Non-classical Crystallization Of Amyloid-like Proteins

Protein amyloid aggregation tends to lead to neurodegenerative disorders such as Parkinson's disease and Alzheimer's disease,on the other hand,protein amyloid aggregation provides excellent novel structures and templates for the design and fabrication of advanced materials.The amyloid protein crystallization is of fundamental importance for understanding and uncovering molecular mechanisms and developing related therapeutics.The thermodynamic difference between amyloid fibrillization and crystallization is hardly diffrentiated.Until now,the acquisition of amyloid crystals has been highly restricted to amyloid-related short peptides;amyloidogenic protein would prefer to form fibrils rather than crystals due to entropy restriction.Therefore,it is still a huge challenge to achieve the amyloid protein crystallization.For this aim,we developed a kinetic driven non-classical amyloid-like protein crystallization pathway through protein unfolding;this non-classical pathway includes two-step nucleation of amyloid-like protein nanocrystals and crystal growth through meso-scale assembly of protein nanocrystals,which can realize the tunable structures and functions of amyloid protein crystals.The research outline is as follows:(1)First,we studied the two-step nucleation process of amyloid-like protein nanocrystals.3(2-carboxyethyl)phosphine(TCEP)was utilized to reduce disulfide bonds in lysozyme,which led to protein unfolding.An amyloid-like transition was confirmed by formation of short-range ordered ?-sheets aggregates between unfolded proteins chains.The kinetic-controlled quasi-equilibrium assembly could impede the over-interactions between unfolded proteins,and also promote protein nanocrystals nucleation by ordered packing of short-range ?-sheets aggregates.The obtained protein nanocrystals have 'core-shell' structures,in which dense crystalline cores were incorporated in amorphous shell consisting of unfolded protein chains.(2)Second,we first discovered the(bio)macromolecular mesocrystals.The acquisition of mesocrystals has been highly restricted to inorganic crystallization with few mesocrystals of small molecular amino acids and organic dyes before our research.The amorphous shell of the nanocrystals as kinetic energy barrier can stabilize protein nanocrystals.However,when this energy barrier was overcome under certain conditions,protein meoscrystals were formed by crystallographic meso-scale assembly of amyloid-like protein nanocrystals,and further fused into large-scale flexible single crystals with hierarchical structures.Besides,crystalline model through short-range ordered ?-sheets packing was built,which were supported by structural characterizations including TEM,SAED,HR-XRD and solution SAXS.(3)Thirdly,we proposed a method to controll the structure and morphology of amyloid protein crystals through mesocrystallization pathway.The protein nanocrystal has a feature of outer flexibility and inner rigidity:the rigid crystalline core with hierarchical multilayered structures could resist the structural fluctuations of ?-sheets,and unfolded chains as flexible 'shell' could release the elastic distortions during further assembly,both of them can balance the ?-sheets structural twisting to reallize the crystalline assembly at high temperature.On the hand,protein nanocrystals,as high level ?-sheets orderd assemblies,have typical heterostructures with 'face' and 'edge',which induce the anisotropic assembly and growth of protein nanocrystals.For instance,the hydrophobic interactions result in 'face-to-face' packing of protein nanocrystals to form lancet-like singe-crystals,while the hydrogen bondings govern a fast'edge-to-edge' extending of protein nanocrystals to form micron-scale poly crystalline platelets,which further stack to form hydrogel film at air/liquid interface.Randomly packed platelet crystals were orderly arranged and fused by a slow drying process of the hydrogel film at ambient condition,through an Evaporation-Induced self-assembly(EISE)process.(4)Forthly,large number of amino acid resides that were exposed on protein nanocrystals surfaces could interact with metal ions,nanoparticles and colloid paricles,and meso-sacle assembly of protein nanocrystals can offer complex hierarchical structures,both of the two points support a platform to design and prepare hybrid materials with complex hierarchical structures.The co-assemby of Au ion-complexed colloid and protein nanocrystals was then achieved to prepared protein-Au soft cryastalline hybrid materials;the interfacial interaction between protein and Au nanoparticles was used to mediate Au nanoparticle assembly to form branching superstructures;the protein nanocrystals also can be used as a template to direct micro/meso porous Co3O4 single-crystalline nanosheets.