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Functional roles of amino acid residues involved in forming the α–helix‐turn–α–helix operator DNA binding motif of tet represser from Tn 10
Author(s) -
Baumeister Ralf,
Müller Gerhard,
Hecht Brigitte,
Hillen Wolfgang
Publication year - 1992
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.340140204
Subject(s) - asparagine , amino acid , alanine , helix turn helix , biochemistry , mutant , serine , leucine , stereochemistry , site directed mutagenesis , helix (gastropod) , chemistry , valine , biology , repressor , gene , transcription factor , enzyme , ecology , snail
Abstract The Tn 10 derived Tet represser contains an amino acid segment with high homology to the α‐helix–turn–α‐helix motif (HTH) of other DNA binding proteins. The five most conserved amino acids in HTH are probably involved in structural formation of the motif. Their functional role was probed by saturation mutagenesis yielding 95 single amino acid replacement mutants of Tet repressor. Their binding efficiencies to tet operator were quantitatively determined in vivo. All functional mutants contain amino acid substitutions consistent with their proposed role in a HTH. In particular, only the two smallest amino acids (serine, glycine) can substitute a conserved alanine in the proposed first α‐helix without loss of activity. The last position of the first α‐helix, the second position in the turn, and the fourth position in the second α‐helix require mostly hydrophobic residues. The proposed C‐terminus of the first α‐helix is supported by a more active asparagine compared to glutamine replacement mutant of the wt leucine residue. The turn is located close to the protein surface as indicated by functional lysine and arginine replacements for valine. A glycine residue at the first position in the turn can be replaced by any amino acid yielding mutants with at least residual tet operator affinity. A structural model of the HTH of Tet repressor is presented. © 1992 Wiley‐Liss, Inc.