Assembly of the Bacteriophage T7 Replisome Probed by Native ESl Top-Down Mass Spectrometry

摘要

Native ESI determines protein stoichiometry of the T7 replisome, demonstrating that gp4 hexamers and heptamers can bind up to three copies of DNA polymerase; the three-copy polymerase is less stable than the one and two polymerase replisome. X-ray crystal structure of the T7 replisome, recently solved, shows that the gp4 heptamer binds three copies of gp5/trx. Two copies of gp5/trx adjacent to each other have a larger interaction area with the gp4 heptamer compared with the third gp5/trx. The third gp5/trx appears to be less stably bound, which is in excellent agreement with the MS data. Comparing different gp4 constructs gives a correlation of sample heterogeneity, as det'd by native ESI, and conditions that give diffracting protein crystals. We, therefore, propose that native ESI is a tool to optimize protein constructs for crystallization. Native ESI reveals oligomeric nature of helicase proteins, surpassing EM and native gels for gp4 oligomers. Native ESIis an efficient method to observe all oligomeric species of the helicase protein.