Construction Of Artificial Light-Harvesting System Based On Protein Self-Assembly

Proteins are one of the most versatile building blocks in nature,which form various ordered and dynamic nanostructures through precise self-assembly to guide all aspects of life.Studying the assembly behavior of proteins,especially manipulating proteins self-assembly in vitro will not only provide insights into the natural protein assembly process and mechanism,but also allow access to the new functional materials.The self-assembled protein frameworks are reminiscent of natural light-harvesting systems,which fully reflect the new application prospects of protein assemblies.By studying the structures and functional mechanism of various light-harvesting complexes,we summarized the following four aspects that lay the foundation and advantages of using protein assemblies for light-harvesting application:?i?Structure.Except for green algae,most light-harvesting antenna complexes contain a protein matrix.In purple photosynthetic bacteria and phycobilisomes,proteins function even in the form of highly ordered assemblies.?ii?Function.Protein is the secret of efficient energy transfer in nature.It can not only help pinpoint the position and orientation of pigment molecules,but also indirectly involve in the regulation of energy transfer process.?iii?Designability.Proteins have well-defined structures and strong designability.By rationally designing proteins,we can easily achieve precise control over the spatial distribution and orientation of the dye molecules.?iv?Dynamic responsiveness.Based on the inherent flexibility of the proteins and the dynamic responsiveness that may be provided by protein assembly strategies,it is expected to simulate the self-regulation behavior of natural light-harvesting system and build more advanced light-harvesting biomimetic materials.In summary,protein assembly holds great promise in the field of light-harvesting simulation.Here,we sought to design efficient artificial light-harvesting systems based on the advantages of protein and assembly strategies.Specifically,we selected self-luminous fluorescent proteins?EBFP2 and EGFP?and thermostable cricoid SP1 protein as building blocks.By rationally designing and modifying these proteins,we aimed to manipulate them assembly to form large-scaled,long-range ordered superstructures.By utilizing the inherent function of the protein modules or introducing functional factors related to the structure of protein,two construction schemes of template-free and templated are described.1.Design and Construction of Two-Dimensional Crystals of Fluorescent ProteinsGreen fluorescent protein?GFP?,as a natural self-luminous macromolecule,has received great attention since its discovery.It has been developed into an important tool for exploring biological structure and function in recent years.However,owing to its highly asymmetric spatial structure and heterogeneous surface,the assembly of GFP is extremely challenging.Here,we have developed a general strategy for preparing two-dimensional?2D?protein nanosheets and successfully constructed highly ordered 2D crystals of fluorescent proteins.We selected EGFP as the basic building block and rhodamine?-?stacking as the driving force.By combining genetic engineering and chemical modification,we endeavored to manipulate EGFP self-assembly to form highly ordered 2D layered structures and thereby lay the foundation for the following applications.To this end,we designed and synthesized a maleimide-functionalized rhodamine B molecule?RhG₂M?.In order to obtain the target 2D structure,we introduced four cysteine lying near the C₄ rotation axes at the lateral surface of EGFP.Since the assembly process is driven by thermodynamics,under low protein concentration conditions,proteins self-assemble to form highly ordered monolayed nanosheets.With the increase of concentration,this orderly assembly behavior is still maintained,and finally perfect band-shaped crystals formed.Not limited to this work,RhG₂M can act as a general tool for site-specific introduction of RhB elements onto the surface of any protein,which also provide the possibility for protein engineering assembly.2.Template-Free Construction of Artificial Light-Harvesting System based onFluorescent ProteinsIn the deep ocean,green light emission is observed in many coelenterates as a result of a nonradiative energy transfer from luciferase to green fluorescent protein?GFP?.This reminds us that the bio-optimized fluorescent protein may have an excellent antiself-quenching property and hold great promise for light-harvesting applications.Here,based on fluorescent proteins,we have developed a new method for constructing a 2D artificial light-harvesting system without relying on any templates.Previously,we have demonstrated that EGFP-4C variant can be assembled to form highly ordered 2D structures under the guidance of reasonable driving forces.Following this mindset,here we employed this variant as the energy acceptor of the light-harvesting system.At the same time,in order to form a complete light-harvesting circuit,another fluorescent protein?EBFP2?was introduced as the energy donor.We designed and synthesized bismaleimide-functionalized PEG molecules EG_n DM.Under the coordinated control of covalent interactions and secondary interactions,proteins self-assembly and extend in orthogonal directions to achieve monolayered and tessellated protein nanoarrays.Thanks to the excellent antiself-quenching property of fluorescent proteins,the coassembled system retains attractive light-harvesting performance even at high local concentrations.More importantly,the distance between adjacent chromophores is continuously adjustable.By making minor changes to the length of the inducing linker,we have achieved significant control over the size of the assembly.And a micron-sized light-harvesting system was finally obtained.Not limited to this work,the special core-shell structure of FPs may be expected to direct the optimization of fluorescent dyes by cladding.3.Construction of Artificial Light-Harvesting System Based on Protein CrystalsProtein crystal has 3D periodic structure,which fully realizes error-free and precise docking of proteins,and therefore is a model of protein assembly.From a functional perspective,its highly condensed periodic structure is clearly an ideal template for building advanced artificial light-harvesting systems.SP1 has a strong designability due to its special C₆ symmetrical structure and excellent thermal stability,which endows itself with a unique advantage in the preparation of functional materials.Herein,we redesigned SP1 crystal reasonably,and used it as a template to construct a novel artificial light-harvesting system.Under different conditions,SP1 variants can self-assemble into rhombic and rod-like crystals in a short time.This method can effectively accelerate the crystallization speed,reduce the crystallization difficulty,and stabilize the 3D structure of protein crystals.In addition,we found that the 2D surface of rhombic crystals can also guide the formation of superlattices of inorganic nanoparticles through electrostatic interactions.By attaching the positively-charged QDs with different light emission to the surface of the rhombic crystal through electrostatic interaction,a complete light-harvesting system was finally constructed.