Lanthanide-binding tags as luminescent probes for studying protein interactions.
Herein, we report a method for studying protein-peptide interactions which exploits the luminescence properties of Tb(III). Lanthanide-binding tags (LBTs) are short peptide sequences comprising 15-20 naturally occurring amino acids that bind Tb(III) with high affinity. These genetically encodable luminescent tags are smaller in size than the Aequorea victoria fluorescent proteins (AFPs) and benefit from the long-lived luminescence lifetime of lanthanides. In this study, luminescence resonance energy transfer (LRET) was used to monitor the interaction between SH2 domains and different phosphopeptides. For the study, the SH2 domains of Src and Crk kinase were each coexpressed with an LBT, and phosphorylated and nonphosphorylated peptides were chemically synthesized with organic fluorophores. The LRET between the protein-bound Tb(III) and the peptide-based organic fluorophore was shown to be specific for the recognition of the SH2 domain and the peptide binding partner. This method can detect differences in binding affinity and can be used to measure the dissociation constant for the protein-peptide interaction. In addition, decay experiments can be used to calculate the distance between a site in the bound peptide and the protein using Förster theory. In all of these experiments, the millisecond luminescence lifetime of Tb(III) can be exploited using time-resolved detection to eliminate background fluorescence from organic fluorophores.
Sculimbrene
Bianca R
BR
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts 02139, USA.
Imperiali
Barbara
B
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D003001:Cloning, Molecular; D004735:Energy Transfer; D004926:Escherichia coli / Q000235:genetics; D049408:Luminescent Agents / Q000737:chemistry*; D008163:Luminescent Measurements; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D011485:Protein Binding; D011506:Proteins / Q000737:chemistry*; D050737:Proto-Oncogene Proteins c-crk / Q000737:chemistry / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics; D013725:Terbium / Q000737:chemistry*; D019061:src-Family Kinases / Q000737:chemistry / Q000235:genetics
D000596:Amino Acids; D049408:Luminescent Agents; D010455:Peptides; D011506:Proteins; D050737:Proto-Oncogene Proteins c-crk; D011993:Recombinant Fusion Proteins; D013725:Terbium; D019061:src-Family Kinases