Construction Of Efficient Artificial Light-harvesting System Based On Protein Nanomaterials

Protein and its assemblies are indispensable to the formation of life,metabolism and reproduction on earth.Natural proteins maintain their three-dimensional structures via intramolecular interactions,and spontaneously assemble into larger,long-range ordered protein nanostructures via multiple covalent or non-covalent interactions,and then participate in various life activities such as biological matter transport,biochemical reaction catalysis and molecular recognition.Most of the energy-transfer process and transfer mechanism in the natural light-harvesting system,which is considered as the "energy converter" in nature,also depends on the interactions between the pigment molecules and proteins or their assembly structures in the environment.In order to better utilize and fully transform the energy from the sun,and to deeply understand the working principle of natural light-harvesting systems,people have been striving to construct efficient artificial light-harvesting systems to explore in a more natural way.Since the pigment molecules in the natural light-harvesting system are usually arranged in an orderly and compact way to achieve efficient light-capture and transfer efficiency,it is one of the effective ways to construct a simulated natural photosynthetic system to distribute the chromophores in a similar form with an appropriate template framework.In the framework of many artificial light-harvesting systems,protein self-assembled nanomaterials show their unique advantages.Firstly,proteins are translated by genetic code,which makes them highly code-able and designable.Using genetic engineering techniques,functional amino acids can be introduced at any desired site to activate functional sites and construct protein nanomaterials.Secondly,protein assembly has a clear and definite structure at the nanoscale,and its building-blocks are precisely arranged,which is one of the most superior factors for good localized fixation and reasonable dispersion of fluorescent chromophores.In addition,the interactions between the proteins and their components can give the protein nanomaterials the characteristics of stimulus response,thus the construction of dynamically adjustable template skeleton is expected to regulate the light capture process.Finally,and crucially,the interaction between protein and pigment molecules is the most essential and primitive interaction in the natural light-harvesting system.Using the scaffold of protein materials arranging chromophores to construct artificial simulated photosynthetic system is an ideal simplified model that is most suitable for nature-bionic.It may,in part,help us to better understand the complex internal processes of natural light-harvesting compounds.Therefore,based on protein nanomaterials,we are expected to realize a series of exploration and simulation of natural light capture system from the aspects of structure,energy transfer process and material transformation.This thesis aims to construct an artificial light-harvesting system based on natural or artificial protein nanomaterials,and realize the catalytic conversion of substances.Specifically,we constructed one-dimensional or two-dimensional protein nano-templates based on the assembly protein nanomaterials formed by natural envelope protein(Csg A)and the chemically stable ring-shaped SP1 protein through rational design,protein fusion and site-directed mutation.The chromophore is anchored in a confined space in order to realize the sequential energy transfer process via covalent or non-covalent interactions,and the collected energy is used for photocatalytic reactions.In addition,the "On" and "Off" switch of the energy transfer of our artificial light-harvesting system was studied based on the stimulus responsiveness of disulfide bonds to the redox environment,and this characteristic was applied to control the catalytic performance of the system.1.Construction of one-dimensional artificial light-harvesting system based on natural biofilm protein self-assemblyAmyloid fibrin of E.coli is a kind of coiled protein fibers secreted and assembled spontaneously by cells.Csg A,one of its subunits,can also achieve exocytosis and self-assembly through simplification and modification.Csg A protein has been proved to be easy to prepare without purification,and its assembly process is spontaneous without any special design or environmental regulation.The assemblies of biofilm proteins possess high stability and orderly arrangement,which makes them ideal one-dimensional protein nanomaterials for constructing artificial light-harvesting systems.Herein,we fused the 13-peptide tag Spy Tag to the terminal of Csg A protein,so that the fusion-protein was expressed,secreted and self-assembled,thus obtaining biofilm nanofibers with uniform array of peptide tags.By fusing the blue or green fluorescent proteins with Spy Catcher proteins which can bind specifically to Spy Tag,the two fluorescent chromophores can be anchored to the constructed one-dimensional protein nanoscffold driven by the combination of the specific recognition pairs,which was characterized by fluorescence microscope and images under UV irradiation.Eosin Y(EY)was added in the system as a dye molecule chromophore.And the composite by means of biological adsorption on the surface of the bacteria,with the confined in fluorescent protein chromophore adjacent to the position.Due to the proper overlap of the spectral band of chromophores,an energy transfer process could be achieved from the blue fluorescent protein to green fluorescent protein,and finally in turn to EY chromophore on the protein one-dimensional scaffold.And the efficiency of the two-step energy transfer reached 62% and 55%,respectively Furthermore,the one-dimensional protein nanomaterial can also be used as a universal template for the construction of bioluminescent materials.2.Construction of a sequential multi-step energy-transfer artificial light-harvesting system based on two-dimensional protein nanosheetsIn natural chloroplasts,accurate and ordered arrangement of pigment molecules on the lamellar thylakoid membrane ensure the efficient harvest and transformation of light energy as the structural prerequisite.Compared with the linear structures,the lamellar two-dimensional structures not only exhibit the advantage of being structurally similar to the natural thylakoid,but also provide more complete transfer paths for energy transfer,avoiding the interruption of energy transfer caused by local defects of the system.Inspired by nature,we chose the SP1 protein with special pore structure as the building-blocks,and constructed the two-dimensional lamellar protein template.SP1 is of C6 symmetric structure and highly thermal stability,which makes it an ideal material for the construction of the 2D protein nanosheets.The large amount of negative charge and nanoscaled-hollow pores on the surface of SP1 provide great possibilities for binding chromophore with matching size and different charge on the surface.Cysteine was designed on its lateral surface so that it could be crosslinked into a two-dimensional layer-liked structure under oxidation conditions.The proteins in the assembly are arranged in order and provide a limited periodic binding site for the fluorescent chromophore.In addition,considering the overlap of spectra of the chromophores and the feasibility of co-assembly,we used two kinds of carbon dots with appropriate size and positive charge on the surface as the donor and the first acceptor,respectively.And then molecule EY was introduced in the system as the second acceptor chromophore,which was covalently modified on SP1 nanorings,to construct an efficient artificial light-harvesting system meeting the requirements of sequential energy transfer.The two-dimensional protein nano-scaffold skillfully arranges chromophores on its surface to avoid undesirable fluorescence quenching.And the efficiency of the two-step energy transfer reached 84% and 76%,respectively.This part of work provides a classical universal structure model for biomimetic and a new idea for understanding the energy transfer process of natural photosynthetic system.3.Redox stimulus-response reversible regulation of artificial light capture system and its applicationNatural photosynthetic systems capture and transfer light energy to other pigment-protein complexes through photosynthetic antennas.Energy is finally transported to reactive centers for utilization and transformation of substances.Actually,the conversion of energy and matter can be considered as our fundamental purpose of the light-harvesting process.In our sequential multi-step artificial light-harvesting system the light can be absorbed and transferred by the carbon dots,and finally reach the second acceptor,EY.As a dye molecule,EY plays excellent photocatalytic performance in organic synthesis,which makes it not only act as the final acceptor of the energy transfer in the artificial light-harvesting system,but also is expected to be a reactive center to realize the catalysis in material conversion under light conditions.In addition,because the disulfide bond,which drives the formation of the protein nanosheets,is a dynamic covalent bond of redox stimulus-response,the light-harvesting system is expected to achieve dynamic artificial regulation.In this chapter,we have successfully constructed a two-dimensional protein template for artificial light-harvesting system to participate in a model reaction of coupled cross-linking hydrogen evolution through photocatalysis.The catalytic yield of the system is as high as 71%,and its performance is much higher than that of the specific catalyst for the reaction--free EY molecule(17% yield)because it broadens the available spectrum.Moreover,the redox stimulus-response of the two-dimensional protein nanomaterials can regulate the fluorescence characteristics of the system and thus control the catalytic performance.Therefore,the catalytic performance of the artificial light-harvesting system can be artificially regulated by changing the external conditions.The realization of this system also provides application potential for the construction of biosensing and intelligent materials.