A new fluorescent biosensor for inositol trisphosphate.
An intracellular second messenger d-myo-inositol-1,4,5-trisphosphate (IP3) is a key biological signaling molecule that controls the cellular Ca2+ concentration. We report the preparation and evaluation of a functionalized protein-based sensor for IP3 by exploring the selective IP3 binding properties of pleckstrin homology (PH) domain. Signal transduction is imparted to the protein by mutation of proximal residues to cysteine and then alkylation of the active site by various fluorophore derivatives. This creates functionalized proteins that show micromolar affinity for IP3, reasonably strong fluorescence emission, and wavelength changes in the fluorophore and selectivity higher than the original PH domain among different inositol phosphate derivatives.
Morii
Takashi
T
Institute of Advanced Energy, Kyoto University, PRESTO, Japan Science and Technology Corporation, Uji, Kyoto 611-0011, Japan.
Sugimoto
Kenji
K
Makino
Keisuke
K
Otsuka
Masami
M
Imoto
Keiji
K
Mori
Yasuo
Y
D000595:Amino Acid Sequence; D015374:Biosensing Techniques; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D015544:Inositol 1,4,5-Trisphosphate / Q000737:chemistry*; D007527:Isoenzymes / Q000737:chemistry*; D008969:Molecular Sequence Data; D054803:Phospholipase C delta; D011487:Protein Conformation; D017434:Protein Structure, Tertiary; D010738:Type C Phospholipases / Q000737:chemistry*
D005456:Fluorescent Dyes; D007527:Isoenzymes; D015544:Inositol 1,4,5-Trisphosphate; D010738:Type C Phospholipases; D054803:Phospholipase C delta