Protein-peptide interactions analyzed with the yeast two-hybrid system.
The yeast two-hybrid system was used to screen a library of random peptides fused to a transcriptional activation domain in order to identify peptides capable of binding to the retinoblastoma protein (Rb). Seven peptides were identified, all of which contain the Leu-X-Cys-X-Glu motif found in Rb-binding proteins, although their activity in the yeast assay varied over a 40-fold range. Mutagenesis of the DNA encoding two of these peptides followed by screening in the two-hybrid system allowed the delineation of residues apart from the invariant Leu, Cys and Glu that affect binding to Rb. Binding affinities of a peptide and one of its variants to Rb, determined by surface plasmon resonance, correlated with results from the two-hybrid assay. This method offers several advantageous features compared to existing technology for screening peptide libraries: in vivo detection of protein-peptide interactions, high sensitivity, the capacity for rapid genetic screening to identify stronger and weaker binding peptide variants, and the use of a simple assay (transcriptional activity) as a means to assess binding affinity.
Yang
M
M
Department of Molecular Genetics and Microbiology, State University of New York at Stony Brook 11794, USA.
Wu
Z
Z
Fields
S
S
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010455:Peptides / Q000235:genetics / Q000378:metabolism*; D011485:Protein Binding; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000235:genetics / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism*
D017931:DNA Primers; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D010455:Peptides; D011993:Recombinant Fusion Proteins; D016160:Retinoblastoma Protein