Browsing by Subject "Binding"
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Item Enzyme immobilization on gold surfaces : effects of surface chemistry and attachment strategies on binding and activity(2023-04-20) Correira, Joshua Manuel; Webb, Lauren J.; Samanta, Devleena; Baiz, Carlos R; Gordon, Vernita; Shear, Jason BFunctional enzymes are the basis for many biotechnological systems, including biosensors, bio-fuel cells, and heterogeneous biocatalysts. In these systems, enzymes are often immobilized on a solid support or surface to capture their catalytic activity. Immobilization has the advantage of improving enzyme stability and reusability but often results in a significant loss of catalytic activity (1-2 orders of magnitude) when compared to the native enzyme. This inactivation is due to direct interactions between the enzyme and the solid support. Here, we developed methods to study enzyme immobilization and resulting inactivation. The aim of this work was to identify optimal surface chemistries and attachment strategies that promote high binding efficiencies while minimizing activity losses. Subsequently, we studied this using three enzymes (acetylcholinesterase (AChE), β-galactosidase (β-gal), horseradish peroxidase (HRP)) immobilized on gold surfaces by direct adsorption, covalent attachment, and DNA-directed attachment. First, AChE was directly adsorbed onto a variety of gold surfaces modified with self-assembled monolayers (SAMs) terminated with -COO⁻, -NH₃⁺, -OH, and -CH₃ functional groups at varying mole % to study the effect of surface hydrophobicity and charge on binding and activity. We found that binding was directly proportional to surface hydrophobicity (r = 0.75) and activity was inversely proportional to surface hydrophobicity (r = -0.62). The highest binding observed was ~40% of a monolayer on the most hydrophobic surfaces and the lowest binding observed was ~10% of a monolayer on the most hydrophilic surfaces. Conversely, on the most hydrophobic surfaces AChE retained <10% of its native activity, and on the most hydrophilic surfaces AChE retained ~40% of its native activity. This illuminated an inherent problem with direct adsorption: high binding and high activity are mutually exclusive. Due to these findings, we next immobilized β-gal and HRP on DNA-functionalized gold surfaces using DNA-DNA interactions, to avoid direct interactions between the enzyme and surface. We found that β-gal retained 62% of its native activity following immobilization, a significant improvement over previous direct adsorption strategies.Item The interaction between NS1B protein of influenza B virus and the ubiquitin-like modifier ISG15 : insights into a unique species specific property of the virus(2009-05) Sridharan, Haripriya; Krug, Robert M.Influenza B virus causes a respiratory disease in people with a compromised immune system. The NS1B protein of influenza B virus is essential for virus growth and plays a crucial role in inhibiting the anti-viral responses mounted by the infected host cell. The N terminal 104 amino acids of NS1B bind a cellular protein called ISG15. ISG15 is an interferon induced 'ubiquitin-like' protein, and upon interferon induction, is conjugated to hundreds of targets. It has been found that both ISG15 and its conjugation inhibit many viruses. The focus of the current study was to characterize the interaction between NS1B and ISG15. Study of a recombinant influenza B virus which encoded a mutant NS1B protein that is unable to bind ISG15 revealed that ISG15 is mis-localized in cells infected with wild type but not the mutant influenza B virus. Further, such a mutant virus is attenuated in growth as compared to wild type virus in human cell lines but is not attenuated in canine cell lines. This result led to the discovery of the species specific nature of the interaction between NS1B and ISG15. Specifically, NS1B was found to bind ISG15 homologs from human and old world monkeys like Rhesus macaques and African green monkeys but not those from mouse or canines. These findings were extended by identifying the hinge between the N and C terminal domains of ISG15 as one of the major determinants of species specificity. These results highlight the importance of using human or primate cell culture models to study the effect of ISG15 on influenza B virus, and raises new possibilities on differences in the function of the ISG15 system in different species.Item Toward a referential view of definite descriptions(2015-05) Woolwine, Cassandra Joan; Dever, Josh; Buchanan, Lawrence RI argue for a referential view of definite descriptions. According to the view that I advocate, definite descriptions are variables. At the syntactic level, they contribute a free variable that must be assigned a referent in order for the sentence to be truth evaluable. When an assignment is provided, through the referential intentions of the speaker, the semantic content of a given use is a singular proposition involving the object that the assignment assigns to the variable. I show that this view has the resources to accommodate uses of definite descriptions that are bound into by external quantifiers. Using the same resources, I show how one can arrive at the dual readings available for sentences containing definite descriptions embedded in modal and belief operators. I discuss the distinction between referential and attributive uses of definite descriptions and explain how the relevant differences are achieved on this view.