Browsing by Subject "Enzymes"
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Item Open Access Elucidating solvent contributions to solution reactions with ab initio QM/MM methods.(J Phys Chem B, 2010-03-04) Hu, Hao; Yang, WeitaoComputer simulations of reaction processes in solution in general rely on the definition of a reaction coordinate and the determination of the thermodynamic changes of the system along the reaction coordinate. The reaction coordinate often is constituted of characteristic geometrical properties of the reactive solute species, while the contributions of solvent molecules are implicitly included in the thermodynamics of the solute degrees of freedoms. However, solvent dynamics can provide the driving force for the reaction process, and in such cases explicit description of the solvent contribution in the free energy of the reaction process becomes necessary. We report here a method that can be used to analyze the solvent contributions to the reaction activation free energies from the combined QM/MM minimum free-energy path simulations. The method was applied to the self-exchange S(N)2 reaction of CH(3)Cl + Cl(-), showing that the importance of solvent-solute interactions to the reaction process. The results were further discussed in the context of coupling between solvent and solute molecules in reaction processes.Item Open Access Exploring the Enzymology of Chlamydial Pathogenesis: An Investigation of Virulence and Energy Metabolism-Associated Enzymes(2021) Dudiak, BrianneChlamydia trachomatis is the obligate intracellular pathogen responsible for the most common bacterial sexually transmitted infection worldwide. While our front-line antibiotics have been historically successful in combatting chlamydial infections, emerging issues including treatment failure and chlamydial persistence necessitate the development of new therapeutic approaches. In this work, an enzyme-focused approach was devised to explore two of the intricate survival strategies of C. trachomatis: virulence and energy metabolism. We sought to employ biochemistry, enzymology, and chemical biology tools to interrogate enzyme functions and inform the design of new antichlamydial agents. To these ends, our efforts focused on characterization of the virulence-associated effector protein chlamydial protease-like activity factor (CPAF) and the futalosine pathway for menaquinone biosynthesis. Mechanistic analyses of CPAF zymogen maturation and peptide hydrolysis were performed that collectively classified CPAF as a serine protease with a catalytic tetrad. Analogs of the natural product salinosporamide A were subsequently explored as CPAF inhibitors. The futalosine pathway was discovered to be a source of novel antichlamydial targets through traditional and chemical genetics analyses in a HeLa cell model of chlamydial infection. The foundation was also established for studying a specific pathway enzyme, CT263, in a continuous coupled enzyme assay. Collectively, this dissertation has progressed our knowledge of several enzymes involved in critical processes for chlamydial pathogenicity and viability. The insights gained on a mechanistic level and in the context of chlamydial infection have laid the groundwork for pursuing virulence and energy metabolism enzymes as antichlamydial targets and for developing inhibitors of their activity, which are much-needed resources to combat this extremely prevalent sexually transmitted infection.
Item Open Access Exploring the Interface Between Macroorganisms and Microorganisms: Biochemical, Ecological, and Evolutionary Contexts(2015) Essock-Burns, TaraThe focus of this dissertation is the extension of the innate immune response in wound healing and non-wound healing contexts. I am interested in interactions at the interface between macroorganisms and microorganisms from marine/aqueous environments. This dissertation explored two aspects of the interactions: 1) the presence and function of macroorganism secretions and 2) the role of secretions in managing microfouling on macroorganism surfaces. Particularly of interest are how barriers are biochemically reinforced to mitigate microfouling and the potential consequences of a breach in those barriers. The innate immune response, an evolutionary conserved system in vertebrates and invertebrates, provides an evolutionary context for developing the hypotheses.
In this dissertation the biochemical composition and uses of crustacean secretions are explored for barnacles, fiddler crabs and blue crabs. Fluids of interest were secretions released during barnacle settlement and metamorphosis and those collected from living adult barnacles, fluids on fiddler crab sensory appendages including dactyl washings and buccal secretions, and fluids from blue crab egg masses. The biochemical composition was determined using a combination of fluorescent probes and confocal microscopy, proteomics, and enzyme-specific substrates with a spectrophotometer.
I demonstrated that self-wounding is inherent to the critical period of settlement and metamorphosis, in barnacles. Wounding occurs during cuticle expansion and organization and generates proteinaceous secretions, which function as a secondary mode of attachment that facilitates the transition to a sessile juvenile. I showed extensive proteomic evidence for components of all categories of the innate immune response, especially coagulation and pathogen defense during attachment and metamorphosis. This work provides insight into wound healing mechanisms that facilitate coagulation of proteinaceous material and expands the knowledge of potential glue curing mechanisms in barnacles.
In order to test macroorganism secretions in a non-wound healing context, I examined fluids sampled from body parts that macroorganisms must keep free of microorganisms. I showed that two types of decapod crustaceans can physically manage microorganisms on most parts of their body, but certain parts are particularly sensitive or difficult to clean mechanically. I examined sensory regions on the fiddler crab, including dactyls that are important for chemoreception and the buccal cavity that is used to remove microorganisms from sand particles, and blue crab egg mass fluids that protect egg masses from fouling through embryo development.
This dissertation explores organismal interactions across scales in size, space, and time. The findings from the barnacle work inform mechanisms of attachment and glue curing, both central to understanding bioadhesion. The work on fiddler crabs and blue crabs contributes to our understanding of chemoreception of feeding and reproductive behaviors.
The work presented here highlights how biological secretions from macroorganisms serve multifaceted roles. In cases of physical breaches of barriers, or wounding, secretions coagulate to obstruct loss of hemolymph and have antimicrobial capabilities to prevent infection by microorganisms. In non-wounding cases, secretions remove microorganisms from surfaces, whether that is on the body of the macroorganism or in the immediate environment.
Item Open Access Sortase as a Tool in Biotechnology and Medicine(2016) Bellucci, JosephWe have harnessed two reactions catalyzed by the enzyme sortase A and applied them to generate new methods for the purification and site-selective modification of recombinant protein therapeutics.
We utilized native peptide ligation —a well-known function of sortase A— to attach a small molecule drug specifically to the carboxy-terminus of a recombinant protein. By combining this reaction with the unique phase behavior of elastin-like polypeptides, we developed a protocol that produces homogenously-labeled protein-small molecule conjugates using only centrifugation. The same reaction can be used to produce unmodified therapeutic proteins simply by substituting a single reactant. The isolated proteins or protein-small molecule conjugates do not have any exogenous purification tags, eliminating the potential influence of these tags on bioactivity. Because both unmodified and modified proteins are produced by a general process that is the same for any protein of interest and does not require any chromatography, the time, effort, and cost associated with protein purification and modification is greatly reduced.
We also developed an innovative and unique method that attaches a tunable number of drug molecules to any recombinant protein of interest in a site-specific manner. Although the ability of sortase A to carry out native peptide ligation is widely used, we demonstrated that Sortase A is also capable of attaching small molecules to proteins through an isopeptide bond at lysine side chains within a unique amino acid sequence. This reaction —isopeptide ligation— is a new site-specific conjugation method that is orthogonal to all available protein-small conjugation technologies and is the first site-specific conjugation method that attaches the payload to lysine residues. We show that isopeptide ligation can be applied broadly to peptides, proteins, and antibodies using a variety of small molecule cargoes to efficiently generate stable conjugates. We thoroughly assessed the site-selectivity of this reaction using a variety of analytical methods and showed that in many cases the reaction is site-specific for lysines in flexible, disordered regions of the substrate proteins. Finally, we showed that isopeptide ligation can be used to create clinically-relevant antibody-drug conjugates that have potent cytotoxicity towards cancerous cells