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Identification of amino acids stabilizing the tetramerization of the single stranded DNA binding protein from Escherichia coli
Author(s) -
Carlini Leslie,
Curth Ute,
Kindler Björn,
Urbanke Claus,
Porter Ronald D
Publication year - 1998
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(98)00655-3
Subject(s) - guanidinium chloride , denaturation (fissile materials) , mutant , histidine , chemistry , escherichia coli , biochemistry , ultracentrifuge , protein quaternary structure , mutation , protein subunit , amino acid , leucine , dna , mutant protein , seqa protein domain , dna replication , enzyme , origin of replication , nuclear chemistry , gene
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 ssb H55Y ( ssb‐1 ) and ssb H55K can be overcome by increasing protein concentration, with the ssb H55K 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 ssb H55K 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.