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@ARTICLE{Gerharz:30967,
      author       = {Gerharz, T. and Reinelt, S. and Kaspar, S. and Scapozza, L.
                      and Bott, M.},
      title        = {{I}dentification of basic amino acid residues important for
                      citrate binding by the periplasmic receptor domain of the
                      sensor kinase {C}it{A}},
      journal      = {Biochemistry},
      volume       = {42},
      issn         = {0006-2960},
      address      = {Columbus, Ohio},
      publisher    = {American Chemical Society},
      reportid     = {PreJuSER-30967},
      pages        = {5917 - 5924},
      year         = {2003},
      note         = {Record converted from VDB: 12.11.2012},
      abstract     = {The sensor kinase CitA and the response regulator CitB of
                      Klebsiella pneumoniae form the paradigm of a subfamily of
                      bacterial two-component regulatory systems that are capable
                      of sensing tri- or dicarboxylates in the environment and
                      then induce transporters for the uptake of these compounds.
                      We recently showed that the separated periplasmic domain of
                      CitA, termed CitAP (encompasses residues 45-176 supplemented
                      with an N-terminal methionine residue and a C-terminal
                      hexahistidine tag), is a highly specific citrate receptor
                      with a K(d) of 5.5 microM at pH 7. To identify positively
                      charged residues involved in binding the citrate anion, each
                      of the arginine, lysine, and histidine residues in CitAP was
                      exchanged for alanine, and the resulting 17 muteins were
                      analyzed by isothermal titration calorimetry (ITC). In 12
                      cases, the K(d) for citrate was identical to that of
                      wild-type CitAP or slightly changed (3.9-17.2 microM). In
                      one case (R98A), the K(d) was 6-fold decreased (0.8 microM),
                      whereas in four cases (R66A, H69A, R107A, and K109A) the
                      K(d) was 38- to >300-fold increased (0.2 to >1 mM). The
                      secondary structure of the latter five proteins in their
                      apo-form as deduced from far-UV circular dichroism (CD)
                      spectra did not differ from the apo-form of wild-type CitAP;
                      however, all of them showed an increased thermostability.
                      Citrate increased the melting point (T(m)) of wild-type
                      CitAP and mutein R98A by 6.2 and 9.5 degrees C,
                      respectively, but had no effect on the T(m) of the four
                      proteins with disturbed binding. Three of the residues
                      important for citrate binding (R66, H69, and R107) are
                      highly conserved in the CitA subfamily of sensor kinases,
                      indicating that they might be involved in ligand binding by
                      many of these sensor kinases.},
      keywords     = {Amino Acid Sequence / Amino Acid Substitution / Amino
                      Acids, Basic: chemistry / Bacterial Proteins: chemistry /
                      Bacterial Proteins: genetics / Bacterial Proteins:
                      metabolism / Base Sequence / Binding Sites / Circular
                      Dichroism / Citric Acid: metabolism / DNA, Bacterial:
                      genetics / Escherichia coli Proteins: chemistry /
                      Escherichia coli Proteins: genetics / Escherichia coli
                      Proteins: metabolism / Kinetics / Klebsiella pneumoniae:
                      genetics / Klebsiella pneumoniae: metabolism / Ligands /
                      Molecular Sequence Data / Mutagenesis, Site-Directed /
                      Periplasm: metabolism / Protein Binding / Protein Kinases:
                      chemistry / Protein Kinases: genetics / Protein Kinases:
                      metabolism / Protein Structure, Tertiary / Sequence
                      Homology, Amino Acid / Spectrometry, Mass, Matrix-Assisted
                      Laser Desorption-Ionization / Thermodynamics / Transcription
                      Factors: chemistry / Transcription Factors: genetics /
                      Transcription Factors: metabolism / Amino Acids, Basic (NLM
                      Chemicals) / Bacterial Proteins (NLM Chemicals) / CitB
                      protein, Klebsiella pneumoniae (NLM Chemicals) / DNA,
                      Bacterial (NLM Chemicals) / Escherichia coli Proteins (NLM
                      Chemicals) / Ligands (NLM Chemicals) / Transcription Factors
                      (NLM Chemicals) / Citric Acid (NLM Chemicals) / Protein
                      Kinases (NLM Chemicals) / dpiB protein, E coli (NLM
                      Chemicals) / J (WoSType)},
      cin          = {IBT-1},
      ddc          = {570},
      cid          = {I:(DE-Juel1)VDB55},
      pnm          = {Biotechnologie},
      pid          = {G:(DE-Juel1)FUEK256},
      shelfmark    = {Biochemistry $\&$ Molecular Biology},
      typ          = {PUB:(DE-HGF)16},
      pubmed       = {pmid:12741850},
      UT           = {WOS:000182934300041},
      doi          = {10.1021/bi0340595},
      url          = {https://juser.fz-juelich.de/record/30967},
}