Study On Self-Assembly And Functionalization Of SP1Protein

In nature, various protein-based self-assemblies exist in the living cells and play their uniquefunctions in maintaining cell shape, improving the metabolism, and keeping the chemical reactionactivity. Owing to the important biological characteristic, the study of protein self-assembly isalways the frontier research topic on protein engineering, molecular biology engineering, andsupramolecular chemistry. First, the designed protein nanostructures have been extensivelyexplored as an important alternative for natural protein nanostructures. Second, due to extendedapplication of many protein monomers, protein assembly provides an opportunity to design andfabricate novel biomedical materials and nanoscale devices. Third, some protein nanostructuresdisplayed obvious advantages in avidity, biodistribution and pharmacokinetics in comparison tosingle proteins in vivo. Further, with the development of analytical techniques, X-ray diffraction,atomic force microscopy, cryogenic electron microscopy, fluorescence spectrophotometer, and gelelectrophoresis, it is facile to study protein interactions in different3D protein nanostructures atthe molecule level. Protein function is tightly coupled to protein structure. The design of proteinself-assembly architectures is highly challenging owing to the chemical and structuralheterogeneity of protein surfaces. Understanding the structural information of protein surfaces andchoosing the appropriate driving forces for self-assembly will promote the engineering ofprotein-based supramolecular nanostructures. The well-defined morphology, recognition capabilityand specific reactivity towards target molecules of the protein make the nanostructures hold greatpotentials for practical applications such as tissue engineering, biomineralization, light harvesting,and drug delivery systems.Here we sought to utilize stable protein one (SP1) as scaffold to prepare high-orderednanostructures. SP1is a ring-like protein consisting of12subunits that are tightly bound to eachother via hydrophobic interaction forming a double-layered6-membered ring. In addition, SP1hasextremely high thermal and chemical stability. For example, its melting temperature is107°C andit exhibits a pronounced resistance to detergents, such as sodium dodecyl sulfate, and proteases.Taking advantage of its unique structure and distinctive chemical characteristics, we constructedvarious nanostructures with two sagnificiant functions: Glutathione Peroxidase mimeticnanoenzyme and light-harvesting system.1. Construction of Highly Stable Artificial Glutathione Peroxidase on SP1NanoringEnzyme as the protein with unique function is always used in constructing nanomaterial. Especially the enzymes which associate with human health and diseases have received muchattention. Among them, an antioxidant selenoenzyme, glutathione peroxidase (GPx), has becomeof great interest subject due to its ability to catalyse the reduction of hydroperoxides (ROOH) bytripeptide glutathione (GSH), and maintains the metabolic balance of reactive oxygen species(ROS) in vivo, thus protecting the biomembranes and other cellular components against oxidativedamage. However, its applications are usually limited due to the intrinsic disadvantages such asinstability, short half-life, and being hard to prepare. So much effort has been invested in trying toconstruct GPx artificial enzymes, such as typical host systems include cyclodextrins, catalyticantibodies and natural enzymes. Recently, as a new development in this regard, the combination ofcomputer, biological, supramolecular and nanoscientific strategies, various nanoenzyme modelswith controlled catalytic activity were well demonstrated. Up to now, some of these artificialselenoenzymes show satisfying enzymatic properties and play important roles against oxidativestress related diseases.However, although many GPxs and protein mimetic enzymes have high catalytic activity, mostof them cannot keep their structure and activity after exposure to extremely high temperatures.SP1is a boiling-stable oligomeric protein. The unique characteristic of SP1offers us a scaffold todesign artificial enzymes against extreme temperature. Here, we reported the successfulconstruction of an efficient antioxidase on the ring-shaped SP1homododecamer. By the methodsof computational design and genetic engineering, the active center of glutathione peroxidase (GPx),selenocysteine (Sec), was introduced to the SP1monomer surface, and the self-assembly propertyof monomer protein led to the ring-shaped SP1with homododecamer catalytic selenium centers(Se-SP1-57Cys). Mass spectrometry (MS), circular dichroism (CD), and transmission electronmicroscope (TEM) analysis confirmed that Sec was successfully incorporated without affectingthe global structure of SP1nanoring. The artificial selenoenzyme exhibited high GPx catalyticactivity and showed a typical ping-pong kinetic mechanism. Moreover, thetemperature-dependence of enzyme activity experiment demonstrated that it had a significantlybroader temperature range and showed high thermostability. Owing to multi-GPx active centers ona SP1oligomer, this selenium-containing nanoenzyme exerted excellent capability to protect cellfrom oxidative damage at the mitochondrial level. This strategy represents a new way to developthermostable artificial nanoenzymes for some specific applications.2. Small Molecule-induced Thermostability Protein Formation of Functional NanotubeWe displayed a novel method for generating protein supramolacular nanotube with naturedboiling SP1: First, small molecule ethylenediamine induced SP1into nanotube framework byelectrostatic interaction. But the nanostructure by the electrostatic self-assembly is instability. Forexample, high ionic strength and different pH will affect the electrostatic force. So in order tostable the nanotube we covalently linked the SP1nanoring into nanotube by the method ofZero-length crosslinking. The catalytic reagents of it are1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and N-Hydroxysulfosuccinimide esters (Sulfo-NHS). Subsequently, we used Se-SP1-57Cys as unit to construct the nanotube by the method of Zero-length crosslinking. Thenanotube with multi-GPx catalytic centers was formed, and the activity was much higher thannative GPx. Combine the methods of computational design, genetic engineering, and chemicaltechnology, the artificial nanoenzyme is the crystallization of multidisciplinary. It is a novel way toconstruct diversified nanomaterial.3. Quantum Dot-Induced Self-Assembly of Cricoid Protein for Light-HarvestingAccording to the analysis of the crystal structure of SP1protein, it can be employed as anappealing building block for electrostatic self-assembly due to its higher symmetric and negativelycharged hydrophilic structure at neutral pH. In particular, most of the acidic amino acids aredistributed on the top and bottom surface of the dodecamer SP1. Herein, we developed a strategyto construct high-ordered protein nanostructures by electrostatic self-assembly of cricoid proteinnanorings and globular quantum dots (QDs). Using multi-electrostatic interactions between12merprotein nanoring SP1and oppositely charged CdTe QDs, highly ordered nanowires with sandwichstructure were achieved by hybridized self-assembly. QDs with different sizes (QD1:3-4nm, QD2:5-6nm, and QD3:~10nm.) would induce the self-assembly protein rings into various nanowires,subsequent bundles, and irregular networks in aqueous solution. Atomic force microscopy,transmission electron microscope, and dynamic light scattering characterizations confirmed thatthe size of QDs and the structural topology of nanoring play critical functions in the formation ofthe superstructures. Furthermore, an ordered arrangement of QDs provides an ideal scaffold fordesigning the light-harvesting antenna. Most importantly, when different sized QDs (e.g. QD1andQD3) self-assembled with SP1, an extremely efficient F rster resonance energy transfer (FRET)was observed on these protein nanowires. The self-assembled protein nanostructures weredemonstrated a promising scaffold for the development of artificial light-harvesting system.