PMID:9688537
| Citation |
Carlini, L, Curth, U, Kindler, B, Urbanke, C and Porter, RD (1998) Identification of amino acids stabilizing the tetramerization of the single stranded DNA binding protein from Escherichia coli. FEBS Lett. 430:197-200 |
|---|---|
| Abstract |
Mutating the histidine at position 55 present at the subunit interface of the tetrameric E. coli single stranded DNA binding (SSB) protein to tyrosine or lysine leads to cells which are UV- and temperature-sensitive. The defects of both ssbH55Y (ssb-1) and ssbH55K can be overcome by increasing protein concentration, with the ssbH55K mutation producing a less stable, readily dissociating protein whose more severe replication and repair phenotypes were less easily ameliorated by protein amplification. In this study we selected and analyzed E. coli strains where the temperature sensitivity caused by the ssbH55K mutation was suppressed by spontaneous mutations that changed the glutamine at position 76 or 110 to leucine. Using guanidinium chloride denaturation monitored by sedimentation diffusion equilibrium experiments in the analytical ultracentrifuge, we demonstrate that the double mutant SSBH55KQ76L and SSBH55KQ110L proteins form more stable homotetramers as compared to the SSBH55K single mutant protein although they are less stable than wild-type SSB. Additionally, the single mutant proteins SSBQ76L and SSBQ110L form tetramers which are more resistant to guanidinium denaturation than wild-type SSB protein. |
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| Keywords |
Amino Acids/physiology; DNA-Binding Proteins/chemistry; DNA-Binding Proteins/genetics; DNA-Binding Proteins/metabolism; Escherichia coli/chemistry; Escherichia coli/genetics; Guanidine; Poly T/metabolism; Protein Binding; Protein Conformation; Protein Denaturation; SOS Response (Genetics); Suppression, Genetic; Temperature; Ultraviolet Rays |
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