014364:Tryptophan
D002712:Chlorides; D004268:DNA-Binding Proteins; D005656:Fungal Proteins; C052130:GAL4 protein, S cerevisiae; D017405:Herpes Simplex Virus Protein Vmw65; D011993:Recombinant Fusion Proteins; D029701:Saccharomyces cerevisiae Proteins; D012997:Solvents; D014157:Transcription Factors; D011193:Potassium Iodide; D002586:Cesium; D010649:Phenylalanine; D014364:Tryptophan; C028019:cesium chloride