Mountain Scholar :: Browsing by Author "Snow, Christopher, advisor"

Browsing by Author "Snow, Christopher, advisor"

Now showing 1 - 8 of 8

Open Access
Engineering stabilized enzymes via computational design and immobilization
(Colorado State University. Libraries, 2016) Johnson, Lucas B., author; Snow, Christopher, advisor; Reardon, Kenneth, committee member; Peebles, Christie, committee member; Peersen, Olve, committee member
The realm of biocatalysis has significantly matured beyond ancient fermentation techniques to accommodate the demand for modern day products. Enzymatically produced goods already influence our daily lives, from sweeteners and laundry detergent to blood pressure medication and antibiotics. Protein engineering has been a major driving force behind this biorevolution, yielding catalysts that can transform non-native substrates and withstand harsh industrial conditions. Although successful in many regards, computational design efforts are still limited by the crude approximations employed in searching a complex energy landscape. Advancements in protein engineering methods will be necessary to develop our understanding of biomolecules and accelerate the next generation of biotechnology applications. Our work employs a combination of computational design and simulation to achieve improved enzyme stability. In the first example, an enzyme used in the production of cellulosic biofuels was redesigned to remain active at high temperature. An initial approach involving consensus sequence analysis, predicted point mutation energy, and combinatorial optimization resulted in a sequence with reduced stability and activity. However, by using recombination methods and molecular dynamics simulations, we were able to identify specific mutations that had a stabilizing or destabilizing effect, and we successfully isolated mutations that benefited enzyme stability. Our iterative approach demonstrated how common design failures could be overcome by careful interpretation and suggested methods for improving future computational design efforts. In the second example, a cellulase was designed to have a high net charge via selected surface mutagenesis. “Supercharged” cellulases were experimentally characterized in various ionic liquids to assess the effect of high ion concentration on enzyme stability and activity. The designed enzymes also provided an opportunity to systematically probe the protein-solvent interface. Molecular dynamics simulations showed how ions influenced protein behavior by inducing minor unfolding events or by physically blocking the active site. Contradictory to previous reports, charged mutations only appeared to alter the affinity of anions and did not significantly change the binding of cations at the protein surface. Understanding the different modes of enzyme inactivation could motivate targeted design strategies for engineering protein resilience in ionic solvents. In addition to the discussed computational design methods, immobilization strategies were identified for capturing enzymes within porous protein crystals. Immobilization offers a generic approach for improving enzyme stability and activity. Our preliminary studies involving horseradish peroxidase and other enzymes suggested protein scaffolds could be employed as an effective immobilization material. Co-immobilizing multiple enzymes within the porous material led to improved product yield via exclusion of off-pathway reactions. Although future studies will be required to assess the potential capabilities of this immobilization strategy in comparison to other materials, preliminary results suggest protein crystals offer a favorable, controlled environment for immobilizing enzymes. The diversity of approaches presented in this thesis emphasizes that there are many options for engineering enzyme stability. Extending the lessons learned from our cellulase engineering to the greater field of rational protein design promotes the concept of biomolecules as designable entities. By establishing the shortcomings of our designs and suggesting routes for improvement, we anticipate our design methods and immobilization strategies will procure continued interest from the biotechnology community. The toolsets we developed for cellulases can be directly transferred to other enzymes and have the potential to impact a range of protein engineering applications.
Embargo
Odor encoder: computational design of a novel allosteric enzyme activation system for providing enhanced olfactory abilities to trained odor detecting sentinel animals
(Colorado State University. Libraries, 2022) Scroggins, Michael, author; Snow, Christopher, advisor; Peebles, Christie, advisor; Gentry-Weeks, Claudia, committee member
From the perfume of a flower, to the aroma of a favorite food, to what for bioengineers is the all-to-familiar smell of E. coli, olfactory senses play in important role in how animals interact with the world around them. An offensive odor can inform us that an object is unsafe to eat or be around, a familiar scent can recall memories of events from decades in our past, and even our natural body odors can affect our mating selection preferences. Yet there are many chemicals, both natural and synthetic, for which we do not possess the ability for olfactory detection. An everyday example of this is the natural gas that we use in our homes and which is naturally odorless, but which is commonly spiked with the odorant tert-butyl mercaptan (TBM) to provide the characteristic sulfuric smell we associate with natural gas. Because of this added odorant we can rapidly detect a leaking gas via the smell of the TBM and address the situation as needed to ensure the safety of ourselves and our community. Unfortunately, there are some hazardous and odorless chemicals which we cannot simply spike with an odorant molecule, and for these situations it would be ideal to have alternative options for facilitating a rapid olfactory detection. Therein lies the goals of the Odor Encoder project; to create enhanced olfactory abilities via a conditionally activated enzyme which produces a smellable product in the presence of a target odorless molecule. The approach to achieving this goal was creation of a genetically modified bacterial organism which could be engineered for conditional expression of an odorant producing enzyme in-situ within the nasal microbiome of trained odor detecting animals. The odorant producing enzyme chosen for this purpose was salicylic acid methyltransferase, a.k.a SAMT, which produces the characteristic odorant molecule methyl salicylate via methylation of salicylic acid. The probiotic E. coli strain Nissle 1917 was selected as the bacterial organism for inoculation of the nasal microbiome, and an expression plasmid was created which could produce both salicylic acid and methyl salicylate from endogenously produced metabolites via dual expression of SAMT along with a salicylate synthase enzyme known as irp9. Conditional production of methyl salicylate was achieved via two methods. The first method involved conditional enzyme expression via use of a riboswitch specific to the small molecule theophylline. The second method involved conditional enzyme activity via constitutive expression of a crippled form of SAMT which may potentially have its enzymatic activity restored via theophylline induced allosteric activation. The allosteric rescue method utilized computational design methods to design novel theophylline-specific allosteric cavities in SAMT, and theophylline induced allosteric reactivation of enzyme activity will be investigated via production and screening of the computationally designed enzyme library.
Open Access
Porous protein microcrystals as a scaffold for nucleic acids and proteins
(Colorado State University. Libraries, 2022) Masri, Mahmoud, author; Snow, Christopher, advisor; Peebles, Christie, committee member; Takamitsu, Kato, committee member
Oral delivery of nucleic acids is restricted by a number of limiting factors, particularly protection of guest DNA and RNA from degradation and hydrolysis within the gastrointestinal tract following ingestion. Highly ordered, self-assembling porous protein crystals have been previously explored for enzyme immobilization, and may offer similar advantages for protection and targeted delivery of therapeutic molecules to cells. We have developed a reproducible method for generating sub-micrometer porous microcrystals from CJ, a putative isoprenoid-binding protein from Campylobacter jejuni, which are non-cytotoxic and capable of passively retaining plasmid DNA and small interfering RNA. Furthermore, we have demonstrated that CJ microcrystals are able to deliver functional plasmid and transfect cells in vitro. In addition to nucleic acids, CJ microcrystals are also capable of adsorbing functional Nanoluciferase, and display chemiluminescent activity following exposure to substrate. The results of this study demonstrate that porous protein microcrystals can serve as a suitable scaffold for RNA, DNA, and functional enzymes, and may represent a viable alternative to spherical nanoparticles and liposomes for therapeutic delivery.
Open Access
Protein crystals as nanotemplating materials
(Colorado State University. Libraries, 2019) Kowalski, Ann, author; Snow, Christopher, advisor; Kipper, Matt, committee member; Peebles, Christie, committee member; Sambur, Justin, committee member
The advancement of nanomaterial development depends on the reliable and scalable synthesis of three dimensional nanostructures and devices. Applications for these materials range from catalysis and energy storage to biomedicine and imaging. Towards the goals of shape-controlled immobilization and synthesis, templating is arising as a promising manufacturing method. With the rise of bionanotechnology, DNA and protein scaffolds can be designed, synthesized, and functionalized to coordinate nanoparticles, enzymes, and other guests in three dimensions, or act as molds for the synthesis of anisotropic nanostructures. Inherently, protein crystals are an attractive target, as they have nearly unlimited designability, intrinsic functionality for a variety of useful materials, and mild reaction conditions. The overarching goal of this work is to explore the feasibility of protein crystals as templates for the creation of biohybrid materials. We show that protein crystals with large solvent channels can strongly adsorb and immobilize gold nanoparticles by reversible metal affinity interactions and that these nanoparticles can serve as nucleation sites for the growth of nanorods within the pores of protein crystals by a variety of gold growth methods. We show that, depending on the method used, gold nanorod synthesis within the crystals can be dependent on the presence of a seed particle. Despite their stability, these crystals can be dissolved to release the gold structures, which can be analyzed by electron microscopy and elemental analysis. A variety of gold nanorod products are formed, from highly anisotropic individual rods, to interconnected rod bundles, to parallel rods embedded within a protein matrix. Additionally, we show that protein crystal pores can be used for the long-term capture of multiple enzymes and that these enzymes retain their activity within the crystal. Product can be separated by a simple washing step, and the immobilized two-enzyme pathway can be used for multiple cycles over several weeks. Rates of product formation are higher for enzymes immobilized within crystals of a high surface-to-volume ratio; thus, the use of micron-sized crystals minimizes transport limitations typically associated with enzyme immobilization. Preliminary work suggests the crystals may also impart significant thermal stability to the embedded enzymes. Porous protein crystals may provide a superior templating method for the development of nanomaterials. Here we further demonstrate the wide variety of applications for protein crystals by revealing their success as scaffolds for immobilization, synthesis, and catalysis.
Open Access
Protein engineering therapeutic strategies and tools
(Colorado State University. Libraries, 2019) Ta, Angeline Ngoc, author; Snow, Christopher, advisor; Henry, Chuck, committee member; Kennan, Alan, committee member; Stasevich, Tim, committee member
Proteins have become an important tool for research development and therapeutics. Proteins complement the use of small molecules as well as overcome challenges that small molecules cannot. The contrasting difference of their diverse functional and structural properties allows for complex processes like molecular recognition and catalysis. Through loops, turns, helixes, and sheets, these structural motifs provide a protein with shape and electrostatics to achieve a particular function. Overall, I describe here two examples of functional proteins where the protein's complex structure plays an important role in the development of new strategies and tools for therapeutics. The first part of this dissertation shows the effects of increased antibody recruitment on targeted cell death through the use of an immunotherapeutic cocktail of cell surface HER2 receptor binding proteins. The second part of this dissertation describes the use of a protein's chiral environment to develop a new artificial metalloenzyme that selectively catalyses synthesis of the most common N-heterocycle found in FDA approved pharmaceuticals.
Open Access
Software for the use of protein fragment recombination and regression in protein structure determination and design
(Colorado State University. Libraries, 2015) Lunt, Mark, author; Snow, Christopher, advisor; Fisk, Nick, committee member; Ben-Hur, Asa, committee member
Recombination of protein structural fragments, in combination with regression-based scoring schemes, provides an alternative to existing iterative strategies for conducting a search over protein conformations. We developed software to define astronomically large combinatorial protein conformation search spaces, and to efficiently search those spaces. We demonstrate that such methods may be applicable to the structure prediction of cytochrome P450 chimeras. More generally, we demonstrate that such methods can be used to produce high-quality protein structural models given only low-resolution X-ray diffraction data.
Open Access
Software to design crosslinks for protein crystal stabilization
(Colorado State University. Libraries, 2015) Sebesta, Jacob Christopher, author; Snow, Christopher, advisor; Fisk, Nick, committee member; Rappe, Anthony, committee member
Programmable materials allow properties as specific locations in the material to be modified through reliable encoding. One class of such materials are protein crystals that allow changes to be made through genetic manipulation. Protein crystals are well-ordered and highly porous materials, but they are also easily dissolved, limiting their utility. Crosslinking techniques previously developed often have a deleterious effects on the crystal order. In this work, we introduce software to design specific crosslinks across protein crystal interfaces using disulfide and dityrosine crosslinks as well as a variety of small molecule crosslinkers used in protein conjugation. The software is a general tool for specific crosslinking that introduces a number of improvements on previous disulfide design software. Several of the disulfide and dityrosine designs were assembled in the lab and one of the disulfide crosslink designs was confirmed using X-ray diffraction.
Open Access
Towards macromolecular scaffold assisted crystallography
(Colorado State University. Libraries, 2017) Huber, Thaddaus, author; Snow, Christopher, advisor; Ackerson, Christopher, committee member; Henry, Charles, committee member; Fisk, Nick, committee member
The current, dominant method for structure determination in atomic detail is X-ray crystallography; but, this method requires a brute force search through non-physiological solution conditions looking for the "needle-in-a-haystack" condition in which the target protein crystallizes. Unfortunately, despite exhaustive screening, most proteins of interest do not form crystals. Other proteins are difficult to obtain in sufficient quantities to make the attempt. Finally, even successful crystals reveal a structure adopted under artificial conditions-a single snapshot that dramatically underrepresents the protein mobility. The motivational insight for this work is the recognition that materials diffract X-rays if they consist of a highly-ordered, repeating lattice, but that the lattice need not be composed only of target protein. Instead of growing conventional protein crystals, we will take the unprecedented step of attaching target proteins (guests) to specific sites within pre-existing, crystalline scaffolds for a new technique called scaffold assisted crystallography. This approach circumvents the haphazard nucleation and growth process that underlies conventional crystallography. Instead, we face novel challenges. We must engineer scaffolds that have very large pores (>10 nm), withstand significant solution condition changes, yet still diffract to high resolution. We must also ensure that guest proteins tightly tethered to the crystalline scaffold adopt a coherent structure visible via X-ray diffraction. Instead of taking on the challenge of de novo design of porous protein crystals, we decided to search the protein databank for a suitable scaffold. Algorithms for identifying highly porous protein crystals are covered (Chapter 1) and a select few representative examples are presented. Constructs for high priority candidates were obtained and crystallization of the targets were attempted. One of the candidates crystallized rapidly and presented a platform for developing methods and identifying roadblocks for second generation scaffolds. Working extensively with a single scaffold, a putative periplasmic protein from Campylobacter jejuni (CJ), allowed for robust method development that enabled highly optimized expression and extensive knowledge of its crystallization space. CJ requires high salt for crystallization. Crystals quickly degrade outside of the growth conditions. Most guest macromolecules will have low solubility in the high salt required to preserve the CJ crystalline lattice. Therefore, methods for chemical crosslinking of CJ crystals were developed to withstand significant solution condition changes, yet still diffract to high resolution. The most ubiquitous crosslinking agent glutaraldehyde effectively stabilized the crystal, but resulted in a dramatic loss of diffraction. Three alternative crosslinkers, formaldehyde, glyoxal, and EDC, were tested for their ability to stabilize CJ crystals. The three alternative crosslinkers all stabilized CJ crystals in challenging conditions (no salt) with little degradation in diffraction quality. The crosslinked crystals were subjected to x-ray diffraction; the resulting electron density demonstrates the first known atomic resolution modifications from formaldehyde, glyoxal, or EDC crosslinks in a protein crystal. In contrast to the weak, noncovalent interactions that hold together typical protein crystals, guest domains can be attached to the host scaffold using strong interactions. For maximum programmability, affinity tags for the desired assembly can be genetically encoded on the guest and scaffold monomers. We demonstrated that non-covalent, metal-mediated capture and genetically encoded histidine tags provide a significant level of control. Loading and release of guest molecules were fine-tuned to spatially segregate multiple guest proteins. Similarly, by controlling the diffusion of crosslinking agents we engineered a crystalline shell that still diffracts well. Scaffold assisted crystallography techniques were demonstrated with small molecule guests in CJ crystals. Guest molecules were installed via a single covalent bond to reduce the conformational freedom and achieve high occupancy structures. We used four different conjugation strategies to attach guest molecules to three different cysteine sites within pre-existing protein crystals. In all but one case, the presence of the adduct was obvious in the electron density. The above methods led to preliminary attempts of scaffold assisted crystallography with macromolecules. Guest mini-proteins variants were obtained with solvent exposed cysteines. These were covalently attached in vitro to CJ with an engineered surface thiol. We attempted to crystallize the resulting CJ-mini-protein conjugates. One of the CJ-mini-protein conjugates crystallized and the structure was determined. While the presence of the guest mini-protein was obvious, the electron density past the attachment point was ambiguous. Still, this result demonstrates feasibility of fusing target proteins to engineered CJ monomers for "chaperoned crystallization". For targets that fail to crystallize when pre-installed, we can perform asynchronous crystallization and by attaching the guest mini-protein to a preformed CJ crystal. Techniques for in crystallo conjugation and quantification are developed. Finally, present strategies for realizing macromolecular scaffold assisted crystallography are presented.

Browsing by Author "Snow, Christopher, advisor"

Results Per Page

Sort Options