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 M urea and then renatured by dialysis against dilute NH4HCO3 buffer. The affinity 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 nondenaturing conditions. While the binding of the 266- and 235-amino-acid (aa) E1A components to TBP showed similar characteristics the larger E1A protein had a higher affinity for CtBP, pRb, and SUG1. Using nuclear magnetic resonance (NMR) spectroscopy it was shown that structural perturbations occurred in the 266-aa protein in the presence of Zn2+ consistent with binding--no such changes were seen for the 235-aa protein. Limited proteolysis of the 266- and 235-aa E1A proteins gave rise to comparable polypeptide products, suggesting overall similarities in structure. However, the different affinities of the 266- and 235-aa proteins for the partner proteins and the differences seen in the NMR spectra from the two proteins suggested structural differences.
Grand
R J
RJ
CRC Institute for Cancer Studies, University of Birmingham, Edgbaston, United Kingdom. R.J.A.Grand@bham.ac.uk
Gash
L
L
Milner
A E
AE
Molloy
D P
DP
Szestak
T
T
Turnell
A S
AS
Gallimore
P H
PH
D017500:Adenovirus E1A Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000260:Adenoviruses, Human / Q000235:genetics / Q000378:metabolism*; D001665:Binding Sites; D010450:Endopeptidases / Q000378:metabolism; D004926:Escherichia coli; D006801:Humans; D011485:Protein Binding; D011489:Protein Denaturation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D015032:Zinc / Q000378:metabolism
D017500:Adenovirus E1A Proteins; D011993:Recombinant Fusion Proteins; D010450:Endopeptidases; D015032:Zinc