Construction Of High-dimensional Protein Self-assembly Based On Supramolecular Driving Forces And Rational Spatial Design

Proteins, which bear the basis of life, are nature‘s evolving ?masterpiece‘. The ubiquity and significance of proteins can be affirmed in all domains of life ranging from viruses, bacteria to eukaryotes. Notably, instead of ?working alone‘, via non-covalent interactions, most natural proteins tend to function in the form of regularly organized protein self-assemblies. The highly synergistic coordination of the protein subunits endows the protein self-assemblies with various advantages over the basic single protein monomers. To take a broad view, the reasons why proteins evolve to self-assembly into quaternary structures can synoptically be ascribed to two aspects:(i) Overall self-assembly structures can provide higher stability and mechanical strengths for cellular protection and molecular movements while preserve the dynamic reversibility due to its supramolecular nature.(ii) Periodic patterns within the assemblies can provide collective behaviors to endow enhanced biological functions and unique physical-chemical properties. Given all the dominant advantages hinted by natural protein self-assembly, therefore, follow the wisdom of nature, manipulating protein building blocks to self-assemble into ordered nanostructures will allow access to novel biomaterials with versatile functionalities. In turn, deep investigation of the complex structural details and the kinetic/thermodynamic control of the self-assembly process will provide insights into natural protein functioning mechanism. Over the years, with the development of supramolecualr chemistry, computational simulation and structural biochemistry, surmounting the heterogeneity and complexity of protein building blocks, various strategies have been created to dictate protein self-assembly. However, facile and programmable construction of sophisticated, high-dimensional protein self-assemblies remains to be a great challenge.Here, we sought to establish a general strategy to construct high-dimensional protein self-assemblies based on the cooperation of suparmolecular driving forces and rational spatial design. Specifically, we chose the glutathione S-transferases(GST) and the second mitochondria-derived activator of caspases(SMAC) as modular building blocks. Via suparmoleuclar tools including metal coordination and host-guest interaction combining with designed ?V‘ shapes, protein superstructures including protein nanorings, protein superlines and protein nanohelixes were constructed.1. Construction of Highly Ordered GST Nanorings via Precise Control over Protein Self-assembly BehaviorThe elegant functionality of protein self-assemblies roots on the sophisticated yet highly-ordered nanostructures. Thus, in the researching field of fabricating protein self-assemblies, the priority for scientists is to guide protein self-associate into ordered architectures. Protein self-assemblies ranging from one-dimensional nanolines, two-dimensional nanorings/nanonetworks, to three dimensional nanocages/nanolattices have been constructed. However, among these efforts, most self-assembly systems cannot provide very precise control over protein self-assembly behavior. While several precise control approaches always need vast modification of protein surface thus need a lot of experimental procedures.Here we endeavored to establish a facile protocol to exert precise control over protein self-assembly behavior and to programmably construct highly-ordered protein nanorings. We utilized GST–a C2 symmetrical enzyme as basic building block and directional metal coordination as driving force. To provide the bending trend for nanoring formation, we designed two pair of metal chelating sites into a ?V‘ shape. Further protein-protein interaction study via computational calculation provided a specific interacting mode where non-specific interfacial interactions and incorporated metal coordination cooperated to exert precise control over protein self-assembly behaviors. Atomic force microscopy(AFM) analysis confirmed our designed mechanism and highly ordered protein nanorings were obtained. Notably, we also obtained ?growing‘ half-ring structures that adopted the same curvature and will finally grow into complete nanorings with identical diameter. This phenomenon further indicated that GST variants self-assembled in a highly programmed way with a bottom-up manner. Moreover, focusing on the interfacial non-specific protein-protein interactions, we can further regulate the diameters of the nanorings via manipulating ion strength of the solution. This work provides a de novo design strategy that may inspire the facile construction of more sophisticated protein superstructures.2. Construction of Host-guest Interaction induced Protein Self-assembly System with Morphological VersatilityTrace back to the basic concept of protein self-assembly we can draw the conclusion that two determintive factors of the final self-assembly structure are the spatial elements of the protein building blocks and utilized driving forces. The alternation of any factor will exert great influence on protein self-assembly results. Follow the first study, we changed the metal coordination into CB[8]-FGG based host-guest interactions. Firstly, to break the spatial constraint and incorporate FGG ligands into any desired location on protein surface, we synthesized a malemide functionalized FGG tag. Not limited to this study, this molecule can act as a general tool for site-specific introduction of FGG elements onto protein surfaces. Compared with the previous study, the incorporated FGG tags featuring ?V‘ shape can give the trend of nanoring formation. However, geometrical factor alone cannot guarantee the predictable nanoring formation. Plus, distinctive form rigid metal-coordination, induced FGG tag is relatively flexible, which increased the uncertainty of the self-assembly process. In addition, the relative large size of CB[8] molecule wedging between protein interfaces eliminated the non-specific protein-protein interactions that can cooperate to exert precise control over self-assembly behavior. In this system, to construct protein nanorings, we referred to the traditional ring-chain competition theory. At low protein concentration, proteins tend to polymerize in a head-to-tail way and form protein nanorings. However, with high protein concentration, most protein building blocks tend to polymerize into large-scale protein nanolines. AFM characterization confirmed both formation of protein nanorings and densely packed nanolines. Further variation of protein/CB[8] ratio can endow more versatility for the system: When self-assembly with excess CB[8] at high protein concentration, GST-FGG variants can self-associate into disc-like aggregates, and some of which even exhibited ?spiral‘ morphology. When treating the CB[8] overloaded mixture with dialysis, the aggregates can slowly transform into evenly dispersed nanolines, some of which can further self-assembly into highly stable ?superline‘ structures with astonishing length induced by electrostatic interactions and host-guest interaction. This work represents a regulatory approach for protein self-assembly morphologies and provides a possible direction for the construction of ?smart‘, dynamic self-assembly systems.3. Design and Construction of Protein Nanohelixes.Three-dimensional nanohelixe represent a very attractive structure. In particular, DNA molecules are knowen for their double-helix structure; Collagen and actin rely on their helix structures to exert their biological task; Alzheimer’s disease related amyloids also feature with helix stuctures. Moreover, fabricated protein nanohelix structures have yet to be constructed. Thus the design and construction of protein nanohelix not only will prompt the researching field to a new level but also may provide insights into natural protein helixes. However, the construction of protein helix is a really arduous task. First, from the top view, protein helix exhibited a ring-formation trend, while form side view it exhibited a successive increment along the axis. Herein, through structural filtration, we selected SMAC as building block. The innate ?V‘-shape and thickness of SMAC will fulfill the requirements of helix formation simultaneously. We further introduced two pair of metal chelating sites on each ?V‘ correlated ?arms‘ of SMAC to pin down the designed self-assembly. AFM characterizations suggested the formation of periodic SMAC filaments. Further mechanism analysis indicated that designed four metal-chelating sites were not fully utilized, which may due to spatial collision and thermodynamic factors. However, the obtained SMAC filaments exhibited further association trend, and some of which can self-twist into helix stuctures. Although we obtained protein helix structure from the hierachical assembly of SMAC filaments, however, our initial design still represents a valuable concept combining rational spatial design and incorporation of decent driving forces. This design concept will be fully realized with the assistance of computational calculation guided protein surface modification in the near future.