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Browsing by Author "Ho, P. Shing, committee member"

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    Characterization of poliovirus 2CATPase bound to bilayer nanodiscs and involvement of the poliovirus 3Dpol thumb α-helix in determining poly(A) tail length
    (Colorado State University. Libraries, 2013) Springer, Courtney Lee, author; Peersen, Olve B., advisor; Ho, P. Shing, committee member; Luger, Karolin, committee member; Kennan, Alan, committee member
    Poliovirus (PV) is a small non-enveloped picornavirus with a ≈7.5 kb long single-stranded, positive-sense RNA genome. Upon infection, the RNA is translated to generate a ≈250 kDa polyprotein that is subsequently cleaved into about a dozen fully processed proteins and several functional intermediates. PV replication occurs in large membrane associated complexes involving the "non-structural" P2 and P3 region proteins and two of these proteins, 2CATPase and 3Dpol, are the subjects of this dissertation. Part I of this work is focused on the 2C protein, an AAA+ family ATPase that plays a key role in host cell membrane rearrangements and virion assembly, but the membrane binding characteristics of 2C and its polyprotein precursors have made it difficult to elucidate their exact roles in virus replication. In this work I show that small lipid bilayers known as nanodiscs can be used to chaperone the in vitro expression of soluble poliovirus 2C and the precursor 2BC and 2BC3AB polyproteins in a membrane bound form. Biochemical analysis shows that the proteins are highly active over a wide range of salt concentrations, exhibit slight lipid headgroup dependence, and show significant stimulation by acetate. Notably, the ATPase activity of the core 2C domain is stimulated ≈60-fold as compared to the larger 2BC3AB polyprotein, with most of this stimulation occurring upon removal of 2B. This data leads to a model wherein the viral replication complex can be assembled with a minimally active form of 2C that then becomes fully activated upon proteolytic cleavage from the adjacent 2B viroporin domain. In Part II of this dissertation, I focus on the role of the viral RNA polymerase, 3Dpol, in maintaining the ≈20-150 nucleotides long 3' poly(A) tail of the viral genome. The length of the tail is important for viral replication and initiation of (-)-strand synthesis, but the means by which the RNA is polyadenylated and how poly(A) tail length is regulated is not well understood. We have identified several mutations in an α-helix of the 3Dpol thumb domain that directly impact poly(A) tail length. Here, I tested the impact of these mutations on reiterative transcription of poly(A), poly(U), and poly(C) templates as well as characterized their effect on 3Dpol initiation, stability, elongation rate, and fidelity. I found that mutations in the thumb have the greatest impact on elongation complex stability and that 3Dpol is able to reiteratively transcribe homopolymeric poly(U) and poly(A), but not poly(C) RNA templates. Interestingly, distinct poly(A) and poly(U) transcripts are generated from 10 nucleotide homopolymers that are 1, 7, or 8 nucleotides longer than the template. Based on these findings, we propose a poly(A) slippage model in which the elongation complex stalls at the end of the homopolymer stretch in the absence of additional nucleotides to promote a single nucleotide slippage. This is followed by a slow structural rearrangement in which 3Dpol slips back to the 3' end of the homopolymer sequence, where it is able to re-transcribe starting from the fifth poly(U) in the template.
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    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.
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    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.
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    Inhibition of a truncated form of human mitochondrial kidney-type glutaminase (hKGA124-551) by bis-2-(5-phenylactamido-1,2,4-thialdiazol-2-yl)ethyl sulfide (BPTES)
    (Colorado State University. Libraries, 2011) Hartwick, Erik William, author; Curthoys, Norman, advisor; Ho, P. Shing, committee member; Peersen, Olve, committee member; Mykles, Donald, committee member
    Mitochondrial glutaminase (GA) catalyzes the hydrolysis of glutamine producing glutamate and an ammonium ion. There are three isoforms of mammalian GA that are essential to hepatic ureagenesis, renal ammoniagenesis, synthesis of the neurotransmitter glutamate, and the catabolism of glutamine. Here we focus on the human KGA isoform that is predominantly expressed in kidney, brain, intestine, and tissues of the immune system. Recent publications suggest that GA is a novel target for developing new cancer therapeutics. These studies have indicated that inhibition of GA by small molecule inhibitors significantly reduces the size of tumors in rats and inhibits growth of transformed cells in culture. A truncated form of human KGA hKGA124-551 that contains amino acids 124-551, was produced to delete the C-terminal sequences that are unique to the KGA and GAC isoforms. This construct was assayed in the presence of (bis-2-(5-phenylactamido-1,2,4-thiadiazol-2-yl)ethyl sulfide (BPTES). BPTES is a potent small molecule inhibitor of mammalian GA that was previously shown to inhibit rat KGA in µM concentrations. In the current study, we adapted the standard GA assay to a microtiter plate format and used it to characterize the inhibition of hKGA124-551 using µM amounts of BPTES. Our data indicate that BPTES is a mixed non-competitive inhibitor at low concentrations of phosphate, but at higher phosphate concentrations the inhibition is predominantly uncompetitive. Lastly, gel filtration and dynamic light scattering experiments were performed to determine if hKGA124-551 oligomers are formed in the presence of BPTES and to characterize the effect of increasing concentrations of phosphate. The data suggest that in low phosphate and in the absence of BPTES, the hKGA124-551 exists as a dimer, but in the presence of BPTES and higher phosphate concentrations the molecular weight shifts to a tetramer or higher oligomer. The combined data indicate that BPTES is a potent lead compound for the development of a therapeutic inhibitor of human GA that may be a potential cancer therapeutic.
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    Investigation of the sequence features controlling aggregation or degradation of prion-like proteins
    (Colorado State University. Libraries, 2017) Cascarina, Sean Micheal, author; Ross, Eric, advisor; Ho, P. Shing, committee member; Di Pietro, Santiago, committee member; Zabel, Mark, committee member
    Protein aggregates result from the conversion of soluble proteins to an insoluble form. In some cases, protein aggregates are capable of catalyzing the conversion of their soluble protein counterparts to the insoluble form, resulting in a mode of molecular self-replication. Many of these infectious proteins, or "prions", have been identified and characterized in yeast. This has led to the development of prediction algorithms designed to identify protein domains capable of forming prions. Recently, a number human proteins with aggregation-prone prion-like domains (PrLDs) have been identified, and mutations within PrLDs have been linked to muscular and neurodegenerative disorders. However, the number and diversity of PrLD mutations linked to disease are currently limited. Therefore, the extent to which a broad assortment of PrLD mutations affect intrinsic aggregation propensity, and how well this correlates with aggregation in a cellular context, has not been systematically examined. In Chapter 2, I present evidence suggesting that our prion aggregation prediction algorithm (PAPA) is capable of predicting the effects of a diverse range of mutations on the aggregation propensity of PrLDs in vitro and in yeast. PAPA was also able to predict the effects of many but not all PrLD mutations when the protein was expressed in Drosophila, but with slightly. Therefore, while great strides have been made in predicting intrinsic aggregation propensity, a more complete understanding of the cellular factors that influence aggregation in vivo may lead to further improvement of prion prediction methods. Many intracellular protein quality control factors specialize in recognizing and degrading aggregation-prone proteins. Therefore, prions must evade or outcompete these quality control systems in order to form and propagate in a cellular context. However, the sequence features that promote degradation versus aggregation of prion domains and PrLDs have not been systematically defined. In Chapter 3, I present evidence that aggregation propensity and degradation propensity can be uncoupled in multiple ways. First, we find that only a subset of classically aggregation-promoting amino acids elicit a strong degradation response in PrLDs. Second, the amino acids that promoted degradation of the PrLDs did not induce degradation of a glutamine/asparagine (Q/N)-rich prion domain, and instead led to a dose-dependent increase in the frequency of spontaneous prion formation, suggesting that protein features surrounding aggregation-prone amino acids can modulate their ultimate effects. Furthermore, degradation suppression correlated with Q/N content of the surrounding prion domain, potentially indicating an underappreciated role for these residues in yeast prion domains. The protein features that foster susceptibility or resistance to degradation are further explored in Chapter 4. We find that Q/N-rich domains resist degradation in a primary sequence-independent manner, and can even exert a dominant degradation-inhibiting effect when coupled to a degradation-prone PrLD. Furthermore, susceptibility to degradation was a relatively de-centralized feature of the PrLD, requiring a large portion of the domain surrounding degradation-promoting amino acids to permit efficient protein turnover. Collectively, these results provide key insights into the relationship between intrinsically aggregation-prone protein features and the ability to aggregate in the context of intracellular protein quality control factors.
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    Long range interaction networks within 3Dpol and the roles they play in picornavirus genome replication and recombination
    (Colorado State University. Libraries, 2020) Watkins, Colleen L., author; Peersen, Olve B., advisor; Cohen, Robert, committee member; Ho, P. Shing, committee member; Wilusz, Jeffrey, committee member
    Picornaviruses contain a single-stranded positive sense RNA genome approximately 7.5kb in length. The genome encodes for a single polyprotein that can future be divided into three functional regions; the P1 region containing the viral capsid proteins, the P2 region whose proteins function primarily in membrane rearrangement during viral replication, and the P3 region which contains four protein responsible for RNA replication. The final protein in the P3 region is 3Dpol, an RNA-dependent RNA polymerase (RdRP) whose structure is analogous to a "right hand" with fingers, palm and thumb domains, and around which this dissertation will be centered. Section one of this work investigates the roles three regions within the fingers domain play in the catalytic cycle of 3Dpol: "The kink" located within the index finger, "the gateway" found on the pinky, and "the sensor", which bridges the two beta-strands of the middle finger. This study demonstrates that the kink residues are involved in RNA binding as mutations to these residues result in decreased initiation time and elongation complex lifetime. The gateway residues are found to act as a molecular stop against which the template-RNA strand positions itself post-translocation, eventually resetting the active site for the next round of nucleotide incorporation. Lastly the sensor residues serve two key functions: 1) A final checkpoint to determine the correct nucleotide has entered the active site, and 2) As a possible source for proton donation to the pyrophosphate leaving group formed during catalysis. The inter-connected nature of the residues investigated in this section give rise to the idea that it is not individual residues alone that control major steps during the catalytic cycle, but instead that long ranging interaction networks within the different polymerase domains are ultimately responsible for controlling different actions carried out by the polymerase. Section two of this work looks at the long-range interaction networks within 3Dpol by dissecting the roles each polymerase domain plays in catalytic cycle. Through generation of chimeric polymerases it was determined that the pinky finger, with some influence by the fingers domain, controls RNA binding, the palm domain dictates nucleotide discrimination, and nucleotide capture and active site closure rates. It was also established that the thumb domain controls translocation, and an interaction between the palm and thumb domains was needed to generate a viable virus, supporting the idea of interface I, a protein-protein interface that was discovered in the first 3Dpol crystal structure. What is most striking about these findings is that unlike other single subunit polymerases that perform translocation by using a large swinging motion within the fingers domain, viral RdRPs use an entirely different domain altogether. The last section of this work deals with viral recombination, an event that is carried out at a low frequency during virus replication. Recombination is proposed to be a mechanism by which mutations can be purged from the genome independent of polymerase fidelity. This study carries out a mechanistic investigation into how mutation of residue 420 from a leucine to an alanine affects polymerase replication kinetics. It also takes a look at the mutation of residue 64 from a glycine to a serine, a previously identified mutation that results in a high-fidelity polymerase, in the presence and absence of L420A. This work revealed that mutations L420A and G64S operate independently of each other by affecting different steps in the catalytic cycle with G64S increases in fidelity predominately from monitoring nucleosugar positioning while L420A affects nucleobase positioning and polymerase grip on the product RNA strand.
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    Role of basic and hydrophobic residues in the poliovirus polymerase elongation complex and the structure of a coxsackievirus polymerase elongation complex
    (Colorado State University. Libraries, 2011) Kortus, Matt, author; Peersen, Olve, advisor; Ho, P. Shing, committee member; Suchman, Erica, committee member
    Picornaviruses encode for and require a viral RNA-dependent RNA polymerase (RdRP) for genome replication. This enzyme synthesizes negative-sense RNA from the infecting positive sense genome producing a replicative intermediate. The negative sense RNA then serves as a template for synthesis of additional positive-sense RNA. To efficiently replicate the genome, RdRPs must form a stable and processive elongation complex (EC) by binding RNA, incorporating the first templating nucleotide, and undergoing a necessary conformational. Upon completion of these steps that comprise initiation, the newly formed EC is capable of rapidly replicating the viral genome. The work presented in this thesis 1) investigates the role that several basic and hydrophobic residues serve in forming and maintaining the poliovirus (PV) EC and 2) presents the crystal structure of a coxsackievirus (CV) EC. To determine the role of that several arginines, lysines, and tyrosines play in the PV polymerase, we assessed whether mutations to these residues affect initiation, elongation, or stability of the EC. The data indicates the basic residues within the fingers domain of the PV polymerase have a major role in binding RNA. In addition, data shows two tyrosine residues in particular are critical for formation and maintenance of the EC. Overall, the data provides evidence the fingers domain interacts with the template RNA in a manner not captured by crystal structures. Finally, we have solved the structure of a CVEC stalled after incorporation of four nucleotides. The CVEC structure closely matches the previously solved PVEC structure. In addition, one crystal form produced an elongation complex trapped in a translocation intermediate state.