Electrostatic Interaction-driven Supercharged Fluorescent Protein Self-assembly

Proteins,as the key units of complex cellular structure and function,are widely presented in living organisms.Among them,most natural proteins spontaneously aggregate into multi-layered structures,such as 1D microtubules,2D bacterial surface layers and 3D viral capsids.These assemblies play important roles in living systems and provide design templates for the construction of new nanomaterials.Contrary to the great achievements in the fields of DNA and RNA assembly,protein assembly is progressing slowly due to their large size and complex interactions.Fluorescent proteins have a relatively rigid β-barrel structure.After surface reconstruction,they can be redesigned to various supercharged fluorescent proteins with positive or negative charges on their surface,and remain the original function.Supercharged fluorescent protein self-assembly based on electrostatic interactions is theoretically possible and expected to expand the toolbox of protein assembly.However,this proposal requires the accurate design of the protein sequence and the exploration of the assembly conditions,since electrostatic interaction-driven protein assembly not only needs the opposite surface charge among proteins,but also demands the accurate surface charge distribution of the proteins and the interaction strength between the interfaces.In order to develop a new method for the self-assembly of supercharged fluorescent protein mediated by electrostatic interaction,this paper intensively studied on the properties of superfolded green fluorescent protein(sf GFP)with stable structure,unique spectral characteristics and surface designability.Using sf GFP as the template,we designed a polarized green fluorescent protein(p GFP)with 15 positive charges at one end and 10 negative charges at the other end by surface reconstruction.The recombinant protein p GFP was heterogeneous expressed and purified,and the optimal conditions for assembly was explored.Cuboid-like structure with the micrometer-level length in the solution was obtained,which realized the ingenious combination of the spectral characteristics of the fluorescent protein with the self-assembly characteristics.By using GROMACS molecular dynamics simulation,protein docking was simulated and interactions between the proteins were analyzed,which help us to understand the assembly process.In addition,p GFP can be used as a template to obtain mutants with different colors,which have the potential to self-assembly.The fluorescent protein mutants that satisfies the overlap of the donor emission spectrum and the acceptor absorption spectrum were constructed as a FRET pair.The specific works are as follows:1.Expression,characterization and self-assembly of p GFP in vitro.The recombinant plasmid p ET28a-pgfp was constructed and p GFP was expressed and purified successfully.After optimization of assembly conditions,it was confirmed that p GFP could spontaneously assembly into micrometer-level cuboid-like structure,with the final protein concentration 1 μM,at 4 ℃ for 2 days.The assembly process driven by electrostatic interaction is reversible by changes of Na Cl concentration and p H in the solution.When the assembly time is extended to 50 days,the average size of the assemblies can grow to 50.2 μm.These properties make it possible to have potential applications in the field of drug release.2.Molecular dynamics simulation study of p GFP self-assembly.Based on the SWISS-MODEL and I-TASSER modelling methods,models of sf GFP and p GFP were constructed and the results were evaluated in aspects of space and energy dimension respectively.Based on the optimal structure models,a rigid docking approach is used to obtain the initial binding models(sf GFP1+1,p GFP1+1 and p GFP2+2)whose geometrically and energy are matched,and GROMACS molecular dynamics simulations were performed on these binding models.The results of binding free energy proved that electrostatic interaction energy is the main energy contribution to promote the aggregation of p GFP molecules,which realized the microscopic interpretation of the self-assembly behavior of p GFP.This part of the works lays the foundation of a universal design based on fluorescent proteins self-assembly.3.Construction and optimization of FRET pairs based on fluorescent protein selfassembly.First,the recombinant plasmids p ET28a-pcfp and p ET28a-pyfp were constructed by site-directed mutagenesis,and p CFP and p YFP were expressed and purified in vitro.Subsequently,the optimal assembly conditions of p CFP and p YFP(the final protein concentration 3 μM,at 4 ℃ for 3 days)were explored based on that of p GFP.The resulting assemblies also exhibits a micrometer-level cuboid-like structure,and molecular simulation was used to analyze their interactions between the two mutants themselves,which can explain their differences in the aspect ratio of the assemblies.Further,the feasibility of constructing a FRET pair based on p CFP and p YFP assemblies were proved.Subsequently,FRET pairs using different hybrid assembly patterns were constructed and compared,and the FRET Ratio value of different experimental groups were optimized to exceed 2.5,and among them the highest value is 21.43(assemble p YFP separately for 30 days,then add p CFP to mix and assemble for 18 days).This work can not only enrich the construction manner of fluorescent protein FRET pairs,but also provide a brand-new idea for designing the subsequent FRET sensing platform.