Mechanism-based fluorescent reporter for protein kinase A detection.
A novel mechanism-based fluorescent reporter was designed for the detection of protein kinase A (PKA), which is known to mediate a variety of cellular responses in most eukaryotic cells. The probe consists of a specific binding peptide sequence, LRRRRFAFC, conjugated with 2'-thioethyl-5-(or -6)-carboxyfluoresceinamide (FAMS; 2) and 5-(or 6-)carboxytetramethylrhodamine (TAMRA) at the cysteine and leucine residues, respectively. In the absence of PKA, the two fluorophores associate by hydrophobic interactions, forming an intramolecular ground-state dimer; this results in fluorescein quenching (>93 %). Upon PKA addition, the reporter reacts with the sulfhydryl functionality at Cys199 through a disulfide-exchange mechanism. FAMS is subsequently released, resulting in significant fluorescence amplification. The remaining peptide sequence, which acts as an inhibitor, is attached covalently to the enzyme. Our results suggest that this type of sensors could have far-reaching applications in the molecular sensing of enzymes.
Law
Benedict
B
Center for Molecular Imaging Research, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129, USA.
Weissleder
Ralph
R
Tung
Ching-Hsuan
CH
D000595:Amino Acid Sequence; D000818:Animals; D017868:Cyclic AMP-Dependent Protein Kinases / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D019793:Fluorescein / Q000737:chemistry; D005456:Fluorescent Dyes / Q000138:chemical synthesis* / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010446:Peptide Fragments / Q000138:chemical synthesis* / Q000737:chemistry; D011485:Protein Binding; D012235:Rhodamines / Q000737:chemistry; D013379:Substrate Specificity
D005456:Fluorescent Dyes; D010446:Peptide Fragments; D012235:Rhodamines; D017868:Cyclic AMP-Dependent Protein Kinases; D019793:Fluorescein