Context-dependent fluorescence detection of a phosphorylated tyrosine residue by a ribonucleopeptide.
Tools for selective recognition and sensing of specific phosphorylated tyrosine residues on the protein surface are essential for understanding signal transduction cascades in the cell. A stable complex of RNA and peptide, a ribonucleopeptide (RNP), provides effective approaches to tailor RNP receptors and fluorescent RNP sensors for small molecules. In vitro selection of an RNA-derived pool of RNP afforded RNP receptors specific for a phosphotyrosine residue within a defined amino-acid sequence Gly-Tyr-Ser-Arg. The RNP receptor for the specific phosphotyrosine residue was successfully converted to a fluorescent RNP sensor for sequence-specific recognition of a phosphorylated tyrosine by screening a pool of fluorescent phosphotyrosine-binding RNPs generated by a combination of the RNA subunits of phosphotyrosine-binding RNPs and various fluorophore-modified peptide subunits. The phosphotyrosine-binding RNP receptor and fluorescent RNP sensor constructed from the RNP receptor not only discriminated phosphotyrosine against tyrosine, phosphoserine, or phosphothreonine, but also showed specific recognition of amino acid residues surrounding the phosphotyrosine residue. A fluorescent RNP sensor for one of the tyrosine phosphorylation sites of p100 coactivator showed a binding affinity to the target site ~95-fold higher than the other tyrosine phosphorylation site. The fluorescent RNP sensor has an ability to function as a specific fluorescent sensor for the phosphorylated tyrosine residue within a defined amino-acid sequence in HeLa cell extracts.
Hasegawa
Tetsuya
T
Institute of Advanced Energy, Institute of Sustainability Science, and Pioneering Research Unit for Next Generation, Kyoto University, Uji, Kyoto 611-0011, Japan.
Hagihara
Masaki
M
Fukuda
Masatora
M
Nakano
Shun
S
Fujieda
Nobutaka
N
Morii
Takashi
T
D020816:Amino Acid Motifs; D001483:Base Sequence; D015374:Biosensing Techniques; D002457:Cell Extracts / Q000737:chemistry; D005453:Fluorescence; D006367:HeLa Cells; D006801:Humans; D010455:Peptides / Q000737:chemistry*; D010766:Phosphorylation; D019000:Phosphotyrosine / Q000032:analysis*; D012265:Ribonucleotides / Q000737:chemistry*
D002457:Cell Extracts; D010455:Peptides; D012265:Ribonucleotides; D019000:Phosphotyrosine