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- All Subjects: Self-assembly
- Creators: Stephanopoulos, Nicholas
Description
Exoelectrogenic organisms transfer electrons from their quinone pool to extracellular acceptors over m-scale distances through appendages known as “biological nanowires”. These structures have been described as cytochrome-rich membrane extensions or pili. However, the components and mechanisms of this long-range electron transfer remain largely unknown. This dissertation describes supramolecular assembly of a tetraheme cytochrome into well-defined models of microbial nanowires and uses those structures to explore the mechanisms of ultra-long-range electron transfer. Chiral-induced-spin-selectivity through the cytochrome is also demonstrated. Nanowire extensions in Shewanella oneidensis have been hypothesized to transfer electrons via electron tunneling through proteinaceous structures that reinforce π-π stacking or through electron hopping via redox cofactors found along their lengths. To provide a model to evaluate the possibility of electron hopping along micron-scale distances, the first part of this dissertation describes the construction of a two-component, supramolecular nanostructure comprised of a small tetraheme cytochrome (STC) from Shewanella oneidensis fused to a peptide domain that self-assembles with a β-fibrillizing peptide. Structural and electrical characterization shows that the self-assembled protein fibers have dimensions relevant to understanding ultralong-range electron transfer and conduct electrons along their length via a cytochrome-mediated mechanism of electron transfer. The second part of this dissertations shows that a model three-component fiber construct based on charge complementary peptides and the redox protein can also be assembled. Structural and electrical characterization of the three-component structure also demonstrates desirable dimensions and electron conductivity along the length via a cytochrome-mediated mechanism.
In vivo, it has been hypothesized that cytochromes in the outer surface conduit are spin-selective. However, cytochromes in the periplasm of Shewanella oneidensis have not been shown to be spin selective, and the physiological impact of the chiral-induced-spin-selectivity (CISS) effect on microbial electron transport remains unclear. In the third part of this dissertation, investigations via spin polarization and a spin-dependent conduction study show that STC is spin selective, suggesting that spin selectivity may be an important factor in the electron transport efficiency of exoelectrogens.
In conclusion, this dissertation enables a better understanding of long-range electron transfer in bacterial nanowires and bioelectronic circuitry and offers suggestions for how to construct enhanced biosensors.
ContributorsNWACHUKWU, JUSTUS NMADUKA (Author) / Jones, Anne K. (Thesis advisor) / Mills, Jeremy (Committee member) / Stephanopoulos, Nicholas (Committee member) / Arizona State University (Publisher)
Created2023
Description
Since the conception of DNA nanotechnology, the field has evolved towards the development of complex, dynamic 3D structures. The predictability of Watson-Crick base pairing makes DNA an unparalleled building block, and enables exceptional programmability in nanostructure shape and size. The work presented in this dissertation focuses on expanding two facets of the field: (1) introducing functionality through the incorporation of peptides to create DNA-peptide hybrid materials, and (2) the development of self-assembling DNA crystal lattices for scaffolding biomolecules. DNA nanostructures have long been proposed as drug delivery vehicles; however, they are not biocompatible because of their low stability in low salt environments and entrapment within the endosome. To address these issues, a functionalized peptide coating was designed to act as a counterion to a six-helix bundle, while simultaneously displaying numerous copies of an endosomal escape peptide to enable cytosolic delivery. This functionalized peptide coating creates a DNA-peptide hybrid material, but does not allow specific positioning or orientation of the peptides. The ability to control those aspects required the synthesis of DNA-peptide or DNA-peptide-DNA conjugates that can be incorporated into the nanostructure. The approach was utilized to produce a synbody where three peptides that bind transferrin with micromolar affinity, which were presented for multivalent binding to optimize affinity. Additionally, two DNA handle was attached to an enzymatically cleavable peptide to link two unique nanostructures. The second DNA handle was also used to constrain the peptide in a cyclic fashion to mimic the cell-adhesive conformations of RGD and PHSRN in fibronectin.
The original goal of DNA nanotechnology was to use a crystalline lattice made of DNA to host proteins for their structural determination using X-ray crystallography. The work presented here takes significant steps towards achieving this goal, including elucidating design rules to control cavity size within the scaffold for accommodating guest molecules of unique sizes, approaches to improve the atomic detail of the scaffold, and strategies to modulate the symmetry of each unique lattice. Finally, this work surveys methodologies towards the incorporation of several guest molecules, with promising preliminary results that constitute a significant advancement towards the ultimate goal of the field.
ContributorsMacCulloch, Tara Lynn (Author) / Stephanopoulos, Nicholas (Thesis advisor) / Borges, Chad (Committee member) / Gould, Ian (Committee member) / Arizona State University (Publisher)
Created2021
Description
In recent years, researchers have employed DNA and protein nanotechnology to develop nanomaterials for applications in the fields of regenerative medicine, gene therapeutic, and materials science. In the current state of research, developing a biomimetic approach to fabricate an extracellular matrix (ECM)-like material has faced key challenges. The difficulty arises due to achieving spatiotemporal complexity that rivals the native ECM. Attempts to replicate the ECM using hydrogels have been limited in their ability to recapitulate its structural and functional properties. Moreover, the biological activities of the ECM, such as cell adhesion, proliferation, and differentiation, are mediated by ECM proteins and their interactions with cells, making it difficult to reproduce these activities in vitro.Thus, the work presented in my dissertation represents efforts to develop DNA and protein-based materials that mimic the biological properties of the ECM. The research involves the design, synthesis, and characterization of nanomaterials that exhibit unique physical, chemical, and mechanical properties. Two specific aspects of the biomimetic system have been to include (1) a modular protein building block to change the bioactivity of the system and (2) to temporally control the self-assembly of the protein nanofiber using different coiled coil mechanisms. The protein nanofibers were characterized using atomic force microscopy, transmission electron microscopy, and super-resolution DNA Point Accumulation for Imaging in Nanoscale Topology. The domains chosen are the fibronectin domains, Fn-III10, Fn-III9-10, and Fn-III12-14, with bioactivity such as cell adhesion and growth factor binding.
To extend this approach, these cys-nanofibers have been embedded in a hyaluronic acid scaffold to enable bioactivity and fibrous morphologies. Nanofiber integration within the HA gel has been shown to promote tunable mechanical properties and architectures, in addition to promoting a temporal display of the protein nanofibers. The hydrogels were characterized using scanning electron microscopy, mechanical compression testing, and fluorescence microscopy. The findings in this dissertation highlight the promise of biomimetic DNA and protein nanomaterials as a versatile approach for developing next-generation materials with unprecedented properties and functions. These findings continue to push the boundaries of what is possible in nanotechnology, leading to new discoveries that will have a significant impact on society.
ContributorsBernal-Chanchavac, Julio (Author) / Stephanopoulos, Nicholas (Thesis advisor) / Jones, Anne (Committee member) / Mills, Jeremy (Committee member) / Arizona State University (Publisher)
Created2023