Jennifer Sniegowski - Maturation efficiency, trypsin sensitivity, and optical properties of Arg96, Glu222, and Gly67 variants of green fluourescent protein

Document created by Jennifer Sniegowski on Aug 22, 2014
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  Publication Details (including relevant citation   information):

  Biochemical and Biophysical Research Communications, 332, (2005),   657-663.


  Spontaneous chromophore biosynthesis in green fluorescent protein   (GFP) is initiated by a main-chain cyclization reaction catalyzed   by the protein fold. To investigate the structural prerequisites   for chromophore formation, we have substituted the conserved   residues Arg96, Glu222, and Gly67. Upon purification, the   variants can be ordered based on their decreasing extent of   chromophore maturation according to the series EGFP, E222Q, R96K,   G67A, and R96M. Arg96 and Glu222 appear to play catalytic roles,   whereas Gly67 is likely important in interior packing to enforce   correct hydrogen bonding to Arg96. The effect of Arg96 can be   partially compensated for by a lysine, but not by a methionine   residue, confirming its electrophilic role. Limited trypsinolysis   data suggest that protein stability is largely unaffected by the   presence of the chromophore, inconsistent with the mechanical   compression hypothesis. Trends in optical properties may be   related to the degree of chromophore charge delocalization, which   is modulated by residue 96.

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