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- Creators: Arizona State University
Description
The overall goal of this research project was to assess the feasibility of investigating the effects of microgravity on mineralization systems in unit gravity environments. If possible to perform these studies in unit gravity earth environments, such as earth, such systems can offer markedly less costly and more concerted research efforts to study these vitally important systems. Expected outcomes from easily accessible test environments and more tractable studies include the development of more advanced and adaptive material systems, including biological systems, particularly as humans ponder human exploration in deep space. The specific focus of the research was the design and development of a prototypical experimental test system that could preliminarily meet the challenging design specifications required of such test systems. Guided by a more unified theoretical foundation and building upon concept design and development heuristics, assessment of the feasibility of two experimental test systems was explored. Test System I was a rotating wall reactor experimental system that closely followed the specifications of a similar test system, Synthecon, designed by NASA contractors and thus closely mimicked microgravity conditions of the space shuttle and station. The latter includes terminal velocity conditions experienced by both innate material systems, as well as, biological systems, including living tissue and humans but has the ability to extend to include those material test systems associated with mineralization processes. Test System II is comprised of a unique vertical column design that offered more easily controlled fluid mechanical test conditions over a much wider flow regime that was necessary to achieving terminal velocities under free convection-less conditions that are important in mineralization processes. Preliminary results indicate that Test System II offers distinct advantages in studying microgravity effects in test systems operating in unit gravity environments and particularly when investigating mineralization and related processes. Verification of the Test System II was performed on validating microgravity effects on calcite mineralization processes reported earlier others. There studies were conducted on calcite mineralization in fixed-wing, reduced gravity aircraft, known as the `vomit comet' where reduced gravity conditions are include for very short (~20second) time periods. Preliminary results indicate that test systems, such as test system II, can be devised to assess microgravity conditions in unit gravity environments, such as earth. Furthermore, the preliminary data obtained on calcite formation suggest that strictly physicochemical mechanisms may be the dominant factors that control adaptation in materials processes, a theory first proposed by Liu et al. Thus the result of this study may also help shine a light on the problem of early osteoporosis in astronauts and long term interest in deep space exploration.
ContributorsSeyedmadani, Kimia (Author) / Pizziconi, Vincent (Thesis advisor) / Towe, Bruce (Committee member) / Alford, Terry (Committee member) / Arizona State University (Publisher)
Created2013
Description
Surface plasmon resonance (SPR) has emerged as a popular technique for elucidating subtle signals from biological events in a label-free, high throughput environment. The efficacy of conventional SPR sensors, whose signals are mass-sensitive, diminishes rapidly with the size of the observed target molecules. The following work advances the current SPR sensor paradigm for the purpose of small molecule detection. The detection limits of two orthogonal components of SPR measurement are targeted: speed and sensitivity. In the context of this report, speed refers to the dynamic range of measured kinetic rate constants, while sensitivity refers to the target molecule mass limitation of conventional SPR measurement. A simple device for high-speed microfluidic delivery of liquid samples to a sensor surface is presented to address the temporal limitations of conventional SPR measurement. The time scale of buffer/sample switching is on the order of milliseconds, thereby minimizing the opportunity for sample plug dispersion. The high rates of mass transport to and from the central microfluidic sensing region allow for SPR-based kinetic analysis of binding events with dissociation rate constants (kd) up to 130 s-1. The required sample volume is only 1 μL, allowing for minimal sample consumption during high-speed kinetic binding measurement. Charge-based detection of small molecules is demonstrated by plasmonic-based electrochemical impedance microscopy (P-EIM). The dependence of surface plasmon resonance (SPR) on surface charge density is used to detect small molecules (60-120 Da) printed on a dextran-modified sensor surface. The SPR response to an applied ac potential is a function of the surface charge density. This optical signal is comprised of a dc and an ac component, and is measured with high spatial resolution. The amplitude and phase of local surface impedance is provided by the ac component. The phase signal of the small molecules is a function of their charge status, which is manipulated by the pH of a solution. This technique is used to detect and distinguish small molecules based on their charge status, thereby circumventing the mass limitation (~100 Da) of conventional SPR measurement.
ContributorsMacGriff, Christopher Assiff (Author) / Tao, Nongjian (Thesis advisor) / Wang, Shaopeng (Committee member) / LaBaer, Joshua (Committee member) / Chae, Junseok (Committee member) / Arizona State University (Publisher)
Created2013
Description
Solution conformations and dynamics of proteins and protein-DNA complexes are often difficult to predict from their crystal structures. The crystal structure only shows a snapshot of the different conformations these biological molecules can have in solution. Multiple different conformations can exist in solution and potentially have more importance in the biological activity. DNA sliding clamps are a family of proteins with known crystal structures. These clamps encircle the DNA and enable other proteins to interact more efficiently with the DNA. Eukaryotic PCNA and prokaryotic β clamp are two of these clamps, some of the most stable homo-oligomers known. However, their solution stability and conformational equilibrium have not been investigated in depth before. Presented here are the studies involving two sliding clamps: yeast PCNA and bacterial β clamp. These studies show that the β clamp has a very different solution stability than PCNA. These conclusions were reached through various different fluorescence-based experiments, including fluorescence correlation spectroscopy (FCS), Förster resonance energy transfer (FRET), single molecule fluorescence, and various time resolved fluorescence techniques. Interpretations of these, and all other, fluorescence-based experiments are often affected by the properties of the fluorophores employed. Often the fluorescence properties of these fluorophores are influenced by their microenvironments. Fluorophores are known to sometimes interact with biological molecules, and this can have pronounced effects on the rotational mobility and photophysical properties of the dye. Misunderstanding the effect of these photophysical and rotational properties can lead to a misinterpretation of the obtained data. In this thesis, photophysical behaviors of various organic dyes were studied in the presence of deoxymononucleotides to examine more closely how interactions between fluorophores and DNA bases can affect fluorescent properties. Furthermore, the properties of cyanine dyes when bound to DNA and the effect of restricted rotation on FRET are presented in this thesis. This thesis involves studying fluorophore photophysics in various microenvironments and then expanding into the solution stability and dynamics of the DNA sliding clamps.
ContributorsRanjit, Suman (Author) / Levitus, Marcia (Thesis advisor) / Lindsay, Stuart (Committee member) / Yan, Hao (Committee member) / Arizona State University (Publisher)
Created2013
Description
Single molecule DNA Sequencing technology has been a hot research topic in the recent decades because it holds the promise to sequence a human genome in a fast and affordable way, which will eventually make personalized medicine possible. Single molecule differentiation and DNA translocation control are the two main challenges in all single molecule DNA sequencing methods. In this thesis, I will first introduce DNA sequencing technology development and its application, and then explain the performance and limitation of prior art in detail. Following that, I will show a single molecule DNA base differentiation result obtained in recognition tunneling experiments. Furthermore, I will explain the assembly of a nanofluidic platform for single strand DNA translocation, which holds the promised to be integrated into a single molecule DNA sequencing instrument for DNA translocation control. Taken together, my dissertation research demonstrated the potential of using recognition tunneling techniques to serve as a general readout system for single molecule DNA sequencing application.
ContributorsLiu, Hao (Author) / Lindsay, Stuart M (Committee member) / Yan, Hao (Committee member) / Levitus, Marcia (Committee member) / Arizona State University (Publisher)
Created2013
Description
The F1Fo ATP synthase is required for energy conversion in almost all living organisms. The F1 complex is a molecular motor that uses ATP hydrolysis to drive rotation of the γ–subunit. It has not been previously possible to resolve the speed and position of the γ–subunit of the F1–ATPase as it rotates during a power stroke. The single molecule experiments presented here measured light scattered from 45X91 nm gold nanorods attached to the γ–subunit that provide an unprecedented 5 μs resolution of rotational position as a function of time. The product of velocity and drag, which were both measured directly, resulted in an average torque of 63±8 pN nm for the Escherichia coli F1-ATPase that was determined to be independent of the load. The rotational velocity had an initial (I) acceleration phase 15° from the end of the catalytic dwell, a slow (S) acceleration phase during ATP binding/ADP release (15°–60°), and a fast (F) acceleration phase (60°–90°) containing an interim deceleration (ID) phase (75°–82°). High ADP concentrations decreased the velocity of the S phase proportional to 'ADP-release' dwells, and the F phase proportional to the free energy derived from the [ADP][Pi]/[ATP] chemical equilibrium. The decreased affinity for ITP increased ITP-binding dwells by 10%, but decreased velocity by 40% during the S phase. This is the first direct evidence that nucleotide binding contributes to F1–ATPase torque. Mutations that affect specific phases of rotation were identified, some in regions of F1 previously considered not to contribute to rotation. Mutations βD372V and γK9I increased the F phase velocity, and γK9I increased the depth of the ID phase. The conversion between S and F phases was specifically affected by γQ269L. While βT273D, βD305E, and αR283Q decreased the velocity of all phases, decreases in velocity due to βD302T, γR268L and γT82A were confined to the I and S phases. The correlations between the structural locations of these mutations and the phases of rotation they affect provide new insight into the molecular basis for F1–ATPase γ-subunit rotation.
ContributorsMartin, James (Author) / Frasch, Wayne D (Thesis advisor) / Chandler, Douglas (Committee member) / Gaxiola, Roberto (Committee member) / Yan, Hao (Committee member) / Arizona State University (Publisher)
Created2012
Description
Sensitivity is a fundamental challenge for in vivo molecular magnetic resonance imaging (MRI). Here, I improve the sensitivity of metal nanoparticle contrast agents by strategically incorporating pure and doped metal oxides in the nanoparticle core, forming a soluble, monodisperse, contrast agent with adjustable T2 or T1 relaxivity (r2 or r1). I first developed a simplified technique to incorporate iron oxides in apoferritin to form "magnetoferritin" for nM-level detection with T2- and T2* weighting. I then explored whether the crystal could be chemically modified to form a particle with high r1. I first adsorbed Mn2+ ions to metal binding sites in the apoferritin pores. The strategic placement of metal ions near sites of water exchange and within the crystal oxide enhance r1, suggesting a mechanism for increasing relaxivity in porous nanoparticle agents. However, the Mn2+ addition was only possible when the particle was simultaneously filled with an iron oxide, resulting in a particle with a high r1 but also a high r2 and making them undetectable with conventional T1-weighting techniques. To solve this problem and decrease the particle r2 for more sensitive detection, I chemically doped the nanoparticles with tungsten to form a disordered W-Fe oxide composite in the apoferritin core. This configuration formed a particle with a r1 of 4,870mM-1s-1 and r2 of 9,076mM-1s-1. These relaxivities allowed the detection of concentrations ranging from 20nM - 400nM in vivo, both passively injected and targeted to the kidney glomerulus. I further developed an MRI acquisition technique to distinguish particles based on r2/r1, and show that three nanoparticles of similar size can be distinguished in vitro and in vivo with MRI. This work forms the basis for a new, highly flexible inorganic approach to design nanoparticle contrast agents for molecular MRI.
ContributorsClavijo Jordan, Maria Veronica (Author) / Bennett, Kevin M (Thesis advisor) / Kodibagkar, Vikram (Committee member) / Sherry, A Dean (Committee member) / Wang, Xiao (Committee member) / Yarger, Jeffery (Committee member) / Arizona State University (Publisher)
Created2012
Description
Spider dragline silk is well known for its outstanding mechanical properties - a combination of strength and extensibility that makes it one of the toughest materials known. Two proteins, major ampullate spidroin 1 (MaSp1) and 2 (MaSp2), comprise dragline silk fibers. There has been considerable focus placed on understanding the source of spider silk's unique mechanical properties by investigating the protein composition, molecular structure and dynamics. Chemical compositional heterogeneity of spider silk fiber is critical to understand as it provides important information for the interactions between MaSp1 and MaSp2. Here, the amino acid composition of dragline silk protein was precisely determined using a solution-state nuclear magnetic resonance (NMR) approach on hydrolyzed silk fibers. In a similar fashion, solution-state NMR was applied to probe the "13"C/"15"N incorporation in silk, which is essential to understand for designing particular solid-state NMR methods for silk structural characterization. Solid-state NMR was used to elucidate silk protein molecular dynamics and the supercontraction mechanism. A "2"H-"13"C heteronuclear correlation (HETCOR) solid-state NMR technique was developed to extract site-specific "2"H quadrupole patterns and spin-lattice relaxation rates for understanding backbone and side-chain dynamics. Using this technique, molecular dynamics were determined for a number of repetitive motifs in silk proteins - Ala residing nanocrystalline &beta-sheet; domains, 3"1"-helical regions, and, Gly-Pro-Gly-XX &beta-turn; motifs. The protein backbone and side-chain dynamics of silk fibers in both dry and wet states reveal the impact of water on motifs with different secondary structures. Spider venom is comprised of a diverse range of molecules including salts, small organics, acylpolyamines, peptides and proteins. Neurotoxins are an important family of peptides in spider venom and have been shown to target and modulate various ion channels. The neurotoxins are Cys-rich and share an inhibitor Cys knot (ICK) fold. Here, the molecular structure of one G. rosea tarantula neurotoxin, GsAF2, was determined by solution-state NMR. In addition, the interaction between neurotoxins and model lipid bilayers was probed with solid-state NMR and negative-staining (NS) transmission electron microscopy (TEM). It is shown that the neurotoxins influence lipid bilayer assembly and morphology with the formation of nanodiscs, worm-like micelles and small vesicles.
ContributorsShi, Xiangyan (Author) / Yarger, Jeffery L (Thesis advisor) / Holland, Gregory P (Thesis advisor) / Levitus, Marcia (Committee member) / Marzke, Robert F (Committee member) / Arizona State University (Publisher)
Created2014
Description
This thesis explores a wide array of topics related to the protein folding problem, ranging from the folding mechanism, ab initio structure prediction and protein design, to the mechanism of protein functional evolution, using multi-scale approaches. To investigate the role of native topology on folding mechanism, the native topology is dissected into non-local and local contacts. The number of non-local contacts and non-local contact orders are both negatively correlated with folding rates, suggesting that the non-local contacts dominate the barrier-crossing process. However, local contact orders show positive correlation with folding rates, indicating the role of a diffusive search in the denatured basin. Additionally, the folding rate distribution of E. coli and Yeast proteomes are predicted from native topology. The distribution is fitted well by a diffusion-drift population model and also directly compared with experimentally measured half life. The results indicate that proteome folding kinetics is limited by protein half life. The crucial role of local contacts in protein folding is further explored by the simulations of WW domains using Zipping and Assembly Method. The correct formation of N-terminal β-turn turns out important for the folding of WW domains. A classification model based on contact probabilities of five critical local contacts is constructed to predict the foldability of WW domains with 81% accuracy. By introducing mutations to stabilize those critical local contacts, a new protein design approach is developed to re-design the unfoldable WW domains and make them foldable. After folding, proteins exhibit inherent conformational dynamics to be functional. Using molecular dynamics simulations in conjunction with Perturbation Response Scanning, it is demonstrated that the divergence of functions can occur through the modification of conformational dynamics within existing fold for β-lactmases and GFP-like proteins: i) the modern TEM-1 lactamase shows a comparatively rigid active-site region, likely reflecting adaptation for efficient degradation of a specific substrate, while the resurrected ancient lactamases indicate enhanced active-site flexibility, which likely allows for the binding and subsequent degradation of different antibiotic molecules; ii) the chromophore and attached peptides of photocoversion-competent GFP-like protein exhibits higher flexibility than the photocoversion-incompetent one, consistent with the evolution of photocoversion capacity.
ContributorsZou, Taisong (Author) / Ozkan, Sefika B (Thesis advisor) / Thorpe, Michael F (Committee member) / Woodbury, Neal W (Committee member) / Vaiana, Sara M (Committee member) / Ghirlanda, Giovanna (Committee member) / Arizona State University (Publisher)
Created2014
Biophysics, rockets, and the state: the making of a scientific discipline in twentieth-century China
Description
This study takes biophysics--a relatively new field with complex origins and contested definitions--as the research focus and investigates the history of disciplinary formation in twentieth-century China. The story of building a scientific discipline in modern China illustrates how a science specialty evolved from an ambiguous and amorphous field into a full-fledged academic discipline in specific socio-institutional contexts. It focuses on archival sources and historical writings concerning the constitution and definition of biophysics in order to examine the relationship between particular scientific styles, national priorities, and institutional opportunities in the People's Republic of China. It argues that Chinese biophysicists exhibited a different style of conceiving and organizing their discipline by adapting to the institutional structure and political economy that had been created since 1949. The eight chapters demonstrate that biophysics as a scientific discipline flourished in China only where priorities of science were congruent with political and institutional imperatives. Initially consisting of cell biologists, the Chinese biophysics community redirected their disciplinary priorities toward rocket science in the late 1950s to accommodate the national need of the time. Biophysicists who had worked on biological sounding rockets were drawn to the military sector and continued to contribute to human spaceflight in post-Mao China. Besides the rocket-and-space missions which provided the material context for biophysics to expand in the late 1950s and early 1960s, Chinese biophysicists also created research and educational programs surrounding biophysics by exploiting the institutional opportunities afforded by the policy emphasis on science's role to drive modernization. Biophysics' tie to nationalistic and utilitarian goals highlights the merits of approaching modern Chinese history from disciplinary, material, and institutional perspectives.
ContributorsLuk, Yi Lai Christine (Author) / Koblitz, Ann Hibner (Thesis advisor) / Maienschein, Jane A (Committee member) / Tillman, Hoyt C (Committee member) / Arizona State University (Publisher)
Created2014
Interactions driving the collapse of islet amyloid polypeptide: implications for amyloid aggregation
Description
Human islet amyloid polypeptide (hIAPP), also known as amylin, is a 37-residue intrinsically disordered hormone involved in glucose regulation and gastric emptying. The aggregation of hIAPP into amyloid fibrils is believed to play a causal role in type 2 diabetes. To date, not much is known about the monomeric state of hIAPP or how it undergoes an irreversible transformation from disordered peptide to insoluble aggregate. IAPP contains a highly conserved disulfide bond that restricts hIAPP(1-8) into a short ring-like structure: N_loop. Removal or chemical reduction of N_loop not only prevents cell response upon binding to the CGRP receptor, but also alters the mass per length distribution of hIAPP fibers and the kinetics of fibril formation. The mechanism by which N_loop affects hIAPP aggregation is not yet understood, but is important for rationalizing kinetics and developing potential inhibitors. By measuring end-to-end contact formation rates, Vaiana et al. showed that N_loop induces collapsed states in IAPP monomers, implying attractive interactions between N_loop and other regions of the disordered polypeptide chain . We show that in addition to being involved in intra-protein interactions, the N_loop is involved in inter-protein interactions, which lead to the formation of extremely long and stable β-turn fibers. These non-amyloid fibers are present in the 10 μM concentration range, under the same solution conditions in which hIAPP forms amyloid fibers. We discuss the effect of peptide cyclization on both intra- and inter-protein interactions, and its possible implications for aggregation. Our findings indicate a potential role of N_loop-N_loop interactions in hIAPP aggregation, which has not previously been explored. Though our findings suggest that N_loop plays an important role in the pathway of amyloid formation, other naturally occurring IAPP variants that contain this structural feature are incapable of forming amyloids. For example, hIAPP readily forms amyloid fibrils in vitro, whereas the rat variant (rIAPP), differing by six amino acids, does not. In addition to being highly soluble, rIAPP is an effective inhibitor of hIAPP fibril formation . Both of these properties have been attributed to rIAPP's three proline residues: A25P, S28P and S29P. Single proline mutants of hIAPP have also been shown to kinetically inhibit hIAPP fibril formation. Because of their intrinsic dihedral angle preferences, prolines are expected to affect conformational ensembles of intrinsically disordered proteins. The specific effect of proline substitutions on IAPP structure and dynamics has not yet been explored, as the detection of such properties is experimentally challenging due to the low molecular weight, fast reconfiguration times, and very low solubility of IAPP peptides. High-resolution techniques able to measure tertiary contact formations are needed to address this issue. We employ a nanosecond laser spectroscopy technique to measure end-to-end contact formation rates in IAPP mutants. We explore the proline substitutions in IAPP and quantify their effects in terms of intrinsic chain stiffness. We find that the three proline mutations found in rIAPP increase chain stiffness. Interestingly, we also find that residue R18 plays an important role in rIAPP's unique chain stiffness and, together with the proline residues, is a determinant for its non-amyloidogenic properties. We discuss the implications of our findings on the role of prolines in IDPs.
ContributorsCope, Stephanie M (Author) / Vaiana, Sara M (Thesis advisor) / Ghirlanda, Giovanna (Committee member) / Ros, Robert (Committee member) / Lindsay, Stuart M (Committee member) / Ozkan, Sefika B (Committee member) / Arizona State University (Publisher)
Created2013