Biochemical characterization of phi29 DNA packaging enzyme gp16
Abstract
DNA packaging as an essential viral replication step has been investigated in the quest for new antiviral drugs. Despite the fact that all of the components involved in phi29 DNA packaging have been examined and well documented, studies of the packaging mechanism of the phi29 virus have been hindered by the hydrophobicity, insolubility, and self-aggregation of its packaging enzyme gp16. A Thioredoxin-fused gp16, ThioHis-gp16, was constructed and was found to have an activity similar wild type gp16. Polyethylene glycol (PEG) or acetone was used in this study to obtain affinity-purified, soluble, and native ThioHis-gp16. The specific activity of this native ThioHis-gp16 was increased three thousand-fold. In such a highly active state, the monomer was determined to be the major functional unit of ThioHis-gp16 in solution. This finding facilitated the stoichiometry study of gp16 in the phi29 DNA-packaging motor. This study also revealed that the N-terminus of gp16 could be extended without obstructing the activity of gp16. The direct interaction of ThioHis-gp16 with ATP was characterized. The order of ThioHis-gp16 binding affinity to the four ribonucleotides is, from high to low, ATP, GTP, CTP, and UTP. The ATP concentration required for 1% activity was 20 μM. The DNA-packaging proteins of herpes virus, adenovirus, pox virus, and other bacteriophages (phi29, T3, T4, P22, P1, psiM2, Sfi21, lambda, phiC31, and T7) all contain a predicted ATP-binding consensus sequence. Mutation studies revealed that changing only one conserved amino acid, whether R17, G24, G27, G29, K30, or I39, in the predicted Walker-A ATP motif of gp16 caused ATP hydrolysis and viral assembly to cease. However, mutations on amino acids G248 and D256 in the predicted conserved Walker-B ATP motif did not affect the function of gp16, which suggests that these two amino acids are not involved in the Walker-B ATP motif. These results will facilitate the study on phi29 DNA packaging motor that involve three pRNA dimers and a 12-subunit connector.
Degree
Ph.D.
Advisors
Guo, Purdue University.
Subject Area
Molecular biology
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