Development And Application Of Fluorescence Probe Based On Lanthanide-binding Tag

Since the discovery of lanthanide ions, because of their long fluorescence lifetime (ms level), fluorescence spectrum with a large Stokes Shift (>200 nm) and fluorescence without polarity, the lanthanide ions have been widely used as fluorescence probe.Lanthanide binding tag (LBT) originated form calmodulin. Calmodulin has an EF-hand structure bind to the calcium specifically. The atomic radius and the physical properties of calcium ion are similar to these of the lanthanide ion. Lanthanide metal ions can interact with calcium ion to combine with calmodulin, the fluorescence quantum yield is very small. Based on the EF-hand structure model combined with calcium,after a series of screening and improvement, Imperiali’s laboratory improved the binding capacity of lanthanide ions to small peptides (KD-nM), introduced tryptophan at the appropriate position to increase the fluorescence quantum yield, and designed a small peptide named lanthanide-binding tag (LBT).LBT composes 15-20 amino acids, which can specifically bind with lanthanide ions. LBT can be inserted into the loop region of the protein, be attached to N-terminal or C-terminal of protein. Rigidification and site-specific tagging could also be achieved by linking an LBT to a single cysteine within the protein via a disulfide bridge. Due to the small size of LBT the protein structure and function would not be affected in most cases.In addition, phosphorescence properties of LBT make it have been widely used in time-resolved fluorescence detection. But the lanthanide ions cannot be excited directly, the indole ring of the tryptophan serves as a sensitizer, to overcome the inherently low absorbance of the lanthanide ions. The measurements based on LBT are conducted with excitation of the sensitizing tryptophan at 280 nm. While clearly advantageous for in vitro studies, the UV light necessary for tryptophan excitation (280 nm) is not ideal in cellular systems.Therefore, based on the wild type LBT (the peptide sequence: YIDTNNDGWIEGDELLA), which includes six coordinating residues (shown in bold) and the tryptophan residue, which is crucial for Ln (III) sensitization and also provides a backbone carbonyl oxygen as a ligand. We introduced mutations on LBT-WT to increase the numbers of indole rings, and to observe the effect on the fluorescence properties of the LBT. In addition, the excitation characteristics of LBT limits it used in living cells. We describe the preparation of new LBT peptides that incorporate fluorescent compound in place of tryptophan, which can sensitize Ln(Ⅲ) luminescence at lower energy.Our study found that introducing indole ring at proper position of LBT can enhance the fluorescence quantum yield of LBT significantly. In five mutants we designed, the quantum yield of LBT-T4W is largest,1.5 times larger than LBT-WT. Besides, we developed combined probe based on LBT-WT, to excite LBT by visible light, made it can be used in living cells. Besides, we used LRET to study the intramolecular interaction of RSV capsid influenced by Ca2+, Mg2+, and Phosphate.In summary, we preserved the advantages fluorescence properties of LBT(ms-level lifetime, characteristics of emission spectrum, the specific binding of the lanthanide metal ions) and made multiple improvements of developed LBT, including: we increased the number of indole rings on LBT to improve the fluorescence quantum yield; we developed the fluorescence probe featuring new sensitizer that is excited at lower energy, mostly expanded the usage of this class of lanthanide-based probes. Our current work is aimed at using the probe in vivo. For our future study, the developed LBT will be more extensively used for biological research. Based on LBT we used LRET to study intramolecular interaction between NTD and CTD of RSV capsid affected by a variety of factors.