REGENERATION OF THE BINDING-PROPERTIES OF ADENOVIRUS-12 EARLY REGION 1A PROTEINS AFTER PREPARATION UNDER DENATURING CONDITIONS

Adenovirus 12 early region 1A (Ad12 E1A) was expressed in Escherichia coli. Protein was purified in good yield in the presence of 8 Fn urea and then renatured by dialysis against dilute NH4HCO3 buffer. The affi nity of this protein for pRb, C-terminal binding protein (CtBP), TATA binding protein (TBP), and SUG1 was similar to, or greater than, that of Ad12 E1A prepared by immunoaffinity chromatography under nondenatur ing conditions. While the binding of the 266- and 235-aminoacid (aa) E 1A components to TBP showed similar characteristics the larger E1A pro tein had a higher affinity for CtBP, pRb, and SUG1. Using nuclear magn etic resonance (NMR) spectroscopy it was shown that structural perturb ations occurred in the 266-aa protein in the presence of Zn2+ consiste nt with binding-no such changes were seen for the 235-aa protein. Limi ted proteolysis of the 266- and 235-aa E1A proteins gave rise to compa rable polypeptide products, suggesting overall similarities in structu re. However, the different affinities of the 266- and 235-aa proteins for the partner proteins and the differences seen in the NMR spectra f rom the two proteins suggested structural differences, (C) 1998 Academ ic Press.