Design And Construction Of Two-Dimensional Self-Assembled Protein Bionanomaterials

In nature,proteins form the most versatile structures in nature,both in terms of structural definition at the nanoscale and their functionality.Tertiary structures of proteins underlie their versatile functionality,such as catalysis,molecular recognition,assembly of cellular scaffolds and many others.Considerable numbers of natural proteins have evolved to form supramolecular structures based on the oligomerization domains,which plays pivotal role in life activities due to the complex and hierarchically ordered nanostructures.Following the wisdom of nature,people aim to construct specific configurational nanostructures based on bioactive proteins,which opens up tremendous possibilities in the precise control of materials and functionalization.Compared with other man-made nanostructures,protein assemblies can form much more detailed as well as asymmetric nanoscale structures and are of significant influence for many technological applications.Over the past decades,protein assembly area has undergone unprecedented and rapid development,where a variety of assembly strategies have been established to construct nanostructures from quasi-zero to three dimensional architectures including protein cages,protein nanotubes,nanorings,nanosheets and crystal nanostructures.These highly ordered protein nanoarrays provide potential applications in biosensors,drug and gene delievery,tissue engineering,vaccine design,biocatalysis and bionanomaterials and also have made great development in functionalization.Notably,two-dimensional?2D?bionanomaterials have attracted extensive attention due to their special structures.S-layer in bacteria,thylakoid membrane in chloroplast are both representative 2D structures in nature and this exquisite planar structure plays an important role in the realization of cellular functions.Based on this,developing novel strategies to construct highly ordered 2D protein nanoarrays and realize their applications of great significance in the unveiling of the natural mystery.This thesis aims at developing appropriate assembly strategies to explore the construction of 2Dprotein sheet nanostructures,precise regulation and further functionalization.Specifically,we utilized cricoid SP1 with highly thermal stability as building block model.Through the combination of rational design and covalent and non-covalent interactions,we have obtained highly orderd single-layered 2D nanosheets and successfully achieve the precise regulation of the size of the assemblies,dynamic control of assembly and disassembly process and artificial light-harvesting system to mimic natural chloroplast.1.Enzyme-Triggered Covalent Protein Assembly to Construct Regular NanoarraysThe concept of covalent self-assembly has received obvious attention in material science andnanotechnology.Here we mainly play emphasis on how to develop covalent assembly in manipulating directional protein assembly and further constructing regular nanoarrays.Protein SP1 possesses strong designability due to its special C₆ symmetry and the innate highly thermal stability endows itself with unique advantage in the field of protein assembly.In view of the fragile microenvironment of protein system,here we established enzyme-triggered covalent self-assembly to construct highly ordered architectures.With the aid of computer simulation and genetic engineering,we introduced Tyr residues to the top and bottom surfaces and lateral surfaces respectively for inducing the directional assembly.Under the single enzyme HRP catalysis,Tyr residues which were symmetrically introduced at the vertical and horizontal sides were enzymatically coupled and finally generated 1D nanotubes and 2D nanosheets.Through controlling charge distribution of SP1 by altering pH,we can regulate the single layer nanosheets into multilayer structures.The excellent stability endowed the self-assembled protein architectures with promising applications.2.Photocontrolled Protein Assembly for Constructing Programmed Two-DimensionalNanomaterialsPrecise self-assembly of proteins with structural heterogeneity,flexibility,and complexity into programmed arrays to mimic the exquisite architectures created by Nature is a great challenge for the development of protein-based functional nanomaterials.Herein,we present a strategy that integrates light stimuli and covalent coupling to prepare size-tunable two-dimensional?2D?protein nanostructures by remote photocontrol.Using Ru?bpy?₃²⁺as a photosensitizer,stable protein one?SP1?was redesigned and self-assembled into nanosheets in the presence of ammonium persulfate?APS?through a rapid and efficient oxidative protein crosslinking reaction.In the design,only a serine-to-tyrosine mutation at position 98 was introduced into SP1 for specific covalent coupling under white light illumination.The chemical and topographical specificities of the photosensitized crosslinking reaction allow control of the direction of protein assembly to form extended 2D nanosheets,which are packed in an orderly manner along the lateral surface of ringshaped SP1S98Y.Notably,the growth of SP1 nanosheets exhibited isotropical characteristics and can be dynamically mediated by illumination time to achieve precise control of the size of the assembled architectures.The subsequent heat treatment further revealed the excellent thermostability of the2D periodic SP1 nanostructures,which may find promising applications in the fabrication of various nanobiomaterials after functionalization.The present work demonstrates that the visible light-triggered crosslinking strategy is a facile and environmentally friendly method for constructing advanced protein architectures through hierarchical self-assembly.3.Regulation of Protein Assembly-Disassembly via Host-Guest InteractionsProtein self-assembly and its reverse process?disassembly?are ubiquitous in nature,which plays pivotal role in many biological activities including cell locomotion,transcription,immune response,and apoptosis.Inspired by the wisdom of nature,the development of regulated assembly/disassembly biosystem is of great significance in the field of diagnosis and therapy.In this part we utilize host-guest interactions to induce protein assembly for the construction of highly ordered two-dimensional sheet nanostructures.As is known that guest molecule MMV⁺can form complex with CB[8]at the ratio of 2:1,we thus design to use this host-guest complexation to trigger protein into ordered growth.Genetic engineering was introduced to obtain SP1S98C with active Cys at the lateral surface,which can be reacted with maleimido viologen?MMV⁺?to form modified SP1S98C-MMV⁺.With the addition of CB[8],host-guest complexation was occurred to guide the isotropic assembly,which finally constructed orderly arranged nanosheets.Furthermore,upon adding excess tripeptide FGG to the assembly system,the assemblies gradually disassembled by the competition of the host-guest complexes,which was due to the higher binding ability of FGG to CB[8]compared with that of MMV⁺.Therefore,we successfully achieve oriented protein assembly and dynamic regulation of assembly/disassembly process with the combination of host-guest interactions and competitive-binding mechanism,which gives potential applications in the field of biological medicine.4.Protein-Baed Artificial Light-Harvesting System to Mimic Natural ChloroplastHighly ordered protein nanoarrays hold great potential in designing and constructing functional bionanomaterials.Herein we combined covalent and noncovalent interactions to prepare a protein-based light-harvesting system to mimic chloroplasts.First utilizing SP1S98Y mutant as building block model,we successfully constructed highly ordered 2D protein nanosheets according to dual enzyme-cooperative?HRP/GOx?nanotechnology.From the charge distribution of SP1,we found that the surface of the protein nanosheets was negatively charged under physiological conditions.We further integrated quantum dots?QDs?possessing optical and electronic properties with the 2D nanosheets.By attaching different sized QDs as donor and acceptor chromophores to the negatively charged surface of SP1-based protein nanosheets via electrostatic interactions,we successfully developed an artificial light-harvesting system,which can achieve pronounced FRET between donors and acceptors.The assembled protein nanosheets structurally resembled the natural thylakoids,and the QDs can achieve pronounced FRET phenomenon just like the chlorophylls.Therefore,the coassembled system was meaningful to explore the photosynthetic process in vitro,as it was designed to mimic the natural chloroplast.