Structural And Functional Study Of Extracellular Oligosaccharide Binding Protein Of ABC Transporter Of Bacillus Licheniformis

Probiotics can use oligosaccharides to promote their own growth and proliferation.The intake of oligosaccharides is the first step for probiotics to use oligosaccharides.Msm-type ABC transporter is an important transporter for probiotics to ingest oligosaccharides.Its oligosaccharide binding protein Msm E can capture oligosaccharides extracellularly and then transfer them to other components of the ABC transporter to complete the oligosaccharide transport.The specific binding of Msm E to oligosaccharides is the key to whether probiotics can ingest oligosaccharides successfully.In view of this,exploring the mechanism of interaction between oligosaccharide binding proteins and substrates can provide important prerequisites for the in-depth analysis of probiotic transport oligosaccharides.In this study,the combination of experimental research and computer simulation was used to study the oligosaccharide binding protein of Bacillus licheniformis.Firstly,the E.coli expression vector p ET15b-Blmsm E was constructed and expressed in E.coli BL21(DE3).The BlMsmE protein was purified by Ni-NTA affinity chromatography and size exclusion chromatograph.Next,the characteristic of the binding of BlMsmE protein to the substrates were studied by isothermal titration calorimetry(ITC)and fluorescence spectroscopy(FS).Finally,molecular mechanics generalized Born surface area(MM-GBSA)and molecular dynamics simulation(MD)were used to study the binding model of oligosaccharide binding protein to different substrates.The following main conclusions are drawn:(1)The pET15a-Blmsm E plasmid was constructed.Using E.coli as the expression system,the BlMsmE protein was successfully expressed.Through purification steps such as Ni-NTA affinity chromatography and size exclusion chromatography,highpurity protein was obtained from the supernatant of E.coli lysate.(2)Isothermal titration calorimetry(ITC)and fluorescence spectroscopy(FS)were used to characterize the binding properties of the protein to the substrate,and protein crystal screening was also performed.The ITC experiment determined that the binding affinities of BlMsmE to the substrates were: galactinol(134n M)> raffinose(170 n M)> stachyose(299 n M)> melibiose(3472 n M)> galactose(16129 n M).Fluorescence spectroscopy tested the interaction of BlMsmE with stachyose,melibiose,and galactinol.The results showed that when BlMsmE interacted with stachyose,galactinol,the fluorescence spectrum changed over a large range,while changed over a small range when interacting with melibiose.Among them,the interaction between BlMsmE and galactinol was first discovered and described in this project.The preliminary screening of protein crystals obtained the condition that BlMsmE crystals appeared in the state of no substrate binding and substrate binding,which were basis for further screening.(3)The details of the binding of BlMsmE to the substrate were explored through molecular mechanics-generalized Born surface area(MM-GBSA)and molecular dynamics simulation.MM-GBSA concluded that the main residues of BlMsmE involved in binding include R307,R375,W383 and V270,and electrostatic energy and van der Waals energy had a major contribution to the binding.Molecular dynamics simulation of the interaction between BlMsmE and substrates found that the interaction between BlMsmE and substrates mainly involved three helical regions hr I,hr II,and hr III,of which the conformational change of hr I was the largest.When substrates(melibiose and stachyose)interacted with BlMsmE,they all could interact with the first half of hr I significantly.When it came to the second half of hr I,stachyose chould interact with this part of the residues,while melibiose could not be extended to this part due to its small size,resulting in the binding affinity of melibiose and BlMsmE weakened rapidly.