FRET-based in vivo Ca2+ imaging by a new calmodulin-GFP fusion molecule.
Intracellular Ca2+ acts as a second messenger that regulates numerous physiological cellular phenomena including development, differentiation and apoptosis. Cameleons, a class of fluorescent indicators for Ca2+ based on green fluorescent proteins (GFPs) and calmodulin (CaM), have proven to be a useful tool in measuring free Ca2+ concentrations in living cells. Traditional cameleons, however, have a small dynamic range of fluorescence resonance energy transfer (FRET), making subtle changes in Ca2+ concentrations difficult to detect and study in some cells and organelles. Using the NMR structure of CaM bound to the CaM binding peptide derived from CaM-dependent kinase kinase (CKKp), we have rationally designed a new cameleon that displays a two-fold increase in the FRET dynamic range within the physiologically significant range of cytoplasmic Ca2+ concentration of 0.05-1 microM.
Truong
K
K
Division of Molecular and Structural Biology, Ontario Cancer Institute and Department of Medical Biophysics, University of Toronto, Toronto, Ontario M5G 2M9, Canada.
Sawano
A
A
Mizuno
H
H
Hama
H
H
Tong
K I
KI
Mal
T K
TK
Miyawaki
A
A
Ikura
M
M
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D020013:Calcium Signaling; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase; D002147:Calmodulin / Q000737:chemistry* / Q000378:metabolism*; D002478:Cells, Cultured; D003593:Cytoplasm / Q000378:metabolism*; D015195:Drug Design; D004735:Energy Transfer; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006624:Hippocampus / Q000166:cytology; D006801:Humans; D008164:Luminescent Proteins / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D008958:Models, Molecular; D009474:Neurons / Q000166:cytology / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011487:Protein Conformation; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000378:metabolism*; D012680:Sensitivity and Specificity; D013050:Spectrometry, Fluorescence; D013329:Structure-Activity Relationship; D014982:Xenopus laevis
D002147:Calmodulin; D008164:Luminescent Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D017346:Protein Serine-Threonine Kinases; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase; D002118:Calcium