Matching Items (55)
Description
Time-resolved serial femtosecond crystallography is an emerging method that allows for structural discovery to be performed on biomacromolecules during their dynamic trajectory through a reaction pathway after activation. This is performed by triggering a reaction on an ensemble of molecules in nano- or microcrystals and then using femtosecond X-ray laser pulses produced by an X-ray free electron laser to collect near-instantaneous data on the crystal. A full data set can be collected by merging a sufficient number of these patterns together and multiple data sets can be collected at different points along the reaction pathway by manipulating the delay time between reaction initiation and the probing X-rays. In this way, these ‘snapshot’ structures can be viewed in series to make a molecular movie, allowing for atomic visualization of a molecule in action and, thereby, a structural basis for the mechanism and function of a given biomacromolecule.
This dissertation presents results towards this end, including the successful implementations of the first diffusive mixing chemoactivated reactions and ultrafast dynamics in the femtosecond regime. The primary focus is on photosynthetic membrane proteins and enzymatic drug targets, in pursuit of strategies for sustainable energy and medical advancement by gaining understanding of the structure-function relationships evolved in nature. In particular, photosystem I, photosystem II, the complex of photosystem I and ferredoxin, and 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase are reported on, from purification and isolation, to crystallogenesis, to experimental design and data collection and subsequent interpretation of results and novel insights gained.
This dissertation presents results towards this end, including the successful implementations of the first diffusive mixing chemoactivated reactions and ultrafast dynamics in the femtosecond regime. The primary focus is on photosynthetic membrane proteins and enzymatic drug targets, in pursuit of strategies for sustainable energy and medical advancement by gaining understanding of the structure-function relationships evolved in nature. In particular, photosystem I, photosystem II, the complex of photosystem I and ferredoxin, and 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase are reported on, from purification and isolation, to crystallogenesis, to experimental design and data collection and subsequent interpretation of results and novel insights gained.
ContributorsCoe, Jesse (Author) / Fromme, Petra (Thesis advisor) / Sayres, Scott (Thesis advisor) / Mujica, Vladimiro (Committee member) / Redding, Kevin (Committee member) / Arizona State University (Publisher)
Created2018
Description
Over the last century, X-ray crystallography has been established as the most successful technique for unravelling the structure-function relationship in molecules. For integral membrane proteins, growing well-ordered large crystals is a challenge and hence, there is room for improving current methods of macromolecular crystallography and for exploring complimentary techniques. Since protein function is deeply associated with its structural dynamics, static position of atoms in a macromolecule are insufficient to unlock the mechanism.
The availability of X-ray free electron lasers presents an opportunity to study micron-sized crystals that could be triggered (using light, small molecules or physical conditions) to capture macromolecules in action. This method of ‘Time-resolved serial crystallography’ answers key biological questions by capturing snapshots of conformational changes associated with multi-step reactions. This dissertation describes approaches for studying structures of large membrane protein complexes. Both macro and micro-seeding techniques have been implemented for improving crystal quality and obtaining high-resolution structures. Well-diffracting 15-20 micron crystals of active Photosystem II were used to perform time-resolved studies with fixed-target Roadrunner sample delivery system. By employing continuous diffraction obtained up to 2 A, significant progress can be made towards understanding the process of water oxidation.
Structure of Photosystem I was solved to 2.3 A by X-ray crystallography and to medium resolution of 4.8 A using Cryogenic electron microscopy. Using complimentary techniques to study macromolecules provides an insight into differences among methods in structural biology. This helps in overcoming limitations of one specific technique and contributes in greater knowledge of the molecule under study.
The availability of X-ray free electron lasers presents an opportunity to study micron-sized crystals that could be triggered (using light, small molecules or physical conditions) to capture macromolecules in action. This method of ‘Time-resolved serial crystallography’ answers key biological questions by capturing snapshots of conformational changes associated with multi-step reactions. This dissertation describes approaches for studying structures of large membrane protein complexes. Both macro and micro-seeding techniques have been implemented for improving crystal quality and obtaining high-resolution structures. Well-diffracting 15-20 micron crystals of active Photosystem II were used to perform time-resolved studies with fixed-target Roadrunner sample delivery system. By employing continuous diffraction obtained up to 2 A, significant progress can be made towards understanding the process of water oxidation.
Structure of Photosystem I was solved to 2.3 A by X-ray crystallography and to medium resolution of 4.8 A using Cryogenic electron microscopy. Using complimentary techniques to study macromolecules provides an insight into differences among methods in structural biology. This helps in overcoming limitations of one specific technique and contributes in greater knowledge of the molecule under study.
ContributorsRoy Chowdhury, Shatabdi (Author) / Fromme, Petra (Thesis advisor) / Ros, Alexandra (Committee member) / Redding, Kevin (Committee member) / Arizona State University (Publisher)
Created2018
Description
In my thesis, I characterize multi-nuclear manganese cofactors in modified reaction
centers from the bacterium Rhodobacter sphaeroides. I characterized interactions
between a variety of secondary electron donors and modified reaction centers. In Chapter
1, I provide the research aims, background, and a summary of the chapters in my thesis.
In Chapter 2 and Chapter 3, I present my work with artificial four-helix bundles as
secondary electron donors to modified bacterial reaction centers. In Chapter 2, I
characterize the binding and energetics of the P1 Mn-protein, as a secondary electron
donor to modified reaction centers. In Chapter 3, I present the activity of a suite of four
helix bundles behaving as secondary electron donors to modified reaction centers. In
Chapter 4, I characterize a suite of modified reaction centers designed to bind and oxidize
manganese. I present work that characterizes bound manganese oxides as secondary
electron donors to the oxidized bacteriochlorophyll dimer in modified reaction centers. In
Chapter 5, I present my conclusions with a short description of future work in
characterizing multiple electron transfers from a multi-nuclear manganese cofactor in
modified reaction centers. To conclude, my thesis presents a characterization of a variety
of secondary electron donors to modified reaction centers that establish the feasibility to
characterize multiple turnovers from a multi-nuclear manganese cofactor.
centers from the bacterium Rhodobacter sphaeroides. I characterized interactions
between a variety of secondary electron donors and modified reaction centers. In Chapter
1, I provide the research aims, background, and a summary of the chapters in my thesis.
In Chapter 2 and Chapter 3, I present my work with artificial four-helix bundles as
secondary electron donors to modified bacterial reaction centers. In Chapter 2, I
characterize the binding and energetics of the P1 Mn-protein, as a secondary electron
donor to modified reaction centers. In Chapter 3, I present the activity of a suite of four
helix bundles behaving as secondary electron donors to modified reaction centers. In
Chapter 4, I characterize a suite of modified reaction centers designed to bind and oxidize
manganese. I present work that characterizes bound manganese oxides as secondary
electron donors to the oxidized bacteriochlorophyll dimer in modified reaction centers. In
Chapter 5, I present my conclusions with a short description of future work in
characterizing multiple electron transfers from a multi-nuclear manganese cofactor in
modified reaction centers. To conclude, my thesis presents a characterization of a variety
of secondary electron donors to modified reaction centers that establish the feasibility to
characterize multiple turnovers from a multi-nuclear manganese cofactor.
ContributorsEspiritu, Eduardo (Author) / Allen, James P. (Thesis advisor) / Jones, Anne K (Committee member) / Redding, Kevin (Committee member) / Arizona State University (Publisher)
Created2019
Description
Transient Receptor Potential (TRP) ion channels are a diverse family of nonselective, polymodal sensors in uni- and multicellular eukaryotes that are implicated in an assortment of biological contexts and human disease. The cold-activated TRP Melastatin-8 (TRPM8) channel, also recognized as the human body's primary cold sensor, is among the few TRP channels responsible for thermosensing. Despite sustained interest in the channel, the mechanisms underlying TRPM8 activation, modulation, and gating have proved challenging to study and remain poorly understood. In this thesis, I offer data collected on various expression, extraction, and purification conditions tested in E. Coli expression systems with the aim to optimize the generation of a structurally stable and functional human TRPM8 pore domain (S5 and S6) construct for application in structural biology studies. These studies, including the biophysical technique nuclear magnetic spectroscopy (NMR), among others, will be essential for elucidating the role of the TRPM8 pore domain in in regulating ligand binding, channel gating, ion selectively, and thermal sensitivity. Moreover, in the second half of this thesis, I discuss the ligation-independent megaprimer PCR of whole-plasmids (MEGAWHOP PCR) cloning technique, and how it was used to generate chimeras between TRPM8 and its nearest analog TRPM2. I review steps taken to optimize the efficiency of MEGAWHOP PCR and the implications and unique applications of this novel methodology for advancing recombinant DNA technology. I lastly present preliminary electrophysiological data on the chimeras, employed to isolate and study the functional contributions of each individual transmembrane helix (S1-S6) to TRPM8 menthol activation. These studies show the utility of the TRPM8\u2014TRPM2 chimeras for dissecting function of TRP channels. The average current traces analyzed thus far indicate that the S2 and S3 helices appear to play an important role in TRPM8 menthol modulation because the TRPM8[M2S2] and TRPM8[M2S3] chimeras significantly reduce channel conductance in the presence of menthol. The TRPM8[M2S4] chimera, oppositely, increases channel conductance, implying that the S4 helix in native TRPM8 may suppress menthol modulation. Overall, these findings show that there is promise in the techniques chosen to identify specific regions of TRPM8 crucial to menthol activation, though the methods chosen to study the TRPM8 pore independent from the whole channel may need to be reevaluated. Further experiments will be necessary to refine TRPM8 pore solubilization and purification before structural studies can proceed, and the electrophysiology traces observed for the chimeras will need to be further verified and evaluated for consistency and physiological significance.
ContributorsWaris, Maryam Siddika (Author) / Van Horn, Wade (Thesis director) / Redding, Kevin (Committee member) / School of Molecular Sciences (Contributor) / Department of English (Contributor) / Barrett, The Honors College (Contributor)
Created2016-05
Description
The objective of this randomized, single-blind crossover study was to examine the effect of vinegar on the blood glucose response to meal ingestion. This study was associated with a companion study Is Apple Cider Vinegar Effective for Reducing Heartburn Symptoms Related to Gastroesophageal Reflux Disease. Glucose meters were utilized to measure blood glucose levels immediately prior to, and at four ½ hour intervals following meal ingestion. Previous studies have demonstrated that vinegar modulates the meal-time glucose response. Hence an alternative hypothesis was used: that a significant difference will be observed between the control and the vinegar groups. The results from the study were not significant likely due to a small sample size. The test meal eaten with a drink composed of vinegar diluted in water appeared to be most effective at decreasing the overall change in postprandial blood glucose. The vinegar drink also played a role in decreasing the peak glucose level at 30 minutes post-meal.
ContributorsPadgitt-Cobb, Lillian Katelyn (Author) / Johnston, Carol (Thesis director) / Redding, Kevin (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2015-05
Description
Hydrogen has the potential to be a highly efficient fuel source. Its current production via steam reformation of natural gas, however, consumes a large amount of energy and gives off carbon dioxide. A newer method has since surfaced: using a microorganism's metabolism to drive hydrogen production. In this study, the conditions for maximum hydrogen production in Heliobacterium modesticaldum were identified and assessed. The cells were grown under varying conditions and their headspaces were sampled using a gas chromatogram to measure the amount of accumulated hydrogen during each condition. Two cell batches were grown under nitrogen-fixing conditions (-NH4+), while the other two cell batches were grown under non-nitrogen-fixing conditions (+NH4+). The headspaces were then exchanged with either nitrogen (N2) or argon (Ar2). It was found that the condition for which the most hydrogen was produced was when the cells were grown under nitrogen-fixing conditions and the headspace was exchanged with argon. These results suggest that most of Heliobacteria modesticaldum's hydrogen production is due to nitrogenase activity rather than hydrogenase activity. Further research is recommended to quantify the roles of nitrogenase, [NiFe] hydrogenase, and [FeFe] hydrogenase.
ContributorsMcmahon, Savanah Dior (Author) / Redding, Kevin (Thesis director) / Ghirlanda, Giovanna (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / School of Music (Contributor)
Created2015-05
Description
F2-isoprostanes are a series of prostaglandin-like compounds derived from the free radical-mediated lipid peroxidation of arachidonic acid, a polyunsaturated fatty acid that is ubiquitously expressed in cell membranes. F2-isoprostanes are biomarkers of oxidative stress, an imbalance between oxidants and antioxidants that can cause damage to DNA, proteins, lipids, and carbohydrates. Increased production of lipid peroxidation products have been implicated in the pathology of a number of conditions and diseases in humans. The objective of this thesis was to (1) optimize the LC/MS/MS F2-isoprostane method currently used in human samples for use in research animals and veterinary medicine, including the use of solid phase extraction, and (2) validate the optimized method in rodent and canine experimental studies. Our optimized method showed that Lyprinol treatment in dogs with osteoarthritis decreases F2-isoprostane levels nearly 2-fold. In addition, adjuvant alpha-tocopherol prevented tumor-induced increased F2-isoprostane levels. Finally, contrary to earlier studies using less specific ELISA F2-isoprostane methods, we demonstrate that unconditioned dogs benefit from low intensity exercise. Our data demonstrate successful optimization of the human LC/MS/MS F2-isoprostane method in rats and canines. Importantly, our results emphasize the need to use the more sensitive and specific LC/MS/MS method as compared to ELISA-based assays in order to distinguish the 15- and 5-series F2-isoprostanes, evidenced in particular by the two canine studies.
ContributorsCorrigan, Devin Connell (Author) / Redding, Kevin (Thesis director) / Anderson, Karen (Committee member) / Mustacich, Debbie (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2015-05
Description
ABSTRACT:
The experiment was conducted to analyze the role of menaquinone (MQ) in heliobacteria’s reaction center (HbRC). Their photosynthetic apparatus is a homodimeric of type I reaction center (1). HbRC contains these cofactors: P800 (special pair cholorphyll), A0 (8-hydroxy-chlorophyll [Chl] a), and FX (iron-sulfur cluster). The MQ factor is bypassed during the electron transfer process in HbRC. Electrons from the excited state of P800 (P800*) are transported to A0 and then directly to Fx. The hypothesis is that when electrons are photoaccumulated at Fx, and without the presence of any electron acceptors to the cluster, they would be transferred to MQ, and reduce it to MQH2 (quinol). Experiments conducted in the past with HbRC within the cell membranes yielded data that supported this hypothesis (Figures 4 and 5). We conducted a new experiment based on that foundation with HbRC, isolated from cell membrane. Two protein assays were prepared with cyt c553 and ascorbate in order to observe this phenomenon. The two samples were left in the glove box for several days for equilibration and then exposed to light in different intensity and periods. Their absorption was monitored at 800 nm for P800 or 554 nm for cyt c553 to observe their oxidation and reduction processes. The measurements were performed with the JTS-10 spectrophotometer. The data obtained from these experiments support the theory that P800+ reduced by the charge recombination of P800+Fx-. However, it did not confirm the reduction of P800+ done by cyt c553¬ which eventually lead to a net accumulation of oxidized cyt c553; instead it revealed another factor that could reduce P800+ faster and more efficient than cyt c553 (0.5 seconds vs several seconds), which could be MQ. More experiments need to be done in order to confirm this result. Hence, the data collected from this experiment have yet to support the theory of MQ being reduced to MQH2 outside the bacterial membranes.
The experiment was conducted to analyze the role of menaquinone (MQ) in heliobacteria’s reaction center (HbRC). Their photosynthetic apparatus is a homodimeric of type I reaction center (1). HbRC contains these cofactors: P800 (special pair cholorphyll), A0 (8-hydroxy-chlorophyll [Chl] a), and FX (iron-sulfur cluster). The MQ factor is bypassed during the electron transfer process in HbRC. Electrons from the excited state of P800 (P800*) are transported to A0 and then directly to Fx. The hypothesis is that when electrons are photoaccumulated at Fx, and without the presence of any electron acceptors to the cluster, they would be transferred to MQ, and reduce it to MQH2 (quinol). Experiments conducted in the past with HbRC within the cell membranes yielded data that supported this hypothesis (Figures 4 and 5). We conducted a new experiment based on that foundation with HbRC, isolated from cell membrane. Two protein assays were prepared with cyt c553 and ascorbate in order to observe this phenomenon. The two samples were left in the glove box for several days for equilibration and then exposed to light in different intensity and periods. Their absorption was monitored at 800 nm for P800 or 554 nm for cyt c553 to observe their oxidation and reduction processes. The measurements were performed with the JTS-10 spectrophotometer. The data obtained from these experiments support the theory that P800+ reduced by the charge recombination of P800+Fx-. However, it did not confirm the reduction of P800+ done by cyt c553¬ which eventually lead to a net accumulation of oxidized cyt c553; instead it revealed another factor that could reduce P800+ faster and more efficient than cyt c553 (0.5 seconds vs several seconds), which could be MQ. More experiments need to be done in order to confirm this result. Hence, the data collected from this experiment have yet to support the theory of MQ being reduced to MQH2 outside the bacterial membranes.
ContributorsNguyen, Phong Thien Huynh (Author) / Redding, Kevin (Thesis director) / Van Horn, Wade (Committee member) / Wachter, Rebekka (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2015-05
Description
Heliobacterium modesticaldum (H. modesticaldum) is an anaerobic photoheterotroph that can fix nitrogen (N2) and produce molecular hydrogen (H2). Recently, the Redding and Jones labs created a microbial photoelectrosynthesis cell that utilized these properties to produce molecular hydrogen using electrons provided by a cathode via a chemical mediator. Although this light-driven creation of fuel within a microbial electrochemical cell was the first of its kind, its production rate of hydrogen was low. It was hypothesized that the injection of electrons into H. modesticaldum was a rate-limiting step in H2 production. Within the H. modesticaldum genome, there is a gene (HM1_0653) that encodes a multi-heme cytochrome c that may be directly involved in this step. From past transcriptomic experiments, this gene is known to be very poorly expressed in H. modesticaldum. Our hypothesis was that increasing its expression with a strong promoter could result in faster electron transfer, and thus, increased H2 production in the photoelectrosynthesis cell. In order to test this hypothesis, different promoters that could lead to high expression in H. modesticaldum were included with a copy of HM1_0653 in various plasmid constructs that were first cloned into E. coli before being conjugated with H. modesticaldum. Cloning in E. coli was possible with the newly derived transformation system and by reducing the copy-number of the vector system. When overexpressed in E. coli, the protein appeared to be expressed, but its purification proved to be difficult. Moreover, conjugation with H. modesticaldum was not achieved. Our results are consistent with the idea that high level overexpression in H. modesticaldum was toxic. An inducible promoter may circumvent these issues and prove more successful in future experiments.
ContributorsSmith, Chelsea Elizabeth (Author) / Redding, Kevin (Thesis director) / Cadillo-Quiroz, Hinsby (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
Description
Photosynthesis is a critical process that fixes the carbon utilized in cellular respiration. In higher plants, the immutans gene codes for a protein that is both involved in carotenoid biosynthesis and plastoquinol oxidation (Carol et al 1999, Josse et al 2003). This plastoquinol terminal oxidase (PTOX) is of great interest in understanding electron flow in the plastoquinol pool. In order to characterize this PTOX, polyclonal antibodies were developed. Expression of Synechococcus WH8102 PTOX in E. coli provided a useful means to harvest the protein required for antibody production. Once developed, the antibody was tested for limit of concentration, effectiveness in whole cell lysate, and overall specificity. The antibody raised against PTOX was able to detect as low as 10 pg of PTOX in SDS-PAGE, and could detect PTOX extracted from lysed Synechococcus WH8102. The production of this antibody could determine the localization of the PTOX in Synechococcus.
ContributorsKhan, Mohammad Iqbal (Author) / Moore, Thomas (Thesis director) / Redding, Kevin (Committee member) / Roberson, Robert (Committee member) / Barrett, The Honors College (Contributor) / Department of Chemistry and Biochemistry (Contributor) / School of Life Sciences (Contributor)
Created2014-05