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Description
Water-soluble, adenosine triphosphate (ATP)-stabilized palladium nanoparticles have been synthesized by reduction of palladium salt in the presence of excess ATP. They have been characterized by electron microscopy, energy dispersive X-ray spectroscopy, ultraviolet-visible (UV-Vis) spectroscopy, and X-ray diffraction in order to determine particle size, shape, composition and crystal structure. The particles were then subsequently attached to a glassy carbon electrode (GCE) in order to explore their electrochemical properties with regard to hydrogen insertion in 1 M sodium hydroxide. The particles were found to be in the size range 2.5 to 4 nm with good size dispersion. The ATP capping ligand allowed the particles to be air-stable and re-dissolved without agglomeration. It was found that the NPs could be firmly attached to the working electrode via cycling the voltage repeatedly in a NP/phosphate solution. Further electrochemical experiments were conducted to investigate the adsorption and absorption of hydrogen in the NPs in 1 M sodium hydroxide. Results for cyclic voltammetry experiments were consistent with those for nanostructured and thin-film palladium in basic solution. Absorbed hydrogen content was analyzed as a function of potential. The maximum hydrogen:Pd ratio was found to be ~0.7, close the theoretical maximum value for β phase palladium hydride.
ContributorsLamb, Timothy (Author) / Buttry, Daniel A (Thesis advisor) / Yarger, Jeffery (Committee member) / Ros, Alexandra (Committee member) / Arizona State University (Publisher)
Created2013
Description
Gold-silver alloy nanoparticles (NPs) capped with adenosine 5'-triphosphate were synthesized by borohydride reduction of dilute aqueous metal precursors. High-resolution transmission electron microscopy showed the as-synthesized particles to be spherical with average diameters ~4 nm. Optical properties were measured by UV-Visible spectroscopy (UV-Vis), and the formation of alloy NPs was verified across all gold:silver ratios by a linear shift in the plasmon band maxima against alloy composition. The molar absorptivities of the NPs decreased non-linearly with increasing gold content from 2.0 x 108 M-1 cm-1 (fÉmax = 404 nm) for pure silver to 4.1 x 107 M-1 cm-1 (fÉmax = 511 nm) for pure gold. The NPs were immobilized onto transparent indium-tin oxide composite electrodes using layer-by-layer (LbL) deposition with poly(diallyldimethylammonium) acting as a cationic binder. The UV-Vis absorbance of the LbL film was used to calculate the surface coverage of alloy NPs on the electrode. Typical preparations had average NP surface coverages of 2.8 x 10-13 mol NPs/cm2 (~5% of cubic closest packing) with saturated films reaching ~20% of ccp for single-layer preparations (1.0 ~ 10-12 mol NPs/cm2). X-ray photoelectron spectroscopy confirmed the presence of alloy NPs in the LbL film and showed silver enrichment of the NP surfaces by ~9%. Irreversible oxidative dissolution (dealloying) of the less noble silver atoms from the NPs on LbL electrodes was performed by cyclic voltammetry (CV) in sulfuric acid. Alloy NPs with higher gold content required larger overpotentials for silver dealloying. Dealloying of the more-noble gold atoms from the alloy NPs was also achieved by CV in sodium chloride. The silver was oxidized first to cohesive silver chloride, and then gold dealloyed to soluble HAuCl4- at higher potentials. Silver oxidation was inhibited during the first oxidative scan, but subsequent cycles showed typical, reversible silver-to-silver chloride voltammetry. The potentials for both silver oxidation and gold dealloying also shifted to more oxidizing potentials with increasing gold content, and both processes converged for alloy NPs with >60% gold content. Charge-mediated electrochemistry of silver NPs immobilized in LbL films, using Fc(meOH) as the charge carrier, showed that 67% of the NPs were electrochemically inactive.
ContributorsStarr, Christopher A (Author) / Buttry, Daniel A (Thesis advisor) / Petuskey, William (Committee member) / Jones, Anne (Committee member) / Arizona State University (Publisher)
Created2014
Description
G protein coupled receptors (GPCRs) mediate various of physiologicalactivities which makes them significant drug targets. Determination of atomic level
structure of GPCRs facilitates the structure-based drug design. The most widely
used method currently for solving GPCR structure is still protein crystallography
especially lipidic cubic phase (LCP) crystallization. LCP could mimic the native
environment of membrane protein which stable the membrane proteins. Traditional
synchrotron source requires large size large size protein crystals (>30 micron) due to
the radiation damage during data collection. However, acquiring large sized protein
crystals is challenging and not guaranteed practically. In this study, a novel method
was developed which combined LCP technology and micro-electron diffraction
(MicroED) technology. LCP-MicroED technology was able to collect complete
diffraction data sets from serval submicron protein crystals and deliver high
resolution protein structures. This technology was first confirmed with soluble
protein crystals, proteinase K and small molecule crystals, cholesterol. Furthermore,
this novel method was applied to a human GPCR target, Î22- adrenergic receptor
(Î22AR). The structure model was successfully built which proved the feasibility of
applying LCP-MicroED method to GPCRs and other membrane proteins. Besides, in
this research, a novel human GPCR target, human histamine 4 receptor(H4R) was
studied. Different constructs were expressed, purified, and characterized. Some key
residuals that affect ligand binding were confirmed.
ContributorsJing, Liang (Author) / Mazor, Yuval (Thesis advisor) / Mills, Jeremy (Committee member) / Wang, Xu (Committee member) / Arizona State University (Publisher)
Created2022
Description
The integrin Mac-1 (αMβ2, CD11b/CD18) is an important adhesion receptorexpressed on macrophages and neutrophils. It plays a crucial role in phagocytosis, cell-cell
fusion, and cell migration. αMβ2 is also the most promiscuous integrin with over 100 known
ligands that span a broad range of physical and chemical attributes, many of which bind to
the inserted (I) domain from the αM subunit. The interaction of αMI-domain with cytokine
pleiotrophin (PTN) were determine. PTN is a cationic protein known to induce Mac-1-
mediated adhesion and migration in cells. The data showed that PTN’s interaction with
αMI-domain contains both divalent cation-dependent and independent mechanisms. In
particular, PTN’s N-terminal domain has weak interactions with the N/C-termini side of
αMI-domain using a metal-independent mechanism. However, stronger interaction is
achieved through the chelation of the divalent cation in the metal ion-dependent adhesion
site of active αMI-domain by PTN’s acidic residues. Although many acidic residues in PTN
can act as the chelator, active αMI-domain’s interaction with PTN’s E98 plays an especially
important role. NOE, chemical shift perturbation (CSP) data, and mutagenesis studies
showed residues near E98 are at the binding interface and the E98 mutation greatly reduced
binding affinity between two proteins. Interestingly, the CSP and MD simulation data
showed the binding interface can be supported by the interaction of PTN’s H95 with the
acidic clusters D242, E244, and D273 from αMI-domain, while PTN’s E66 form
electrostatic interaction with R208 and K245 from αMI-domain. The determined
recognition motif of αMI-domain for its ligands is (H/R/K)xxE. The ability to accommodate
the longer distance between E and (H, R, K) compared to the zwitterionic motif RGDii
explained how αMβ2 can interact with a large repertoire of ligands and be versatile in its
functional portfolio.
ContributorsNguyen, Hoa Thi Thanh (Author) / Wang, Xu (Thesis advisor) / Van Horn, Wade (Committee member) / Ugarova, Tatiana (Committee member) / Arizona State University (Publisher)
Created2024
Description
The physiological phenomenon of sensing temperature is detected by transient
receptor (TRP) ion channels, which are pore forming proteins that reside in the
membrane bilayer. The cold and hot sensing TRP channels named TRPV1 and TRPM8
respectively, can be modulated by diverse stimuli and are finely tuned by proteins and
lipids. PIRT (phosphoinositide interacting regulator of TRP channels) is a small
membrane protein that modifies TRPV1 responses to heat and TRPM8 responses to cold.
In this dissertation, the first direct measurements between PIRT and TRPM8 are
quantified with nuclear magnetic resonance and microscale thermophoresis. Using
Rosetta computational biology, TRPM8 is modeled with a regulatory, and functionally
essential, lipid named PIP2. Furthermore, a PIRT ligand screen identified several novel
small molecular binders for PIRT as well a protein named calmodulin. The ligand
screening results implicate PIRT in diverse physiological functions. Additionally, sparse
NMR data and state of the art Rosetta protocols were used to experimentally guide PIRT
structure predictions. Finally, the mechanism of thermosensing from the evolutionarily
conserved sensing domain of TRPV1 was investigated using NMR. The body of work
presented herein advances the understanding of thermosensing and TRP channel function
with TRP channel regulatory implications for PIRT.
receptor (TRP) ion channels, which are pore forming proteins that reside in the
membrane bilayer. The cold and hot sensing TRP channels named TRPV1 and TRPM8
respectively, can be modulated by diverse stimuli and are finely tuned by proteins and
lipids. PIRT (phosphoinositide interacting regulator of TRP channels) is a small
membrane protein that modifies TRPV1 responses to heat and TRPM8 responses to cold.
In this dissertation, the first direct measurements between PIRT and TRPM8 are
quantified with nuclear magnetic resonance and microscale thermophoresis. Using
Rosetta computational biology, TRPM8 is modeled with a regulatory, and functionally
essential, lipid named PIP2. Furthermore, a PIRT ligand screen identified several novel
small molecular binders for PIRT as well a protein named calmodulin. The ligand
screening results implicate PIRT in diverse physiological functions. Additionally, sparse
NMR data and state of the art Rosetta protocols were used to experimentally guide PIRT
structure predictions. Finally, the mechanism of thermosensing from the evolutionarily
conserved sensing domain of TRPV1 was investigated using NMR. The body of work
presented herein advances the understanding of thermosensing and TRP channel function
with TRP channel regulatory implications for PIRT.
ContributorsSisco, Nicholas John (Author) / Van Horn, Wade D (Thesis advisor) / Mills, Jeremy H (Committee member) / Wang, Xu (Committee member) / Yarger, Jeff L (Committee member) / Arizona State University (Publisher)
Created2018
Description
Glycosaminoglycans (GAGs) are long chains of negatively charged sulfated polysaccharides. They are often found to be covalently attached to proteins and form proteoglycans in the extracellular matrix (ECM). Many proteins bind GAGs through electrostatic interactions. GAG-binding proteins (GBPs) are involved in diverse physiological activities ranging from bacterial infections to cell-cell/cell-ECM contacts. This thesis is devoted to understanding how interactions between GBPs and their receptors modulate biological phenomena. Bacteria express GBPs on surface that facilitate dissemination and colonization by attaching to host ECM. The first GBP investigated in this thesis is decorin binding protein (DBP) found on the surface of Borrelia burgdorferi, causative pathogens in Lyme disease. DBPs bind GAGs of decorin, a proteoglycan in ECM. Of the two isoforms, DBPB is less studied than DBPA. In current work, structure of DBPB from B. burgdorferi and its GAG interactions were investigated using solution NMR techniques. DBPB adopts a five-helical structure, similar to DBPA. Despite similar GAG affinities, DBPB has its primary GAG-binding site on the lysine-rich C terminus, which is different from DBPA. Besides GAGs, GBPs in ECM also interact with cell surface receptors, such as integrins. Integrins belong to a big family of heterodimeric transmembrane proteins that receive extracellular cues and transmit signals bidirectionally to regulate cell adhesion, migration, growth and survival. The second part of this thesis focuses on αM I-domain of the promiscuous integrin αMβ2 (Mac-1 or CD11b/CD18) and explores the structural mechanism of αM I-domain interactions with pleiotrophin (PTN) and platelet factor 4 (PF4), which are cationic proteins with high GAG affinities. After completing the backbone assignment of αM I-domain, paramagnetic relaxation enhancement (PRE) experiments were performed to show that both PTN and PF4 bind αM I-domain using metal ion dependent adhesion site (MIDAS) in an Mg2+ independent way, which differs from the classical Mg2+ dependent mechanism used by all known integrin ligands thus far. In addition, NMR relaxation dispersion analysis revealed unique inherent conformational dynamics in αM I-domain centered around MIDAS and the crucial C-terminal helix. These dynamic motions are potentially functionally relevant and may explain the ligand promiscuity of the receptor, but requires further studies.
ContributorsFeng, Wei (Biologist) (Author) / Wang, Xu (Thesis advisor) / Yarger, Jeff L (Committee member) / Ghirlanda, Giovanna (Committee member) / Arizona State University (Publisher)
Created2019
Description
Cancer is a major public health challenge and the second leading cause of death in the United States. Large amount of effort has been made to achieve sensitive and specific detection of cancer, and to predict the course of cancer. Glycans are promising avenues toward the diagnosis and prognosis of cancer, because aberrant glycosylation is a prevalent hallmark of diverse types of cancer. A bottom-up “glycan node analysis” approach was employed as a useful tool, which captures most essential glycan features from blood plasma or serum (P/S) specimens and quantifies them as single analytical signals, to a lung cancer set from the Women Epidemiology Lung Cancer (WELCA) study. In addition, developments were performed to simplify a relatively cumbersome step involved in sample preparation of glycan node analysis. Furthermore, as a biomarker discovery research, one crucial concern of the glycan node analysis is to ensure that the specimen integrity has not been compromised for the employed P/S samples. A simple P/S integrity quality assurance assay was applied to the same sample set from WELCA study, which also afford the opportunity to evaluate the effects of different collection sites on sample integrity in a multisite clinical trial.
Here, 208 samples from lung cancer patients and 207 age-matched controls enrolled in the WELCA study were analyzed by glycan node analysis. Glycan features, quantified as single analytical signals, including 2-linked mannose, α2‐6 sialylation, β1‐4 branching, β1‐6 branching, 4-linked GlcNAc, and outer-arm fucosylation, exhibited abilities to distinguish lung cancer cases from controls and predict survival in patients.
To circumvent the laborious preparation steps for permethylation of glycan node analysis, a spin column-free (SCF) glycan permethylation procedure was developed, applicable to both intact glycan analysis or glycan node analysis, with improved or comparable permethylation efficiency relative to some widely-used spin column-based procedures.
Biospecimen integrity of the same set of plasma samples from WELCA study was evaluated by a simple intact protein assay (ΔS-Cysteinylated-Albumin), which quantifies cumulative exposure of P/S to thawed conditions (-30 °C). Notable differences were observed between different groups of samples with various initial handling/storage conditions, as well as among the different collection sites.
Here, 208 samples from lung cancer patients and 207 age-matched controls enrolled in the WELCA study were analyzed by glycan node analysis. Glycan features, quantified as single analytical signals, including 2-linked mannose, α2‐6 sialylation, β1‐4 branching, β1‐6 branching, 4-linked GlcNAc, and outer-arm fucosylation, exhibited abilities to distinguish lung cancer cases from controls and predict survival in patients.
To circumvent the laborious preparation steps for permethylation of glycan node analysis, a spin column-free (SCF) glycan permethylation procedure was developed, applicable to both intact glycan analysis or glycan node analysis, with improved or comparable permethylation efficiency relative to some widely-used spin column-based procedures.
Biospecimen integrity of the same set of plasma samples from WELCA study was evaluated by a simple intact protein assay (ΔS-Cysteinylated-Albumin), which quantifies cumulative exposure of P/S to thawed conditions (-30 °C). Notable differences were observed between different groups of samples with various initial handling/storage conditions, as well as among the different collection sites.
ContributorsHu, Yueming (Ph.D.) (Author) / Borges, Chad R (Thesis advisor) / Ros, Alexandra (Committee member) / Wang, Xu (Committee member) / Arizona State University (Publisher)
Created2019
Description
Glycosaminoglycans (GAGs) are a class of complex biomolecules comprised of linear, sulfated polysaccharides whose presence on cell surfaces and in the extracellular matrix involve them in many physiological phenomena as well as in interactions with pathogenic microbes. Decorin binding protein A (DBPA), a Borrelia surface lipoprotein involved in the infectivity of Lyme disease, is responsible for binding GAGs found on decorin, a small proteoglycan present in the extracellular matrix. Different DBPA strains have notable sequence heterogeneity that results in varying levels of GAG-binding affinity. In this dissertation, the structures and GAG-binding mechanisms for three strains of DBPA (B31 and N40 DBPAs from B. burgdorferi and PBr DBPA from B. garinii) are studied to determine why each strain has a different affinity for GAGs. These three strains have similar topologies consisting of five α-helices held together by a hydrophobic core as well as two long flexible segments: a linker between helices one and two and a C-terminal tail. This structural arrangement facilitates the formation of a basic pocket below the flexible linker which is the primary GAG-binding epitope. However, this GAG-binding site can be occluded by the flexible linker, which makes the linker a negative regulator of GAG-binding. ITC and NMR titrations provide KD values that show PBr DBPA binds GAGs with higher affinity than B31 and N40 DBPAs, while N40 binds with the lowest affinity of the three. Work in this thesis demonstrates that much of the discrepancies seen in GAG affinities of the three DBPAs can be explained by the amino acid composition and conformation of the linker. Mutagenesis studies show that B31 DBPA overcomes the pocket obstruction with the BXBB motif in its linker while PBr DBPA has a retracted linker that exposes the basic pocket as well as a secondary GAG-binding site. N40 DBPA, however, does not have any evolutionary modifications to its structure to enhance GAG binding which explains its lower affinity for GAGs. GMSA and ELISA assays, along with NMR PRE experiments, confirm that structural changes in the linker do affect GAG-binding and, as a result, the linker is responsible for regulating GAG affinity.
ContributorsMorgan, Ashli M (Author) / Wang, Xu (Thesis advisor) / Allen, James (Committee member) / Yarger, Jeffery (Committee member) / Arizona State University (Publisher)
Created2015
Description
Waste heat energy conversion remains an inviting subject for research, given the renewed emphasis on energy efficiency and carbon emissions reduction. Solid-state thermoelectric devices have been widely investigated, but their practical application remains challenging because of cost and the inability to fabricate them in geometries that are easily compatible with heat sources. An intriguing alternative to solid-state thermoelectric devices is thermogalvanic cells, which include a generally liquid electrolyte that permits the transport of ions. Thermogalvanic cells have long been known in the electrochemistry community, but have not received much attention from the thermal transport community. This is surprising given that their performance is highly dependent on controlling both thermal and mass (ionic) transport. This research will focus on a research project, which is an interdisciplinary collaboration between mechanical engineering (i.e. thermal transport) and chemistry, and is a largely experimental effort aimed at improving fundamental understanding of thermogalvanic systems. The first part will discuss how a simple utilization of natural convection within the cell doubles the maximum power output of the cell. In the second part of the research, some of the results from the previous part will be applied in a feasibility study of incorporating thermogalvanic waste heat recovery systems into automobiles. Finally, a new approach to enhance Seebeck coefficient by tuning the configurational entropy of a mixed-ligand complex formation of copper sulfate aqueous electrolytes will be presented. Ultimately, a summary of these results as well as possible future work that can be formed from these efforts is discussed.
ContributorsGunawan, Andrey (Author) / Phelan, Patrick E (Thesis advisor) / Buttry, Daniel A (Committee member) / Mujica, Vladimiro (Committee member) / Chan, Candace K. (Committee member) / Wang, Robert Y (Committee member) / Arizona State University (Publisher)
Created2015
Description
Deoxyribonucleic acid (DNA) has emerged as an attractive building material for creating complex architectures at the nanometer scale that simultaneously affords versatility and modularity. Particularly, the programmability of DNA enables the assembly of basic building units into increasingly complex, arbitrary shapes or patterns. With the expanding complexity and functionality of DNA toolboxes, a quantitative understanding of DNA self-assembly in terms of thermodynamics and kinetics, will provide researchers with more subtle design guidelines that facilitate more precise spatial and temporal control. This dissertation focuses on studying the physicochemical properties of DNA tile-based self-assembly process by recapitulating representative scenarios and intermediate states with unique assembly pathways.
First, DNA double-helical tiles with increasing flexibility were designed to investigate the dimerization kinetics. The higher dimerization rates of more rigid tiles result from the opposing effects of higher activation energies and higher pre-exponential factors from the Arrhenius equation, where the pre-exponential factor dominates. Next, the thermodynamics and kinetics of single tile attachment to preformed “multitile” arrays were investigated to test the fundamental assumptions of tile assembly models. The results offer experimental evidences that double crossover tile attachment is determined by the electrostatic environment and the steric hindrance at the binding site. Finally, the assembly of double crossover tiles within a rhombic DNA origami frame was employed as the model system to investigate the competition between unseeded, facet and seeded nucleation. The results revealed that preference of nucleation types can be tuned by controlling the rate-limiting nucleation step.
The works presented in this dissertation will be helpful for refining the DNA tile assembly model for future designs and simulations. Moreover, The works presented here could also be helpful in understanding how individual molecules interact and more complex cooperative bindings in chemistry and biology. The future direction will focus on the characterization of tile assembly at single molecule level and the development of error-free tile assembly systems.
First, DNA double-helical tiles with increasing flexibility were designed to investigate the dimerization kinetics. The higher dimerization rates of more rigid tiles result from the opposing effects of higher activation energies and higher pre-exponential factors from the Arrhenius equation, where the pre-exponential factor dominates. Next, the thermodynamics and kinetics of single tile attachment to preformed “multitile” arrays were investigated to test the fundamental assumptions of tile assembly models. The results offer experimental evidences that double crossover tile attachment is determined by the electrostatic environment and the steric hindrance at the binding site. Finally, the assembly of double crossover tiles within a rhombic DNA origami frame was employed as the model system to investigate the competition between unseeded, facet and seeded nucleation. The results revealed that preference of nucleation types can be tuned by controlling the rate-limiting nucleation step.
The works presented in this dissertation will be helpful for refining the DNA tile assembly model for future designs and simulations. Moreover, The works presented here could also be helpful in understanding how individual molecules interact and more complex cooperative bindings in chemistry and biology. The future direction will focus on the characterization of tile assembly at single molecule level and the development of error-free tile assembly systems.
ContributorsJiang, Shuoxing (Author) / Yan, Hao (Thesis advisor) / Liu, Yan (Thesis advisor) / Hayes, Mark (Committee member) / Wang, Xu (Committee member) / Arizona State University (Publisher)
Created2016