Ca2+ indicators based on computationally redesigned calmodulin-peptide pairs.
The binding interface of calmodulin and a calmodulin binding peptide were reengineered by computationally designing complementary bumps and holes. This redesign led to the development of sensitive and specific pairs of mutant proteins used to sense Ca(2+) in a second generation of genetically encoded Ca(2+) indicators (cameleons). These cameleons are no longer perturbed by large excesses of native calmodulin, and they display Ca(2+) sensitivities tuned over a 100-fold range (0.6-160 microM). Incorporation of circularly permuted Venus in place of Citrine results in a 3- to 5-fold increase in the dynamic range. These redesigned cameleons show significant improvements over previous versions in the ability to monitor Ca(2+) in the cytoplasm as well as distinct subcellular localizations, such as the plasma membrane of neurons and the mitochondria.
Palmer
Amy E
AE
Department of Pharmacology and Howard Hughes Medical Institute, University of California, San Diego, 9500 Gilman Drive, La Jolla, California 92093, USA.
Giacomello
Marta
M
Kortemme
Tanja
T
Hires
S Andrew
SA
Lev-Ram
Varda
V
Baker
David
D
Tsien
Roger Y
RY
D000595:Amino Acid Sequence; D002118:Calcium / Q000737:chemistry / Q000378:metabolism*; D002147:Calmodulin / Q000737:chemistry / Q000378:metabolism*; D002462:Cell Membrane / Q000378:metabolism; D003600:Cytosol / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry / Q000378:metabolism*; D011485:Protein Binding; D013347:Subcellular Fractions / Q000378:metabolism
D002147:Calmodulin; D010455:Peptides; D002118:Calcium