Identification of amino acids stabilizing the tetramerization of the single stranded DNA binding protein from Escherichia coli.
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.
Department of Biochemistry and Molecular Biology, The Pennsylvania State University, University Park 16802, USA.
L
L
Carlini
U
U
Curth
B
B
Kindler
C
C
Urbanke
R D
RD
Porter
D000596:Amino Acids / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000737:chemistry* / Q000235:genetics; D019791:Guanidine; D011071:Poly T / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D011489:Protein Denaturation; D013014:SOS Response, Genetics; D013489:Suppression, Genetic; D013696:Temperature; D014466:Ultraviolet Rays
D000596:Amino Acids; D004268:DNA-Binding Proteins; D011071:Poly T; D019791:Guanidine