Fluorescence complementation via EF-hand interactions.
Fluorescence complementation technology with fluorescent proteins is a powerful approach to investigate molecular recognition by monitoring fluorescence enhancement when non-fluorescent fragments of fluorescent proteins are fused with target proteins, resulting in a new fluorescent complex. Extension of the technology to calcium-dependent protein-protein interactions has, however, rarely been reported. Here, a linker containing trypsin cleavage sites was grafted onto enhanced green fluorescent protein (EGFP). Under physiological conditions, a modified fluorescent protein, EGFP-T1, was cleaved into two major fragments which continue to interact with each other, exhibiting strong optical and fluorescence signals. The larger fragment, comprised of amino acids 1-172, including the chromophore, retains only weak fluorescence. Strong green fluorescence was observed when plasmid DNA encoding complementary EGFP fragments fused to the EF-hand motifs of calbindin D9k (EF1 and EF2) were co-transfected into HeLa cells, suggesting that chromophore maturation and fluorescence complementation from EGFP fragments can be accomplished intracellularly by reassembly of EF-hand motifs, which have a strong tendency for dimerization. Moreover, an intracellular calcium increase upon addition of a calcium ionophore, ionomycin in living cells, results in an increase of fluorescence signal. This novel application of calcium-dependent fluorescence complementation has the potential to monitor protein-protein interactions triggered by calcium signalling pathways in living cells.
Chen
Ning
N
Department of Chemistry, Center for Drug Design and Advanced Biotechnology, Georgia State University, Atlanta, GA 30302, USA.
Ye
Yiming
Y
Zou
Jin
J
Li
Shunyi
S
Wang
Siming
S
Martin
Amy
A
Wohlhueter
Robert
R
Yang
Jenny J
JJ
D000595:Amino Acid Sequence; D020013:Calcium Signaling; D003001:Cloning, Molecular; D020832:EF Hand Motifs; D049452:Green Fluorescent Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D008856:Microscopy, Fluorescence / Q000379:methods; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D054730:Protein Interaction Domains and Motifs; D011993:Recombinant Fusion Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D014357:Trypsin / Q000378:metabolism
D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D014357:Trypsin