Browsing by Author "William Miller, Committee Member"

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A comparative analysis of Bacillus subtilis and Bacillus anthracis AbrB using Circular Dichroism and NMR Spectroscopy
(2009-04-20) Perry, Nicole Alana; William Miller, Committee Member; John Cavanagh, Committee Chair; Dennis Brown, Committee Member
The transition state regulators are DNA-binding proteins found in many bacterial species and are involved in the regulation of processes related to the organismÃ¢â‚¬â„¢s defense. Each transition state regulator binds multiple DNA targets with affinity in the nanomolar range, in spite of not recognizing a consensus sequence. Structural studies of the N-terminal DNA-binding domain have revealed a mechanism by which the proteins bind DNA, however, to date no NMR structure of a full-length transition state regulator exists. AbrB is the most well-characterized transition state regulator, and homologs are found in both Bacillus subtilis and Bacillus anthracis. The sequences of AbrB from B. subtilis and B. anthracis are overall 85% identical, and understanding the sequence differences from a structural standpoint is key to understanding the function of each protein. The far-UV circular dichroism spectra of each were deconvoluted using a number of programs, and the estimates of secondary structure content compared to the predictions by PSIPRED. Multiple NMR experiments were performed in order to assign the backbone chemical shifts of AbrB from both B. subtilis and B. anthracis. Chemical shift analysis has recently proven to be a powerful tool to investigate protein structure, providing information on backbone dihedral angles, aromatic ring orientation and other parameters. A comparative analysis of the homologous proteins was undertaken by determining the change in chemical shifts (ÃŽâ€ ÃŽÂ´) of conserved residues. The Chemical Shift Index (CSI) was used to determine the secondary structure of AbrB based on chemical shift information. Results show that the N-terminal DNA-binding domain adopts the same structure as observed in the NMR structure of the isolated AbrB DNA-binding domain. The appearance of the TROSYs indicates that neither AbrBBA nor AbrBBS adopts a largely random coil structure, and likely contains the C-terminal ÃŽÂ±-helix predicted by PSIPRED.
Host Cytokines and Immune Responses in Pregnancy Associated Transmission of Arrested Hookworm Larvae
(2006-09-30) Trivedi, Shweta; Prema Arasu, Committee Chair; William Miller, Committee Member; Paul Mozdziak, Committee Member; Scott Laster, Committee Member
Over one billion people worldwide are infected with blood-feeding intestinal hookworms. The life cycle of A. duodenale (humans) and A. caninum (dogs) includes the propensity for L3 to undergo a temporary state of developmental arrest in the host. In female hosts, tissue-arrested L3 reactivate during pregnancy and are transmitted to neonates through milk. During pregnancy, transforming growth factor (TGF)-β2 is upregulated in the mammary gland. Studies in C. elegans show that TGF-β and insulin-like signaling pathways regulate larval arrest and reactivation. We previously utilized an in vitro assay to demonstrate that recombinant human TGF-β stimulates a feeding response in tissue-arrested A. caninum L3. We hypothesize that host expression of TGF-β and pregnancy hormones such as estrogen and prolactin signal arrested L3 to reactivate. To facilitate in vivo analyses, we utilized a mouse model of A. caninum infection. Mice were utilized because L3 do not develop into adults but arrest in somatic tissues, and reactivate during the periparturient period. We investigated TGF-β1, TGF-β2 and IGF-1 transcript and serum cytokine profiles during late pregnancy, early lactation and mid-lactation to correlate levels with transmammary transmission of L3 to nursing pups. An in vitro co-culture system was developed to mimic in vivo conditions and assess effects of TGF-β and, estrogen and prolactin on larval reactivation. A. caninum L3 were co-incubated with skeletal muscle or mammary epithelial cells and larval reactivation was measured. Additionally, immune responses were assessed as by measuring serum and transcript levels of IFN-γ and IL-4 in skeletal muscle, mammary gland and spleen to dissect the effects of pregnancy and A. caninum infection in the mouse. Our findings suggest that host-derived TGF- β1 and IGF-1, but not TGF- β2, are important in reactivation and transmission of arrested A. caninum larvae. Also, a Th2-like response characterized by increased IL-4 transcript levels was observed in skeletal muscle, while a mixed Th1⁄Th2 profile was observed in mammary gland when comparing infection with A. caninum versus pregnancy/lactation in BALB⁄c mice.
Isolating HIV NCp7 Peptide Mimics that Target the Anticodon Stem Loop Region of Human Transfer RNA Lysine3 in a Modification Dependent Manner
(2010-07-30) Graham, William; Michael Goshe, Committee Chair; William Miller, Committee Member; John Cavanagh, Committee Member
Kinetic Folding Studies of Apaf-1 CARD and Procaspase-3
(2008-11-13) Milam, Sara Lis; Carol Hall, Committee Member; William Miller, Committee Member; A. Clay Clark, Committee Chair; Michael Goshe, Committee Member
Apoptosis regulates the balance between cell growth and death. Apoptosis consists of two main signaling pathways, extrinsic and intrinsic. We have examined the kinetic folding mechanism of two proteins, Apaf-1 CARD and procaspase-3, which play a major part in these signaling cascades. The caspase recruitment domain (CARD) of Apaf-1 (apoptotic protease activating factor) is the 97 amino acid N-terminal domain involved in protein-protein interactions, allowing for the activation of procaspase-9. Apaf-1 CARD consists of six antiparallel Î±-helices arranged in a Greek key topology. Single and sequential mixing stopped-flow studies showed that Apaf-1 CARD folds and unfolds rapidly and suggest a folding mechanism that contains parallel channels with two unfolded conformations folding to the native conformation. KINSIM simulations show that a slow folding phase is described by a third conformation in the unfolded ensemble that interconverts with one or both unfolded species. Overall, the native ensemble is formed rapidly upon refolding. Procaspase-3, like all other caspases, exists in the cell as an inactive zymogen and is the final step in the apoptotic pathway. Once activated it cleaves numerous substrates, leading to the dismantling of the cell. The refolding pathway of homodimeric procaspase-3 is complex, consisting of multiple monomeric intermediates with a slow rate of dimerization. The refolding and unfolding burst phase revealed multiple species formed within milliseconds of folding. Kinetic data support the hypothesis of two native conformations, one of which is enzymatically active. Collectively, these results demonstrate that dimerization is an important aspect in both folding and activation of procaspase-3. Overall, the kinetic folding data for Apaf-1 CARD and procaspase-3 provide an improved picture of the function and regulation of apoptosis. These studies propose new targets for therapeutic design to combat diseases associated with apoptosis.
Protein Labeling Strategies for Improving the Efficiency of Structure Determination by NMR
(2006-04-01) Rogers, Constance Ann; John Cavanagh, Committee Chair; William Miller, Committee Member; Michael Goshe, Committee Member
Rapid and efficient methods for preparing isotopically labeled recombinant proteins and refining solved structures via NMR are presented. The former approach was developed for 2H⁄13C⁄15N isotopic labeling and specific protonation of the methyl groups of isoleucine, leucine, and valine (ILV) residues of rat brain calbindin D28K, a calcium sensor and buffer. This protocol produces cell mass using unlabeled rich media followed by exchange into labeled media at high cell density. Allowing for a short period for growth recovery and unlabeled metabolite clearance, the cells were induced. Additionally, the solution structure of LuxU, a subunit of the quorum sensing circuit of Vibrio harveyi, has been refined using residual dipolar coupling (RDC). In slightly anisotropic environments, large one-bond internuclear dipolar interactions no longer average to zero, and therefore can provide information on the average orientation of the corresponding vectors relative to the magnetic field. This ordering was induced by introducing Pf1 filamentous bacteriophage into a solution of 13C15N isotopically labeled LuxU.
Structural and functional characterization of bacterial response and neuroprotective proteins
(2009-09-14) Hobbs, Carey Anne; John Cavanagh, Committee Chair; William Miller, Committee Member; David Bird, Committee Member; Dennis Brown, Committee Member; Kenneth Tomer, Committee Member
The studies described here involve the structural and functional characterization of two proteins, calbindin-D28k and ComAC. Calbindin-D28k is a calcium binding protein that plays a unique role in eukaryotic cells, acting as both a calcium buffer and sensor. Upon binding calcium, calbindin-D28k transitions from an ordered apo- state into a disordered state as calcium is bound. Once fully loaded with four calcium ions it again takes on an ordered state. The high resolution NMR solution structure of holo- calbindin-D28k has been solved. The structure of the apo- calbindin-D28k, however, remains to be solved. In the absence of a high resolution structure of apo- calbindin-D28k it has not been possible to compare the three-dimensional structure of the two states, making it impossible to analyze how this change affects the function of calbindin-D28k. This study uses differential surface modification analyzed by mass spectrometry to probe the tertiary structural changes that occur upon calcium binding. This comparison provides the first look at the conformational change of calbindin-D28k upon calcium binding. In the second study, the structure and function of the C-terminal DNA binding domain of ComA (ComAC) from Bacillus subtilis was investigated. ComA is a response regulator protein involved in the two-component signal transduction system regulating the development of genetic competence. The protein can be divided into two distinct domains, an N-terminal regulatory domain and a C-terminal effector domain. The C-terminal domain, ComAC, is a DNA binding domain responsible for binding to target operons, including the srfA operon. The transcriptional activation of the srfA operon ultimately controls the expression of the genes responsible for expressing the protein machinery that bind to and take up exogenous DNA. This research determined the high resolution solution structure of ComAC using NMR. This structure was then used in conjunction with SPR, NMR DNA titration studies and molecular modeling to analyze and determine how ComAC interacts with its cognate DNA promoter sequences.
Synchronization of Carbohydrate and Protein Metabolism by Ruminal Microbes in a Continuous Culture
(2002-11-25) Mohney, Kathryn Suzanne; Vivek Fellner, Committee Chair; William Miller, Committee Member; Jack Odle, Committee Co-Chair
A major factor in maximizing microbial protein synthesis is the availability of energy and protein in the diet. Our objective was to determine the effect of fermentable carbohydrate and protein on microbial fermentation. Diets were formulated using three ingredients, soybean meal (SBM), ground corn (GC) and soybean hulls (SBH). Corn and SBH were used in ratios of 60:20, 40:40 or 20:60, respectively to prepare high, medium or low non-fibrous carbohydrate (NFC) diets. Soybean meal was included either unextruded (control) or extruded at low, medium or high temperature. Degradability of the N fractions in the control, low, medium and high soybean meal were 97, 80, 80 and 60%, respectively. Diets were arranged as a 3 x 4 factorial (3 levels of corn/soybean hulls and 4 levels of protein) and analyzed as a completely randomized block design. There were no statistically significant interactions seen between NFC and protein sources. Total volatile fatty acids were affected (P<0.01) by the NFC with 78.5, 63.2 and 71.5 mM with increasing NFC levels. The NFC level affected the acetate and butyrate whereas the protein source had an effect on the propionate. Molar ratios of acetate, propionate and butyrate averaged 60.1, 31.0 and 6.79, respectively. Varying the level of fermentable carbohydrate had a negative linear affect on ruminal pH (P<0.01). Extrusion did not alter pH greatly P>0.67). Higher extrusion temperatures altered ammonia concentrations when compared to control or low extrusion. In the low NFC diets, the medium and high extrusion increased (P<0.10) ammonia concentration (29.8 and 32.6 mg/dl, respectively) when compared with control and the low (18.9 and 23.4 mg/dl, respectively). Methane concentration averaged 308 nmoles/ml and was affected by both the NFC treatment and protein source. The high and medium NFC diets increased (P<0.01) bacterial nitrogen percentage (9.0 and 9.5%, respectively) compared to the low NFC diet (8.7%). Data suggest that the fermentability of the structural carbohydrates in SBH was similar to the high starch corn diets. Furthermore, large differences in protein degradability did not seem to have a major impact on microbial fermentation.