2006
DOI: 10.1021/ja0577692
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Self-Reporting Fluorescent Substrates of Protein Tyrosine Kinases

Abstract: A new mechanistic principle by which protein tyrosine kinase substrates fluorescently report the introduction of a phosphate moiety has been developed. NMR was used to establish that tyrosine phosphorylation induces the disruption of pi-pi stacking interactions of the tyrosine moiety with a proximal fluorophore on the peptide substrate. We have demonstrated that (1) the peptide substrates described in this study are useful for a wide variety of different tyrosine kinases, (2) physiological concentrations of AT… Show more

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Cited by 75 publications
(80 citation statements)
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“…49 In a recent study involving mammalian cells, dansylalanine has also been incorporated into neural stem cells using an orthogonal tyrosyl tRNA/TyrRS pair from E. fold. 55 The increase in fluorescence was attributed to a disruption of a π-π interaction between tyrosine and pyrene, corroborated by 2D nOe NMR data showing marked differences in the observed nOes between the pyrene and tyrosine protons in nonphosphorylated and phosphorylated states. 55 The pyrene amino acid approach was further applied in a protein kinase detection assay using a pyrene-containing peptide substrate for a serine kinase (PKA).…”
Section: Probing Interactions With Solvatochromic Flaasmentioning
confidence: 66%
See 1 more Smart Citation
“…49 In a recent study involving mammalian cells, dansylalanine has also been incorporated into neural stem cells using an orthogonal tyrosyl tRNA/TyrRS pair from E. fold. 55 The increase in fluorescence was attributed to a disruption of a π-π interaction between tyrosine and pyrene, corroborated by 2D nOe NMR data showing marked differences in the observed nOes between the pyrene and tyrosine protons in nonphosphorylated and phosphorylated states. 55 The pyrene amino acid approach was further applied in a protein kinase detection assay using a pyrene-containing peptide substrate for a serine kinase (PKA).…”
Section: Probing Interactions With Solvatochromic Flaasmentioning
confidence: 66%
“…55 The increase in fluorescence was attributed to a disruption of a π-π interaction between tyrosine and pyrene, corroborated by 2D nOe NMR data showing marked differences in the observed nOes between the pyrene and tyrosine protons in nonphosphorylated and phosphorylated states. 55 The pyrene amino acid approach was further applied in a protein kinase detection assay using a pyrene-containing peptide substrate for a serine kinase (PKA). In the assay, generate a response to kinase activity, the Imperiali group has developed a fluorescent amino acid of which the emission is derived from metal chelation, termed chelationenhanced fluorescence (CHEF, Figure 6).…”
Section: Probing Interactions With Solvatochromic Flaasmentioning
confidence: 66%
“…Leads display up to a 5-fold enhancement in fluorescence in response to phosphorylation [7]. An examination of the non-phosphorylated sensor and its phosphorylated counterpart by 2D NOESY NMR spectroscopy revealed dramatic differences in nuclear Overhauser enhancements (nOes).…”
Section: "Self-reporting" Protein Kinase Sensorsmentioning
confidence: 99%
“…[1,2] Recently, we described a strategy for the design of PTK reporters that relies upon the phosphorylation-induced disruption of noncovalent interactions between the kinasetargeted Tyr residue and a proximal pyrene moiety. [3] However, the latter, like all other PTK sensors described to date, is immediately susceptible to phosphorylation. The inability to control sensing activity precludes an assessment of intracellular kinase action as a function of the cell cycle in particular, and environmentally-stimulated behaviors in general.…”
mentioning
confidence: 97%
“…With this limitation in mind, we constructed a small peptide library containing several fluorophores positioned on the (L)-2,4-diaminobutanoic acid 1 (Dab) and the (L)-2,3-diaminopropionic acid 2 (Dap) at the Y−2 position and the Y+3 positions, respectively, of a known Src kinase substrate. [3] Oregon green, [4] cascade blue, [6] and cascade yellow [6] are relatively bright fluorophores with high quantum yields, good resistance to photobleaching and distinct spectral properties. The cascade yellow Y−2 derivative 3 exhibits a 2.7-fold enhancement in fluorescence intensity upon phosphorylation.…”
mentioning
confidence: 99%