Construction Of Supramolecular Biomaterials Based On Protein Fragment Reconstitution

In natural life,protein is the basic unit to construct architectures and realize biological functions.Through various weak interactions between different molecules,protein monomers are able to assemble into complex structures in an orderly manner.For the changes in the external environment,these naturally occurred assemblies can achieve dynamic transformation in structure,so as to regulate their biological functions to maintain the life activities of the organisms.In recent decades,the field of the protein self-assembly has attracted more and more attention.The study of protein self-assembling behaviors deepens the understanding of the assembling mechanism of natural proteins,at the same time,provides a new method for developing protein-based biomaterials.Different driving methods and design strategies have been used to build protein assemblies orderly,a series of remarkable research results have been obtained.Among these,supramolecular interactions as a kind of non-covalent interactions are often used as an efficient driving force to induce orderly binding of building blocks and eventually to obtain complex assemblies due to their special properties such as specific binding affinity,dynamic regulation and universal applicability.Common supramolecular interactions such as hydrogen bond interactions,electric charge effect,metal ligand coordination,?-?conjugation,hydrophilic and hydrophobic interactions,and host-guest interactions etc.have been used to control assembly formation by regulating monomer connections.Protein fragment reconstitution is a natural phenomenon based on supramolecular interactions.When some proteins are divided into two parts at a specific location,the fragments can reconstitute into native folded domains through specially recognition and the resulting reconstitutions have similar properties and functions with the original ones.The discovery of this phenomenon provides a robust tool for constructing the protein assembling system induced by supramolecular interactions and opens up a new avenue to develop functional biomimetic biomaterials.Our study is to use the properties of protein fragment reconstitution to achieve the construction and precise regulation of dynamic protein assemblies,moreover,to fabricate functional biomimetic materials by simulating tissues and proteins in living organisms.We designed and build a dual responsive protein fragment reconstitution system which would respond to both redox potential and temperature.GN and GC fragments served as building blocks and driving force combining with the other supramolecular interactions and covalent connections to realize the construction guidance,dynamic adjusting and functionalization towards protein self-assembling through the precise control of environment.1.Construct ultra-high molecular weight titin mimicking based on supramolecular self-assembly strategyNatural elastomeric proteins are placed under mechanical stress during a wide range of biological processes and act as molecular springs to provide the required mechanical properties for tissues.One common feature of these elastin proteins is that they are composed of individually folded protein domains in tandem.As the largest protein in cells,muscle protein titin consists of more than 34,000 amino acid residues and with an ultra-high molecular weight(MW)of more than 3.5 MDa,which is responsible for the passive elasticity of muscles.Titin is formed by hundreds of individually folded globular domains,so it can be regarded as a protein polymer with globular proteins as macromolecular monomers.It's still challenging to design titin mimicking protein polymers with ultra-high molecular weight like titin.The complementation nature of GL5CC,a mutant of small protein GB1,is when split into two fragments,G_N and G_C can specifically recognize and reconstitute into a complete structural domain through supramolecular interactions.Taking advantage of this special method,we developed a new supramolecular assembly strategy based on protein fragment reconstitution to construct supramolecular assemblies with ultra-high MW.We found that the well-designed bifunctional protein macromolecule assembled efficiently and orderly through the head-to-tail connection resulted in linear assemblies with ultra-high MW,and an average MW of 0.5 MDa was obtained.This kind of high MW linear assemblies is very closed to titin and provides building blocks for constructing biological materials with improved physical and mechanical properties.2.Dynamic multidimensional protein self-assembly driven by host-guest chemistry and mutually exclusive proteinIn living cells,protein assemblies are the most important working machines,maintaining a wide variety of life activities.Natural protein assemblies are dynamic and respond to changes in the environment to perform specific biological functions.Inspired by nature,scientists have paid a lot of efforts to explore the artificial protein nanostructures and made important progress.Protein assemblies with different morphologies,such as nanowires,nanotubes,nanosheet and nanocages,from one-dimensional to three-dimensional assembly structures have been successfully constructed through rational design and precise control of protein interactions.Meanwhile,protein assemblies that respond to different conditions such as pH,redox potential,temperature,light and ions concentrations are also being developed.However,due to the limitation and uncontrollability of the primitive structure of natural proteins,it is difficult to construct stimuli-responsive protein assemblies with reversible morphologies.After reasonable design and careful consideration,we solved this problem by introducing a mutually exclusive protein(MEP)based on protein fragment reconstitution and thermodynamic stability.Using genetic engineering,we obtained a novel stimuli-responsive fusion protein containing the MEP domain,which can form dynamic protein assemblies driven by the supramolecular interactions of CB[8].By adjusting the assembly conditions to the oxidation or reduction state,the conformation of the building blocks changed significantly,thus resulting in the dynamic transformation of the assembly morphology from one-dimensional nanowires to two-dimensional nanorings.This assembly system has the potential application as a biomaterial because the morphology can be precisely controlled.3.Construction of functional protein hydrogels with dynamic ligand reversible decoration based on protein fragment reconstitutionThe extracellular matrix(ECM)is an important component of tissues in most mammals and is responsible for many fine biological processes on the cellular level.By taking charge of intercellular communications,ECM can manipulate a variety of cell behaviors such as adhesion,migration,differentiation,apoptosis and so on.Hydrogels constructed from synthetic polymers or recombinant proteins are often used as materials to simulate ECM for different applications in biomedical fields due to their high water content and adjustable physical,chemical and mechanical properties.Because cell behaviors are often dynamic,which hydrogels act as a controlled manner to bind and release functional signaling molecules reversibly is critical for their biological applications.To achieve this goal,a"blank plate"of hydrogels formed by bioinert polymers has been constructed and can be decorated by desired signal molecules by various biological coupling methods.This method has been widely used in the synthesis of polymer hydrogels and achieved great success.However,its application in protein-based hydrogels is rather limited.To solve this problem,we first time developed the strategy by using the reversible bioconjugation from protein fragment reconstitution to reversibly presenting and releasing ligand molecules on and from protein hydrogels.Due to the dual responsive characteristics of redox potential and temperature from applied reconstituted protein fragments,the modification behaviors of ligand to protein hydrogels can be controlled by changing the environment.So that the biochemical properties of hydrogels are adjusted to adapt to different applications.This method is also suitable for different proteins even the synthesis polymer hydrogels.There is a wide range of application prospects in the field of biomimetic material fabrication because this ECM simulation system facilitates the investigation of cell behaviors.