Related Experiment Videos
Phosphorylation and dephosphorylation alter the structure of D2 hybrid T antigen
Abstract:
D2 hybrid T antigen is a protein closely related to simian virus 40 large T antigen and is synthesized in large quantities in cells infected with Ad2+D2, an adenovirus-simian virus 40 hybrid. We have analyzed the effects of phosphorylation on the structure and DNA binding of this protein. On nondenaturing pore-gradient gels, the purified protein migrated with an apparent molecular weight of 135,000, with a minor band at 330,000 molecular weight. In vitro phosphorylation catalyzed by the protein kinase activity associated with the protein resulted in a structural change so that most of it migrated with an apparent molecular weight of 740,000. Treatment of the phosphorylated form of the protein with alkaline phosphatase (which removed 95% of the phosphate) caused the disappearance of the 740,000-molecular-weight form and reappearance of the smaller forms. Partial tryptic digestion showed that D2 T antigen has two major regions of phosphorylation, only one of which was phosphorylated in vitro. The region phosphorylated in vitro was responsible for the aggregation of D2 T antigen and was tentatively assigned to the N-terminal part of the protein. As shown by protein blotting onto nitrocellulose filters, it was mainly the form of 740,000 molecular weight that bound to simian virus 40 DNA. However, sucrose gradient analyses showed that only a fraction of the in vitro-phosphorylated protein bound to DNA, suggesting that aggregation alone is not sufficient for binding.
Insights
Phosphorylation significantly alters the structure of D2 hybrid T antigen, promoting aggregation and enhancing its binding to simian virus 40 DNA. This modification is crucial for the protein's DNA interaction.
Area of Science:
- Molecular Biology
- Virology
- Protein Biochemistry
Background:
- D2 hybrid T antigen, structurally similar to simian virus 40 large T antigen, is produced during Ad2+D2 hybrid virus infection.
- Understanding protein phosphorylation is key to elucidating viral protein function and host-cell interactions.
Purpose of the Study:
- To investigate the impact of phosphorylation on the structural conformation and DNA-binding capabilities of D2 hybrid T antigen.
- To identify the specific phosphorylation sites and their role in protein aggregation and DNA interaction.
Main Methods:
- Purification and structural analysis of D2 hybrid T antigen using nondenaturing pore-gradient gel electrophoresis.
- In vitro phosphorylation assays using endogenous protein kinase activity and dephosphorylation with alkaline phosphatase.
- Tryptic digestion to map phosphorylation sites and protein blotting for DNA-binding studies.
- Sucrose gradient centrifugation to analyze DNA-binding fractions.
Main Results:
- In vitro phosphorylation induced a significant structural change in D2 T antigen, increasing its apparent molecular weight from 135,000 to 740,000.
- Dephosphorylation reversed this structural change, confirming the role of phosphorylation in aggregation.
- The N-terminal region was identified as the site of in vitro phosphorylation, responsible for protein aggregation.
- The aggregated, phosphorylated form (740,000 MW) exhibited enhanced binding to simian virus 40 DNA, though only a fraction of the phosphorylated protein effectively bound.
Conclusions:
- Phosphorylation is a critical regulatory mechanism for D2 hybrid T antigen structure and function.
- While phosphorylation-induced aggregation facilitates DNA binding, it is not the sole determinant for stable interaction with simian virus 40 DNA.
- Further research is needed to understand the precise mechanisms governing D2 T antigen DNA binding.