Nano-ölcek metal yüzeylerde moleküler tanıma esaslı kendiliğinden montaj olabilen protein tasarımı

In recent years, a wealth of novel, nature-inspired materials have been explored across a wide range application in medicine and technology. Hierarchical organization is one of the key components that are reflected in the diverse structure and function in biological materials. In mimicking these functions in our engineering approaches have been challenging due to the complexity of these organization. Biological self-assembly principle based upon precise molecularscale recognition is the key in Nature`s design. Biological machinery facilitates self-organization and self-regulation that we observe in biological materials. Proteins are perceived as crucial interaction components in this machinery owing to their versatile characteristics. The protein assemblies may be the key in making next generation materials mimicking Nature`s design. Peptides as smaller domains of proteins become crucial components in constructing new types of materials having the versatility that we observe in Nature. Inorganic material specific peptides have attracted increasing interest in the recent years as novel bio-surface functionalization agents. Such functionalization can recruit biological cues to the material's surface and promote the otherwise challenging coupling of distinct materials. Their modularity also offers a unique opportunity to link them to numerous other functional proteins. Herein, we provide the details for their design and recombinant incorporation into functional proteins and enzymes to achieve addressable self-organization. In the scope of this dissertation, we first constructed an AuBP1 peptide genetically fused to the C-terminus of maltose-binding protein (MBP) using two different linkers to produce MBP-AuBP1 hetero-functional constructs in order to accomplish the biological self-assembly on gold surfaces. Using various spectroscopic techniques, surface plasmon resonance (SPR), and localized surface plasmon resonance (LSPR), we verified the binding of MBP-AuBP1 fusion protein on gold substrates. The AuBP1 peptide tag were demonstrated to direct the organization of recombinant MBP protein onto various gold surfaces via efficient control of the organic–inorganic interface at the molecular level. Furthermore, using a combination of soft-lithography and self-assembly techniques, AuBP1 peptide tag controlled the formation of protein assemblies onto gold nanoparticle arrays with high molecular packing density and patterning efficiency following a series of simple, reproducible steps. This model system offers layer-by-layer assembly capability upon specific AuBP1. In our second design, we developed multifunctional monitoring molecule through genetic fusion of gold binding peptide (AuBP2) to a red fluorescence protein, DsRed-Monomer. AuBP2 was utilized as a material specific linker to construct novel bio-imaging, drug delivery, and targeting bio-nano hybrid systems. The AuBP2c peptide tag was enabled the self-immobilization of the DsRed protein onto a variety of nanoscale gold surfaces. Functional DsRed monomer generated by protein engineering methods using AuBP2 allowed variety of properties for biological sensing as well as analytical applications. The red emission of DsRed protein offered new opportunities at the nano- to micro-scale of a material surface such as multicolor labeling and fluorescence resonance energy transfer (FRET) applications. Our designed fusion protein with a fluorescent tag provided quick and accurate visualization of the self-immobilized proteins on the gold surface. Additionally, DsRed constructs have a selective and reversible binding affinity to copper. This unique binding property of DsRed protein to copper ions and gold nanoparticles results in tunable quenching of its fluorescence activity. Given the importance of gold nanoparticles and copper ions in several application areas, DsRed-AuBP2 was proposed as a bio-sensing modality through its bi-functional properties, fluorescence and self-assembly. We also tested the efficiency of the DsRed-AuBP2 protein as an integrated component in nanofiber system. The fibers were formed in the presence of the protein provided the integration of gold nanoparticles along the fiber length through Au-binding peptide tag. These nanofibers that feature gold nanoparticles with proteins did not affect the red fluorescence property of the protein, rather protein allowed the visualization of the protein construct along the fibers. Engineering proteins that can decorate gold nanoparticles well integrated into PEO polymer fibers offers several opportunities to create versatile structural templates for metallization in formation of conducting nanofibers. Our final set of design included a fusion enzyme that is designed to have gold binding activity. Formate dehydrogenase, FDH, was fused to an AuBP2 fusion tag to construct FDH-AuBP2. This fusion enzyme was utilized to develop a circuit-based electrode system that provided verification of the redox catalytic ability of the self-immobilized enzyme on gold electrode by subsequent addition of formate. We achieved this validation following the self-immobilization of FDH-AuBP2 onto a gold electrode surface and monitored the conversion of formate to CO2 electrochemically. The circuit-based system we constructed consisted of two gold electrodes submerged in a buffer solution. Prolonged catalytic activity of the enzyme was observed by subsequent addition of format into solution. The engineered multi-functional biomolecular constructs developed were demonstrated to self-organize, forming functional hybrid hierarchical entities. However, no all-encompassing solution exists to control the orientation of the enzymes on surfaces using inorganic binding peptide tags. Each peptide and enzyme system requires detailed evaluation following genetic conjugation to investigate the performance of the chimeric functions. Our results demonstrate the engineered inorganic binding peptides as a promising platform technology by their role in producing bi-hybrid nanostructured systems including fluorescent biosensors for metals, bioelectrodes for biobatteries, and nanofibers for conductive nanomaterial. Based upon an in-depth investigation of the limited research currently published in this growing field, we have developed a strategy for the design of new protein constructs as well as their over-expression as recombinant products. Moreover, we provide a valuable approach to generate bioactive nanostructures for the engineering protein complexes by co-expression, purification, and characterization, coupled with bionanotechnological applications. Our engineering approach to conjugate proteins with inorganic binding peptides to create nanodevices propels this technology forward, one step closer to becoming a reality. Our fabrication process is based upon a bio-enabled self-assembly technique and is, herein, proven capable for engineering multilayered protein and nanometallic assemblies that utilize modular goldbinding peptides, such as AuBP1 and AuBP2. Overall, the reported findings in this dissertation demonstrate a great potential to design novel nanostructured materials that incorporate the engineered proteins that are designed in the scope of this thesis. Our multi-functional engineered proteins display high binding affinity and specificity to various gold surfaces at the nano- to micro scale under ambient conditions. These novel platforms can be used for a variety of purposes such as preparing protein micro/nano-arrays, biosensors, biobatteries, and reagents for molecular imaging and targeting. Our established model constitutes biological routes for biofabrication of various protein arrays, plasmon-active nanometallic assemblies, and devices that feature controlled organization, packing density, and architecture.