Browsing by Author "Snow, Christopher D., advisor"

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Open Access
Characterization and application of a novel composite nanomaterial comprised of porous protein crystals and synthetic DNA
(Colorado State University. Libraries, 2022) Stuart, Julius D., author; Snow, Christopher D., advisor; Kennan, Alan J., committee member; Shores, Matthew P., committee member; De Long, Susan K., committee member
Composite nanomaterials are systems comprised of multiple components boasting enhanced properties over those exhibited by the individual constituents when isolated. Such systems are highly tunable, allowing one to vary component types (e.g., polymer, metal, ceramic) for influencing performance in various contexts. Moreover, composite nanomaterials can be further modified using biofunctionalization for use in biological settings. Composite nanomaterials have been tested in applications including, but not limited to, textile, defense, food, energy and biomedical engineering. A sub-domain within composite nanomaterials involves porous protein crystals soaking, or, separately, encapsulating various guest molecules. Porous protein crystals are ordered, insoluble assemblies forming a network of nanopores capable of allowing inward diffusion of guest molecules. Moreover, recombinant protein variants can be engineered for probing guest molecule binding to host crystal nanopores further highlighting the tunability of this novel composite nanomaterial. The goal of this work is to evaluate a novel composite nanomaterial comprised of host porous protein crystals and guest double stranded DNA. We show that guest DNA loads into host crystals predominantly along the axial nanopores. Equilibrium adsorption isotherm results suggest guest DNA unbinds from host crystals relatively slowly. Computational modeling and Fluorescence Recovery After Photobleaching (FRAP) studies suggest intra-nanopore guest diffusion is attenuated relative to bulk diffusion. We also show that porous protein crystals loading with synthetic DNA barcodes can be used for tracking mosquitoes. Fluorescently labeled crystals can be ingested by mosquito larvae and adults, followed by detection using fluorescence confocal microscopy. Crystal-bound DNA can be liberated from host crystals by incubation with solution containing deoxynucleotide triphosphates (dNTPs). Previously ingested barcode-loaded crystals can be recovered using standard molecular biological techniques. Lastly, we show a DNA barcode sequence construction strategy that is modular, economical and scalable. Computational sequence design and scoring allowing identification of top candidates for experimental validation. Analysis of next-generation sequencing datasets informs barcode construction specificity while simultaneously reinforcing the multiplexing capabilities boasted by modular DNA barcodes.
Open Access
Characterizing porous protein crystal materials for applications in nanomedicine and nanobiotechnology
(Colorado State University. Libraries, 2018) Hartje, Luke Fredrick, author; Snow, Christopher D., advisor; Ho, P. Shing, committee member; Peersen, Olve B., committee member; McCullagh, Martin, committee member
Protein crystals are biologically derived, self-assembling, porous structures that have been used for decades in structure determination via X-ray diffraction. Recently, however, there has been increased interest in utilizing protein crystals for their unique material properties—most notably, their highly ordered porous structure, innate biocompatibility, and chemical plasticity. The diverse topologies of protein crystals and the relative ease with which their chemical properties can be altered via genetic mutation or chemical modification offers a wider and more dynamic design palette than existing chemically-synthesized nanoporous frameworks. These traits make protein crystals an attractive new material for applications in nanomedicine and nanobiotechnology. The intent of this project is to demonstrate the application potential of porous protein crystal materials for use in nanostructured devices. This work highlights our efforts to: experimentally and computationally investigate macromolecular transport and interaction energies within a large-pore protein crystal environment using time-lapse confocal microscopy, bulk equilibrium adsorption, and hindered diffusion simulation; assess the cytocompatibility of various cross-linking chemistries for the production of biostable protein crystal materials for use in biologically sensitive environments; and create multifunctional textiles by covalently attaching various cross-linked protein crystals to cellulose fibers in woven cotton fabrics. By pursuing this research, we hope to better understand porous protein crystal materials and leverage that knowledge to design advanced nanostructured devices for applications in medicine and biotechnology.
Open Access
Engineered co-crystals as scaffolds for structural biology
(Colorado State University. Libraries, 2022) Orun, Abigail R., author; Snow, Christopher D., advisor; Ackerson, Christopher, committee member; Kim, Seonah, committee member; Ho, P. Shing, committee member
Biomolecules, like protein and DNA, serve as the foundation of life. The structure of biomolecules can give insight to their functions. X-ray crystallography is a cornerstone of structural biology, revealing atomic-level details of macromolecular structures. Even with advances in X-ray diffraction technology, haphazard and tedious crystal preparation remains the bottleneck of routine structure determination. An alternative to the crystal growth challenge is a scaffold crystal. Hypothetically, if one had a high-quality crystal already prepared with large enough pores for diffusion of a macromolecule, a biomolecule of interest could join the scaffold crystal for scaffold- assisted X-ray diffraction. An ideal scaffold crystal must be highly porous for guest addition, modular for installation of various guest molecules, and robust in changing solution conditions. A crystal with guest anchoring sites for post-crystallization guest addition may provide a high-throughput technique for guest DNA-binding protein structure determination. The overarching goal of this work is to design a novel scaffold crystal capable of scaffold-assisted X-ray crystallography. The scaffold crystals we designed are co- crystals of DNA and DNA-binding protein. In the co-crystal, the DNA serves as the anchoring point for guest DNA-binding guest targets while the protein acts as connective tissue to hold the DNA structure together. The scaffold co-crystal we engineered, Co-Crystal 1 (CC1), is the first example of a porous host crystal for DNA-binding guests. Ultimately, the expanded co-crystals may serve as a revolutionary figurative "lens" for routine structure determination. In addition to scaffold crystal development, we advanced methods to enhance scaffold stability and solution-independence, thereby augmenting the bioconjugation toolkit for crystals containing stacking DNA-DNA junctions. Specifically, we optimized a known bioconjugation technique, carbodiimide chemical DNA ligation, templated by crystals with stacking DNA junctions. Furthermore, crystal crosslinking chemistries were optimized to provide crystal strength at both the nanoscale and the macroscale. Post- crosslinking, co-crystal nanostructures were preserved as assessed using X-ray diffraction and co-crystal macrostructures were bolstered in harsh solution conditions. The crosslinking chemistry and protocol guidelines may advance the progress of DNA crystals and protein-DNA co-crystals utility in biomedical applications and structural biology. We are on the cusp of using designed co-crystals to host guest DNA-binding proteins for structural biology, bio-sensing, and bio-therapeutic delivery. Successful engineering of a designed porous co-crystal will open numerous application possibilities and scientific questions. For example, a future study could focus on quantifying guest protein diffusion rates and adsorption strength inside the porous scaffold crystals. The technology presented here may advance the study of DNA-binding proteins and advance our understanding of key proteins for cancer and disease.
Embargo
Interdisciplinary techniques in protein binding prediction and crystal engineering
(Colorado State University. Libraries, 2024) DeRoo, Jacob Benjamin, author; Reynolds, Melissa, advisor; Snow, Christopher D., advisor; Reardon, Ken, committee member; Zabel, Mark, committee member
This dissertation explores the integration of interdisciplinary methods such as advanced robotic automation, machine learning, and hybrid materials synthesis to dual protein engineering challenges: predicting protein-peptide binding specificity and the preparation of crystalline protein materials. The first chapter introduces a computational pipeline, PAbFold, based on AlphaFold2, designed to predict linear antibody epitopes from a given antigen sequence. This method provides a rapid and cost-effective alternative to traditional experimental techniques for epitope mapping, significantly lowering the financial barrier for laboratories. By accurately identifying binding sites on target proteins, PAbFold enhances the understanding of antibody-antigen interactions, facilitating the development of diagnostic and therapeutic antibodies in a more accessible manner. The second chapter presents an innovative approach to protein crystallization scale-up utilizing the Opentrons 2 liquid handling robot. This automation not only reduces manual labor and variability in crystallization experiments but also makes high-throughput crystallization more accessible to a broader range of laboratories by decreasing costs. Traditional high throughput protein crystallization liquid handling robots are priced around $75,000; the Opentrons 2 costs around $15,000. By employing Python scripts for precise control of the Opentrons 2, the study demonstrates successful crystallization of model and non-model proteins, highlighting the potential of automated systems in structural biochemistry to democratize access to high-quality protein crystals. The third chapter delves into the creation of hybrid materials by combining metal-organic frameworks (MOFs) with porous protein crystals. The research demonstrates the feasibility of embedding MOF domains within protein crystals, potentially opening new avenues for applications in catalysis, gas storage, and chemical warfare agent detoxification. By developing a new class of hybrid materials, this work contributes to making advanced structural biochemical research. Together, these chapters illustrate a modern interdisciplinary approach that embraces machine learning and automation in service of the engineering of peptide-binding proteins and crystalline protein materials. The integration of automation, computational predictions, and hybrid materials offers a promising path toward more efficient and innovative solutions in biochemical research, while significantly lowering the cost barriers, thereby increasing accessibility for researchers worldwide.