The Escherichia coli single strand binding (SSB) protein is an essential protein required for DNA replication and involved in recombination and a number of repair processes. It is a stable homotetramer in solution; however the ssb-1 mutation (His-55 to Tyr) destabilizes the tetramer with respect to monomers and this defect seems to explain the observed phenotype (Williams, K.R., Murphy, J.B., and Chase, J.W. (1984) J. Biol. Chem. 259, 11804-11811). We report a quantitative study of the SSB-1 monomer-tetramer equilibrium in vitro as a function of temperature, pH, NaCl, MgCl2, urea, and guanidine hydrochloride concentrations. The self-assembly equilibrium was monitored by the increase in intrinsic protein fluorescence anisotropy accompanying the formation of the tetramer. The experimental isotherms indicate that SSB-1 dimers are not highly populated at equilibrium, hence the formation of the tetramer is well-described as a one-step association of four monomers. At 25°C, pH 8.1, the monomer concentration for 50% tetramer dissociation is (M(T))( 1/2 ) = 0.87 μM, corresponding to a monomer-tetramer equilibrium constant, K(T) = 3 ± 1 x 1018 M-3. The tetramerization constant, K(T), is highly dependent upon temperature and pH, with ΔH0 = -51 ± 7 kcal/mol (pH 8.1) and ΔH(o) = -37 ± 5 kcal/mol (pH 6.9). There is no effect of NaCl on the monomer-tetramer association in the range from 0.20 to 1.0 M; however, MgCl2 decreases the stability of the SSB-1 tetramer. In the presence of high concentrations of the single-stranded oligonucleotide, dT(pT)15, the tetramerization constant is slightly increased indicating that binding of the oligonucleotide to the SSB-1 monomer promotes the assembly process, although not dramatically. The large negative ΔH0 that is associated with formation of the tetramer provides a likely explanation for the temperature sensitivity of the ssb-1 mutation.
|Original language||English (US)|
|Number of pages||11|
|Journal||Journal of Biological Chemistry|
|State||Published - 1991|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology