Matching Items (6)
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
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
Description
The photosynthetic reaction center is a type of pigment-protein complex found widely in photosynthetic bacteria, algae and higher plants. Its function is to convert the energy of sunlight into a chemical form that can be used to support other life processes. The high efficiency and structural simplicity make the bacterial reaction center a paradigm for studying electron transfer in biomolecules. This thesis starts with a comparison of the primary electron transfer process in the reaction centers from the Rhodobacter shperoides bacterium and those from its thermophilic homolog, Chloroflexus aurantiacus. Different temperature dependences in the primary electron transfer were found in these two type of reaction centers. Analyses of the structural differences between these two proteins suggested that the excess surface charged amino acids as well as a larger solvent exposure area in the Chloroflexus aurantiacus reaction center could explain the different temperature depenence. The conclusion from this work is that the electrostatic interaction potentially has a major effect on the electron transfer. Inspired by these results, a single point mutant was designed for Rhodobacter shperoides reaction centers by placing an ionizable amino acid in the protein interior to perturb the dielectrics. The ionizable group in the mutation site largely deprotonated in the ground state judging from the cofactor absorption spectra as a function of pH. By contrast, a fast charge recombination assoicated with protein dielectric relaxation was observed in this mutant, suggesting the possibility that dynamic protonation/deprotonation may be taking place during the electron transfer. The fast protein dielectric relaxation occuring in this mutant complicates the electron transfer pathway and reduces the yield of electron transfer to QA. Considering the importance of the protein dielectric environment, efforts have been made in quantifying variations of the internal field during charge separation. An analysis protocol based on the Stark effect of reaction center cofactor spectra during charge separation has been developed to characterize the charge-separated radical field acting on probe chromophores. The field change, monitored by the dynamic Stark shift, correlates with, but is not identical to, the electron transfer kinetics. The dynamic Stark shift results have lead to a dynamic model for the time-dependent dielectric that is complementary to the static dielectric asymmetry observed in past steady state experiments. Taken together, the work in this thesis emphasizes the importance of protein electrostatics and its dielectric response to electron transfer.
ContributorsGuo, Zhi (Author) / Woodbury, Neal W (Thesis advisor) / Lindsay, Stuart M (Committee member) / Ross, Robert (Committee member) / Ozkan, Banu S (Committee member) / Moore, Thomas A. (Committee member) / Arizona State University (Publisher)
Created2012
Description
Richard Feynman said “There’s plenty of room at the bottom”. This inspired the techniques to improve the single molecule measurements. Since the first single molecule study was in 1961, it has been developed in various field and evolved into powerful tools to understand chemical and biological property of molecules. This thesis demonstrates electronic single molecule measurement with Scanning Tunneling Microscopy (STM) and two of applications of STM; Break Junction (BJ) and Recognition Tunneling (RT). First, the two series of carotenoid molecules with four different substituents were investigated to show how substituents relate to the conductance and molecular structure. The measured conductance by STM-BJ shows that Nitrogen induces molecular twist of phenyl distal substituents and conductivity increasing rather than Carbon. Also, the conductivity is adjustable by replacing the sort of residues at phenyl substituents. Next, amino acids and peptides were identified through STM-RT. The distribution of the intuitive features (such as amplitude or width) are mostly overlapped and gives only a little bit higher separation probability than random separation. By generating some features in frequency and cepstrum domain, the classification accuracy was dramatically increased. Because of large data size and many features, supporting vector machine (machine learning algorithm for big data) was used to identify the analyte from a data pool of all analytes RT data. The STM-RT opens a possibility of molecular sequencing in single molecule level. Similarly, carbohydrates were studied by STM-RT. Carbohydrates are difficult to read the sequence, due to their huge number of possible isomeric configurations. This study shows that STM-RT can identify not only isomers of mono-saccharides and disaccharides, but also various mono-saccharides from a data pool of eleven analytes. In addition, the binding affinity between recognition molecule and analyte was investigated by comparing with surface plasmon resonance. In present, the RT technique is applying to chip type sequencing device onto solid-state nanopore to read out glycosaminoglycans which is ubiquitous to all mammalian cells and controls biological activities.
ContributorsIm, Jong One (Author) / Lindsay, Stuart M (Thesis advisor) / Zhang, Peiming (Committee member) / Ros, Robert (Committee member) / Chamberlin, Ralph (Committee member) / Arizona State University (Publisher)
Created2016
Description
This dissertation describes work on three projects concerning the design and implementation of instrumentation used to study potential organic electronic devices. The first section describes the conducting atomic force microscope (CAFM) in the study of the mechanical and electronic interactions between DNA bases and nucleosides. Previous STM data suggested that an STM tip could recognize single base pairs through an electronic interaction after a functionalized tip made contact with a self assembled monolayer then was retracted. The conducting AFM was employed in order to understand the mechanical interactions of such a system and how they were affecting electrical responses. The results from the conducting AFM showed that the scanning probe system was measuring multiple base-pair interactions, and thus did not have single base resolution. Further, results showed that the conductance between a single base-nucleoside pair is below the detection limit of a potential commercial sequencing device. The second section describes the modifications of a scanning probe microscope in order to study the conductance of single organic molecules under illumination. Modifications to the scanning probe microscope are described as are the control and data analysis software for an experiment testing the single molecule conductance of an organic molecule under illumination. This instrument was then tested using a novel charge-separation molecule, which is being considered for its potential photovoltaic properties. The experiments showed that the instrumentation is capable of detecting differences in conductance upon laser illumination of the molecule on a transparent conductive surface. The third section describes measurements using the illuminated CAFM, as well as the design and construction of an illuminated mercury drop electrode apparatus. Both instruments were tested by attempting to observe photovoltaic behavior in a novel self-organized film of the charge-separation molecules mentioned in the previous paragraph. Results and calculations show that the conducting AFM is not a useful tool in the examination of these organic photovoltaics, while the mercury drop apparatus measured photovoltaic effects in the film. Although photovoltaic effects were measurable with the mercury drop electrode, it was found that the film exhibited very low photon-to-electron conversion efficiency (IPCE).
ContributorsKibel, Ashley Ann (Author) / Lindsay, Stuart M (Thesis advisor) / Chamberlin, Ralph (Committee member) / Moore, Thomas (Committee member) / Ozkan, Sefika (Committee member) / Sankey, Otto (Committee member) / Arizona State University (Publisher)
Created2010
Description
In eukaryotes, DNA is packed in a highly condensed and hierarchically organized structure called chromatin, in which DNA tightly wraps around the histone octamer consisting of one histone 3-histone 4 (H3-H4) tetramer and two histone 2A- histone 2B (H2A-H2B) dimers with 147 base pairs in an almost two left handed turns. Almost all DNA dependent cellular processes, such as DNA duplication, transcription, DNA repair and recombination, take place in the chromatin form. Based on the critical importance of appropriate chromatin condensation, this thesis focused on the folding behavior of the nucleosome array reconstituted using different templates with various controllable factors such as histone tail modification, linker DNA length, and DNA binding proteins. Firstly, the folding behaviors of wild type (WT) and nucleosome arrays reconstituted with acetylation on the histone H4 at lysine 16 (H4K16 (Ac)) were studied. In contrast to the sedimentation result, atomic force microscopy (AFM) measurements revealed no apparent difference in the compact nucleosome arrays between WT and H4K16 (Ac) and WT. Instead, an optimal loading of nucleosome along the template was found necessary for the Mg2+ induced nucleosome array compaction. This finding leads to the further study on the role of linker DNA in the nucleosome compaction. A method of constructing DNA templates with varied linker DNA lengths was developed, and uniformly and randomly spaced nucleosome arrays with average linker DNA lengths of 30 bp and 60 bp were constructed. After comprehensive analyses of the nucleosome arrays' structure in mica surface, the lengths of the linker DNA were found playing an important role in controlling the structural geometries of nucleosome arrays in both their extended and compact forms. In addition, higher concentration of the DNA binding domain of the telomere repeat factor 2 (TRF2) was found to stimulate the compaction of the telomeric nucleosome array. Finally, AFM was successfully applied to investigate the nucleosome positioning behaviors on the Mouse Mammary Tumor Virus (MMTV) promoter region, and two highly positioned region corresponded to nucleosome A and B were identified by this method.
ContributorsFu, Qiang (Author) / Lindsay, Stuart M (Thesis advisor) / Yan, Hao (Committee member) / Ghirlanda, Giovanna (Committee member) / Arizona State University (Publisher)
Created2010