Impact of nonnatural amino acid mutagenesis on the in vivo function and binding modes of a transcriptional activator

J Am Chem Soc. 2009 Oct 14;131(40):14240-2. doi: 10.1021/ja904378z.


Protein-protein interactions play an essential role in cellular function, and methods to discover and characterize them in their native context are of paramount importance for gaining a deeper understanding of biological networks. In this study, an enhanced nonsense suppression system was utilized to incorporate the nonnatural amino acid p-benzoyl-L-phenylalanine (pBpa) throughout the transcriptional activation domain of the prototypical eukaryotic transcriptional activator Gal4 in vivo (S. cerevisiae). Functional studies of the pBpa-containing Gal4 mutants suggest that this essential binding interface of Gal4 is minimally impacted by these substitutions, with both transcriptional activity and sensitivity to growth conditions maintained. Further supporting this are in vivo cross-linking studies, including the detection of a key binding partner of Gal4, the inhibitor protein Gal80. Cross-linking with a range of pBpa-containing mutants revealed a Gal4 x Gal80 binding interface that extends beyond that previously predicted by conventional strategies. Thus, this approach can be broadened to the discovery of novel binding partners of transcription factors, information that will be critical for the development of therapeutically useful small molecule modulators of these protein-protein interactions.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Benzophenones / chemistry
  • Benzophenones / metabolism
  • DNA-Binding Proteins / chemistry
  • DNA-Binding Proteins / genetics*
  • DNA-Binding Proteins / metabolism
  • Models, Molecular
  • Molecular Sequence Data
  • Phenylalanine / analogs & derivatives*
  • Phenylalanine / chemistry
  • Phenylalanine / genetics
  • Phenylalanine / metabolism
  • Saccharomyces cerevisiae / genetics
  • Saccharomyces cerevisiae / metabolism
  • Saccharomyces cerevisiae Proteins / chemistry
  • Saccharomyces cerevisiae Proteins / genetics*
  • Saccharomyces cerevisiae Proteins / metabolism
  • Transcription Factors / chemistry
  • Transcription Factors / genetics*
  • Transcription Factors / metabolism


  • 4-benzoylphenylalanine
  • Benzophenones
  • DNA-Binding Proteins
  • GAL4 protein, S cerevisiae
  • Saccharomyces cerevisiae Proteins
  • Transcription Factors
  • Phenylalanine