Self-assembly Mechanism Of Hydrogels Made Of Escherichia Coli Lipoate-protein Ligase A

In previous studies of our group,we observed that lipoate-protein ligase A(Lpl A)from Escherichia coli undergoes reversible sol-gel transition at a high concentration(> 1.0 m M)in vitro.Different from traditional linear gel proteins and phase-separable proteins containing intrinsically disordered regions(IDRs),Lpl A is the first globular protein reported to independently form stable hydrogels under native conditions.In addition,the gel-state catalytic activity of Lpl A makes it promising in the development of microphase biochemical reaction systems,biosensors,and smart drug delivery systems.In this study,Lpl A was prepared by E.coli p ET protein expression system.It was verified that neither His-tag nor ammonium sulfate precipitation method affects the gelation of purified Lpl A.In several common neutral low-salt buffer solutions,the sensitivity of the solution environment of Lpl A hydrogel was tested.The suitable p H range for Lpl A gelation was about5.5～7.5.In rheological tests,the complex modulus and shear yield point of Lpl A hydrogels were measured.In the temperature sweep mode,the lower critical solution temperature of the hydrogel was determined to be about13.5°C.With the aid of scanning electron microscopy,the cross-linked structure of the gel at the micron scale was observed,and it was confirmed that the gelation of Lpl A was essentially induced by the microscopic phase separation of the protein.In order to further explore the potential intermolecular interactions during the phase separation of Lpl A protein,we performed single-site mutation on some residues on the surface of Lpl A molecule to find and verify the key residues that affect the orderly assembly of Lpl A.Several possible conformations and assembly mechanisms of Lpl A molecule were determined and discussed by carrying out calculations of electrostatic potential energy surface of protein molecule surface,molecular docking simulation and X-ray small-angle scattering characterization.The intermolecular electrostatic interaction,dipole interaction and hydrophobic interaction are found to play a leading role in the assembly of Lpl A molecules.