Calcium indicators based on calmodulin-fluorescent protein fusions.
Calmodulin (CaM) is an ubiquitous protein involved in Ca2+-mediated signal transduction. On Ca2+ influx, CaM acquires a strong affinity to various cellular proteins with one or more CaM recognition sequences, resulting in the onset or termination of Ca2+-regulated cascades. Through nuclear magnetic resonance and crystallographic structural studies of these Ca2+-CaM complexes, we have gained a deep understanding of CaM target recognition mechanisms. One immediate application is the creation of protein-based Ca2+ sensors using CaM complexes and green fluorescent proteins, previously named "chameleon." The major advantage of chameleons is that they can be expressed in single cells and targeted to the specific organelles or tissues to measure localized Ca2+ changes. This chapter describes the methods involved in cloning chameleons, characterizing their biochemical and biophysical properties, and imaging them in single cells using a digital fluorescence microscope.
Truong
Kevin
K
Institute of Biomaterials and Biomedical Engineering, Department of Electrical and Computer Engineering, University of Toronto, Ontario, Canada.
Sawano
Asako
A
Miyawaki
Atsushi
A
Ikura
Mitsuhiko
M
D002118:Calcium / Q000378:metabolism*; D002147:Calmodulin / Q000235:genetics* / Q000378:metabolism; D003001:Cloning, Molecular; D031541:Fluorescence Resonance Energy Transfer; D008164:Luminescent Proteins / Q000235:genetics*; D008958:Models, Molecular; D011487:Protein Conformation; D015202:Protein Engineering / Q000379:methods*; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification
D002147:Calmodulin; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D002118:Calcium