Structural Study Of ?-galactosidase In Complex With Small Molecule Fluorescent Probes

?-galactosidase(?-gal)as a typical enzyme has been proven to be an important biomarker for studying cell senescence.Since senescence-associated ?-galactosidase(SA-?-gal)is currently the most widely used biomarker of cell aging in vitro and in vivo,it is important to monitor ?-gal activity at the subcellular level in real time.In recent years,many studies have mainly studied cell aging by using fluorescent probes for visual imaging in biological systems.The main purpose of this study is to analyze the main interactions between ?-galactosidase and fluorescent probe molecules through their three-dimensional crystal structures,providing a basis for the future design and optimization of fluorescent probe molecules.In this thesis,E.coli ?-gal mutant E537 Q expression strain from the active site amino acid Glu537 in the “TIM” domain was constructed: sumo-?-gal E537Q-p ET21a/BL21(DE3)as well as human ?-galactosidase(human ?-gal)expression strain through the Bac-to-Bac baculovirus expression system: human ?-gal-p Fast Bac/DH10 Bac.Afterwards,E.coli ?-gal E537 Q protein with a purity greater than 95% was successfully obtained through further expression and purification.Co-crystals of E.coli ?-gal E537 Q in complex with three fluorescent probes(GY1,GY2,KSL01)by soaking method were obtained respectively,Xray diffraction data were collected and the crystal structures of the three complexes were successfully resolved.Analysis and comparison of complex structures indicate that E.coli ?-gal is a homo-tetramer,but the way of packing of E.coli ?-gal monomers is different from that reported(PDB ID: 1jz7);Comparing the interaction modes of E.coli ?-gal with the three probes it was found that besides galactosyl binding,there is a ?-? stacking interaction between Trp999 and the benzyl ring of the probe and it is speculated that this interaction is very important to stabilize the binding of the probe to E.coli ?-gal.Besides,the benzyl ring as a linker leads the fluorophores of the probes towards the outside of the narrow binding pocket,preventing steric hindrance and promoting the binding of the probes to ?-gal.By comparing the electron density of the probe molecules at the active site it was found that GY1 and KSL01 are more flexible in structure and show higher B-factor.Because human ?-gal was expressed but purification unsuccessful,the complex structures of human ?-gal with probes were analyzed through molecular docking.Combined with the catalytic mechanism and the mode of interaction,it is concluded that human ?-gal may recognize these probes through a similar network of interactions.The application of fluorescent probes for the recognition and visualization of ?-galactosidase in biological systems has been extensively studied,but our work is the first one to reveal the mechanism by which the enzyme E.coli ?-gal recognizes fluorescent probes based on the crystal structures,which provides a structural basis for the design and optimization of future fluorescent probes.