Retroviral Integrase: Difference between revisions
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The remarkable stability of the integrase-DNA complexes were determined by observing the ''in vitro'' strand transfer reactions, which were classified into three modes of deproteination migration: (1) single concerted events: linearized target plasmid; (2) multiple concerted events: smear; (3) half-site events: open circular DNA. Further characterization of the PFV intasome also exhibited structural substantiality which implied strong protein-protein and protein-DNA interactions despite prolonged incubation under high ionic strength conditions. Comprehensive crystallization assays effected a viable crystal configuration that diffracted X-rays to 2.9 Angstroms resolution. A three-dimensional structure was ultimately determined. The asymmetric unit contained a single integrase dimer with a stably bound viral DNA molecule, and a pair of integrase dimers consociated with symmetry, which formed an oblong tetramer. The dimer interface is stabilized by intermolecular amino terminal and catalytic core domains (inner subunit-outer subunit) interactions. The overall shape of the oblong tetramer is unique albeit bearing semblances to previously reported HIV-1 integrase complexes. | The remarkable stability of the integrase-DNA complexes were determined by observing the ''in vitro'' strand transfer reactions, which were classified into three modes of deproteination migration: (1) single concerted events: linearized target plasmid; (2) multiple concerted events: smear; (3) half-site events: open circular DNA. Further characterization of the PFV intasome also exhibited structural substantiality which implied strong protein-protein and protein-DNA interactions despite prolonged incubation under high ionic strength conditions. Comprehensive crystallization assays effected a viable crystal configuration that diffracted X-rays to 2.9 Angstroms resolution. A three-dimensional structure was ultimately determined. The asymmetric unit contained a single integrase dimer with a stably bound viral DNA molecule, and a pair of integrase dimers consociated with symmetry, which formed an oblong tetramer. The dimer interface is stabilized by intermolecular amino terminal and catalytic core domains (inner subunit-outer subunit) interactions. The overall shape of the oblong tetramer is unique albeit bearing semblances to previously reported HIV-1 integrase complexes. | ||
The protein-DNA complexes were formed using the full-length, wild-type PFV IN and synthetic dsDNA that modeled the viral end. | |||
The intasome was crystallized utilizing the vapor-drop hanging diffusion technique. The reservoir solution consisted on 1.35 M ammonium sulphate, 25% (v/v) glycerol, 4.8% (v/v) 1,6-hexanediol, and 50mM 2-(N-morpholino) ethanesulphonic acid (MES) at pH 6.5. The protein-DNA crystals were also soaked in the presence of MK0518, GS9137, Mg(II), and/or Mn(II). The crystal structure was solved using molecular replacement. | |||
==Overall Architecture & Components== | ==Overall Architecture & Components== | ||
