Phage phi29 protein p6 is in a monomer-dimer equilibrium that shifts to higher association states at the millimolar concentrations found in vivo.
Protein p6 from Bacillus subtilis phage phi29 (Mr = 11 800) binds in vitro to DNA forming a large nucleoprotein complex in which the DNA wraps a multimeric protein core. The high intracellular abundance of protein p6 together with its ability to bind the whole phi29 DNA in vitro strongly suggests that it plays a role in viral genome organization. We have determined by sedimentation equilibrium analysis that protein p6 (1-100 microM range), in the absence of DNA, is in a monomer-dimer equilibrium, with an association constant (K2) of approximately 2 x 10(5) M-1. The intracellular concentration of protein p6 (approximately 1 mM) was estimated measuring the number of copies per cell (7 x 10(5)) and the cell volume (1 x 10(-15) L). At concentrations around 1 mM, protein p6 associates into oligomers. This self-association behavior is compatible with a dimer-hexamer model (K2,6 = 3.2 x 10(8) M-2) or with an isodesmic association of the dimer (K = 950 M-1), because the apparent weight-average molecular mass (Mw,a) does not reach saturation at the highest protein concentrations. The sedimentation coefficients of protein p6 monomer and dimer were 1.4 and 2.0, respectively, compatible with translational frictional ratios (f/fo) of 1.15 and 1.30, which slightly deviate from the hydrodynamics of a rigid globular protein. Taking together these results and considering the structure of the nucleoprotein complex, we speculate that the observed oligomers of protein p6 could mimic a scaffold on which DNA folds to form the nucleoprotein complex in vivo.
Abril
A M
AM
Centro de Biología Molecular "Severo Ochoa" (CSIC-UAM), Universidad Autónoma, Cantoblanco, 28049 Madrid, Spain.
Salas
M
M
Andreu
J M
JM
Hermoso
J M
JM
Rivas
G
G
D017103:Bacillus Phages / Q000737:chemistry*; D001412:Bacillus subtilis; D004247:DNA / Q000378:metabolism; D019281:Dimerization; D007700:Kinetics; D008958:Models, Molecular; D008970:Molecular Weight; D009698:Nucleoproteins / Q000737:chemistry; D011487:Protein Conformation; D014764:Viral Proteins / Q000737:chemistry*
D009698:Nucleoproteins; D014764:Viral Proteins; D004247:DNA