Use of acetone to attain highly active and soluble DNA packaging protein Gp16 of Phi29 for ATPase assay.
All the well-defined DNA-packaging motors of the dsDNA viruses contain one pair of nonstructural DNA-packaging enzymes. Studies on the mechanism of virus DNA packaging have been seriously hampered by their insolubility. Phi29's DNA-packaging enzyme, gp16, is also hydrophobic, insoluble, and self-aggregating. This article describes approaches to obtain affinity-purified, soluble, and highly active native gp16 with the aid of polyethylene glycol or acetone. The specific activity of this native gp16 was increased 3400-fold when compared with the traditional method. This unique approach made the ATP-gp16 interaction study feasible. Gp16 binds strongly to ATP, binds to ADP with a lower efficiency, and binds very weakly to AMP. The order of gp16-binding efficiency to the four ribonucleotides is, from high to low, ATP, GTP, CTP, and UTP. The ATP concentration level required to produce 50% of maximum virus yield exhibited during in vitro phi29 assembly is around 45 microM, which is close to the gp16 and ATP dissociation constant of 65 microM. Mutation studies revealed that changing only one conserved amino acid, whether R(17), G(24), G(27), G(29), K(30), or I(39), in the predicted Walker-A ATP motif of gp16 caused ATP hydrolysis and viral assembly to cease, while such mutation did not affect gp16's binding to ATP. However, mutation on amino acids G(248) and D(256) did not affect the function of gp16 in DNA packaging.
Huang
Lisa P
LP
Department of Pathobiology, Purdue University, West Lafayette, IN 47907, USA.
Guo
Peixuan
P
D000096:Acetone / Q000737:chemistry*; D000244:Adenosine Diphosphate / Q000378:metabolism; D000249:Adenosine Monophosphate / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D020816:Amino Acid Motifs; D017103:Bacillus Phages / Q000737:chemistry*; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000378:metabolism*; D009154:Mutation; D011092:Polyethylene Glycols / Q000737:chemistry; D011485:Protein Binding; D012995:Solubility; D013816:Thermodynamics; D014764:Viral Proteins / Q000737:chemistry* / Q000378:metabolism*
D004268:DNA-Binding Proteins; D014764:Viral Proteins; D000096:Acetone; D011092:Polyethylene Glycols; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D004247:DNA; D000251:Adenosine Triphosphatases