Protein Labeling And Interaction Study With Paramagnetic And Luminescence Lanthanide Probe

BackgroundsThe paramagnetic gadolinium (Gd) and luminescent terbium (Tb) are both lanthanide, which belong to the rare earth elements. In biomedicine study, with the help of liquid NMR and fluorescence techniques, the paramagnetic and luminescent lanthanide probes are used to label proteins and to study the protein-protein interaction.There is a complex network of phosphorylation reactions in the PTS of E.coli. One canonical pathway is that, the phosphoryl group from PEP is transferred first to EI, then to HPr and last to EⅡA. EⅡAs correspond to the transport of PTS sugar. For example, EⅡAGlc, EⅡAMtl and EⅡAMan are involved in the utilization of glucose, mannitol and mannose, respectively. The phosphorylation reaction is completed based on the protein complexes, most of which with weak bindings. These transient complexes could be detected by PRE NMR techniques. As is the network of cell signaling and predicted in STRING database, there may be a interaction between EI and EⅡA. Therefore, the interaction between EI and EⅡA could be studied with PRE NMR technique based on paramagnetic Gd probe.Besides NMR, the fluorescence technique is also wildly used, especially in in-vivo experiments, for its real-time observation. However, there are often many problems for traditional fluorescence, such as the specificity, stability of labeling and the background fluorescence of bio-samples. The sensitized Tb luminescence is green and has a ms-lifetime, so the background fluorescence with a ns-lifetime could be filtered by setting a time delay.CysLTR including two subtypes CysLT1R and CylLT2R belongs to G protein-coupled receptor. CysLTR play an important role in the ischemic neuronal injury in the central nervous system. However, limited by the available techniques, a lot of unknown questions are to be solved, such as the protein structure, oligomer state, assembly, and ligand signaling. Therefore, the first important things to do is to find a proper labeling method.AimsUsing Gd3+based PRE NMR technique to study the interaction between EI and EIIA, to prove that there is a non-canonical pathway in PTS of E.coli. And then, label hCysLTR in living cells with luminescent Tb probe.Methods and ResultsPartⅠ Using paramagnetic Gd probe to study the non-canonical pathway in PTS in E.coliNMR titrations between EIN and EIIAMtl are performed, and a Gd3+-EDTA-benzyl-acetate paramagnetic probe was introduced, one site at a time, to E69C mutant of EIIAMtl and to E97C, D144C mutants of EIIAGlc, to perform a PRE NMR experiment. We detected the weak binding between EIN and EIIAMtl, and fitted the Kd to～13mM. Also, the possible interface for interaction was. analysed.We discovered that the phosphorylation of EIIA could be accomplished by El alone, from NMR experiments. Resulted from competitive binding towards EIN and EIIA, addition of HPr-H15A obstructs the phosphorylation of EIIA by EI. Therefore, the direct transfer of phosphoryl group between El and EIIA could be accomplished without HPr.Finally, we measured the growth curves and sugar utilizations of AptsH and AptsH-H15A strains. The results show that EIIA can be directly phosphorylated by EI in vivo, and the interaction between El and EIIA accounts for physiological functions.Part Ⅱ Labeling hCysLTR in living cells with luminescence lanthanide probeWe engineered LBT on the N-terminal and into3extracellular loop regions of hCysLT1R and hCysLT2R. The hCysLTR with LBT mutation are expressed in HEK-293cells after transiently transfection.We measured the exogenous agonist LTD4-enhanced intracellular calcium with Fluo-4, to identify the function of CysLTR with LBT mutation. The results showed that LTD4increased intracellular calcium release in the loop-LBT mutation transfected HEK293cells, while the N-terminal LBT had an influence on the receptor functions. Also, Tb3+of concentration0.1nM to1μM had no effects on morphology and viability of HEK-293cells. Therefore, the loop LBT binding Tb3+as the luminescence probe could be used to labeling hCysLTR.At last, we have observed the sensitized luminescence of Tb in living HEK-293cells via multi-photon microscope.ConclusionsWe have detected the weak binding and phosphoryl group transfer between El and EIIA, with Gd3+based PRE NMR techniques, which indicates that there is a non-canonical pathway in PTS of E.coli.With luminescent Tb3+, we have made the labeling of hCysLTR in living HEK-293cells, which will be the basis of hCysLTR structure and function studies.