Abstract
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.
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.