Biotin tagging deletion analysis of domain limits involved in protein-macromolecular interactions. Mapping the tau binding domain of the DNA polymerase III alpha subunit.
The tau subunit dimerizes DNA polymerase III via interaction with the alpha subunit, allowing DNA polymerase III holoenzyme to synthesize both leading and lagging strands simultaneously at the DNA replication fork. Here, we report a general method to map the limits of domains required for heterologous protein-protein interactions using surface plasmon resonance. The method employs fusion of a short biotinylation sequence at either the NH2 or COOH terminus of the protein to be immobilized on streptavidin-derivatized biosensor chips. Inclusion of a hexahistidine sequence permits rapid purification and separation of the fusion protein from the endogenous Escherichia coli biotin carboxyl carrier protein. Ten deletions of the alpha subunit were constructed and purified by Ni2+-nitrilotriacetic acid chromatography and, when required, monomeric avidin chromatography. Each alpha deletion protein was captured by streptavidin immobilized on a Pharmacia Biosensor BIAcore chip, and the tau binding activity of each alpha deletion was analyzed using surface plasmon resonance. The tau subunit bound very tightly to a full-length amino-terminal fusion of the biotinylation sequence with alpha (KD approximately 70 pm). Four additional NH2-terminal alpha deletion proteins (60, 240, 360, and 542 residues deleted) retained strong binding activity to the tau subunit (KD = 0.19-0.39 nM), whereas deletion of 705 residues or more from the NH2 terminus of the alpha subunit abolished tau binding activity. Full-length alpha that contained a carboxyl-terminal fusion with the biotinylation sequence bound tau strongly (KD = 0.37 nM). However, deletion of 48 amino acids from the COOH terminus totally eliminated tau binding. These results indicate that the COOH-terminal half of the alpha subunit is involved in tau interaction.
Kim
D R
DR
Department of Biochemistry, Biophysics, and Genetics, University of Colorado Health Sciences Center, Denver, Colorado 80262, USA.
McHenry
C S
CS
D000595:Amino Acid Sequence; D001665:Binding Sites; D015374:Biosensing Techniques; D001710:Biotin; D004258:DNA Polymerase III / Q000737:chemistry*; D004261:DNA Replication; D004926:Escherichia coli / Q000201:enzymology; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D011485:Protein Binding; D011993:Recombinant Fusion Proteins; D016415:Sequence Alignment; D017384:Sequence Deletion; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D014157:Transcription Factors
D046911:Macromolecular Substances; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D001710:Biotin; C074904:DNA polymerase III, alpha subunit; C041273:transcription factor tau; D004258:DNA Polymerase III