Characterization of the self association of Avian sarcoma virus integrase by analytical ultracentrifugation.
Retroviral integration protein (IN) has been shown to be both necessary and sufficient for the integration of reverse-transcribed retroviral DNA into the host cell DNA. It has been demonstrated that self-assembly of IN is essential for proper function. Analytical ultracentrifugation was used to determine the stoichiometry and free energy of self-association of a full-length IN in various solvents at 23.3 degrees C. Below 8% glycerol, an association stoichiometry of monomer-dimer-tetramer is observed. At salt concentrations above 500 mM, dimer is the dominant species over a wide range of protein concentrations. However, as physiological salt concentrations are approached, tetramer formation is favored. The addition of glycerol to 500 mM NaCl, 20 mM Tris (pH 8.4), 2 mM beta-mercaptoethanol significantly enhances dimer formation with little effect on tetramer formation. Furthermore, as electrostatic shielding is increased by increasing the ionic strength or decreasing the cation size, dimer formation is strengthened while tetramer formation is weakened. Taken together, the data support a model in which dimer formation includes favorable buried surface interactions which are opposed by charge-charge repulsion, while favorable electrostatic interactions contribute significantly to tetramer formation.
Coleman
J
J
Department of Biochemistry, University of New Hampshire, Durham, New Hampshire 03824, USA.
Eaton
S
S
Merkel
G
G
Skalka
A M
AM
Laue
T
T
D001358:Avian Sarcoma Viruses / Q000201:enzymology*; D019281:Dimerization; D005990:Glycerol; D006863:Hydrogen-Ion Concentration; D019426:Integrases / Q000737:chemistry*; D008623:Mercaptoethanol; D009994:Osmolar Concentration; D011487:Protein Conformation; D012191:Retroviridae Proteins / Q000737:chemistry; D012492:Salts; D012995:Solubility; D055672:Static Electricity; D013696:Temperature; D014461:Ultracentrifugation
D012191:Retroviridae Proteins; D012492:Salts; D008623:Mercaptoethanol; D019426:Integrases; D005990:Glycerol