Overexpression, Purification and Biophysical Studies of the Carboxy Terminal Transactivation Domain of Vmw65 from Herpes Simplex Virus Type 1

Abstract

In order to facilitate a biophysical analysis of the carboxy terminal acidic transactivation domain (AAD) of Vmw65 from Herpes Simplex Virus Type 1 (HSV-1), an overexpression system in Escherichia coli was constructed and optimized to produce milligram quantities of this polypeptide. Purification of the polypeptide was facilitated by creating a fusion protein to glutathione S-transferase (GST) from Schizosoma japonicum using a commercially available vector. Upon thrombin digestion of the fusion protein, the carrier and AAD products were resolved by anion-exchange chromatography. With typically 15 mg of AAD available from a 12 litre culture, several biophysical studies were initiated. Circular dichroism and fluorescence spectroscopy both described a polypeptide with an extended structure reminicent of a random-coil; that is, it did not possess substantial quantities of known elements of secondary structure such as a-helicies and β-sheets under physiological conditions. A new structure high in α-helical content was induced upon addition of trifluoroethanol to mimic a hydrophobic milieu. Ultracentrifugation data supported the spectroscopic observations by describing an extended, monomeric polypeptide. The ultimate goal of the study, a teritiary structure, was sought by attempting to crystallize AAD with popular salts and organic solvents. Biologically, the described random-coil structure of AAD could be relevant to its role as a promoter and stablizer of the transcriptional pre-initation complex, the determining step in gene expression. A structurally labile domain would support AAD’s ability to interact with several targets including TFIID and TFIIB, though not necessarily by similar mechanisms. The requirement for a drastic conformational change such as a random-coil to α-helical transition currently remains unclear though observations made in this study of AAD in trifluoroethanol have shown that a conformational change is indeed possible. With a means of producing large quantities of AAD, the opportunity now arises to study its interaction with available cloned targets. The ensuing biophysical studies will then provide a greater understanding of AAD’s important role in gene expression.