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ASU Electronic Theses and Dissertations


This collection includes most of the ASU Theses and Dissertations from 2011 to present. ASU Theses and Dissertations are available in downloadable PDF format; however, a small percentage of items are under embargo. Information about the dissertations/theses includes degree information, committee members, an abstract, supporting data or media.

In addition to the electronic theses found in the ASU Digital Repository, ASU Theses and Dissertations can be found in the ASU Library Catalog.

Dissertations and Theses granted by Arizona State University are archived and made available through a joint effort of the ASU Graduate College and the ASU Libraries. For more information or questions about this collection contact or visit the Digital Repository ETD Library Guide or contact the ASU Graduate College at gradformat@asu.edu.


Resource Type
Subject
Date Range
2010 2019


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 …

Contributors
Fu, Qiang, Lindsay, Stuart M, Yan, Hao, et al.
Created Date
2010

A novel small metal-binding protein (SmbP), with only 93 residues and no similarity to other known proteins, has been isolated from the periplasm of Nitrosomonas europaea. It is characterized by its high percentage (17%) of histidines, a motif of ten repeats of seven residues, a four α-helix bundle structure, and a high binding affinity to about six equivalents of Cu2+. The goal of this study is to investigate the Cu2+ binding sites in SmbP and to understand how Cu2+ stabilizes the protein. Preliminary folding experiments indicated that Cu2+ greatly stabilizes SmbP. In this study, protein folding data from circular dichroism …

Contributors
Yan, Qin, Francisco, Wilson A, Allen, James, et al.
Created Date
2010

In oxygenic photosynthesis, Photosystem I (PSI) and Photosystem II (PSII) are two transmembrane protein complexes that catalyze the main step of energy conversion; the light induced charge separation that drives an electron transfer reaction across the thylakoid membrane. Current knowledge of the structure of PSI and PSII is based on three structures: PSI and PSII from the thermophilic cyanobacterium Thermosynechococcus elonagatus and the PSI/light harvesting complex I (PSI-LHCI) of the plant, Pisum sativum. To improve the knowledge of these important membrane protein complexes from a wider spectrum of photosynthetic organisms, photosynthetic apparatus of the thermo-acidophilic red alga, Galdieria sulphuraria and …

Contributors
Thangaraj, Balakumar, Fromme, Petra, Shock, Everett, et al.
Created Date
2010

The metalloenzyme quercetin 2,3-dioxygenase (QueD) catalyzes the oxidative decomposition of the aromatic compound, quercetin. The most recently characterized example is a product of the bacterium Bacillus subtilis (BsQueD); all previous examples were fungal enzymes from the genus Aspergillus (AQueD). AQueD contains a single atom of Cu(II) per monomer. However, BsQueD, over expressed in Escherichia coli, contains Mn(II) and has two metal-binding sites, and therefore two possible active sites per monomer. To understand the contribution of each site to BsQueD's activity, the N-terminal and C-terminal metal-binding sites have been mutated individually in an effort to disrupt metal binding. In wild type …

Contributors
Bowen, Sara, Francisco, Wilson A, Allen, James, et al.
Created Date
2010

Enzymes which regulate the metabolic reactions for sustaining all living things, are the engines of life. The discovery of molecules that are able to control enzyme activity is of great interest for therapeutics and the biocatalysis industry. Peptides are promising enzyme modulators due to their large chemical diversity and the existence of well-established methods for library synthesis. Microarrays represent a powerful tool for screening thousands of molecules, on a small chip, for candidates that interact with enzymes and modulate their functions. In this work, a method is presented for screening high-density arrays to discover peptides that bind and modulate enzyme …

Contributors
Fu, Jinglin, Woodbury, Neal W, Johnston, Stephen A, et al.
Created Date
2010

Protein crystallization has become an extremely important tool in biochemistry since the first structure of the protein Myoglobin was solved in 1958. Survival of motor neuron protein has proved to be an elusive target in regards to producing crystals of sufficient quality for X-ray diffraction. One form of Survival of motor neuron protein has been found to be a cause of the disease Spinal Muscular Atrophy that currently affects 1 in 6000 live births. The production, purification and crystallization of Survival of motor neuron protein are detailed. The Fenna-Matthews-Olson (FMO) protein from Pelodictyon phaeum is responsible for the transfer of …

Contributors
Larson, Chadwick Robert, Allen, James P, Francisco, Wilson, et al.
Created Date
2010

In an effort to begin validating the large number of discovered candidate biomarkers, proteomics is beginning to shift from shotgun proteomic experiments towards targeted proteomic approaches that provide solutions to automation and economic concerns. Such approaches to validate biomarkers necessitate the mass spectrometric analysis of hundreds to thousands of human samples. As this takes place, a serendipitous opportunity has become evident. By the virtue that as one narrows the focus towards "single" protein targets (instead of entire proteomes) using pan-antibody-based enrichment techniques, a discovery science has emerged, so to speak. This is due to the largely unknown context in which …

Contributors
Oran, Paul, Nelson, Randall, Hayes, Mark, et al.
Created Date
2011

A major goal of synthetic biology is to recapitulate emergent properties of life. Despite a significant body of work, a longstanding question that remains to be answered is how such a complex system arose? In this dissertation, synthetic nucleic acid molecules with alternative sugar-phosphate backbones were investigated as potential ancestors of DNA and RNA. Threose nucleic acid (TNA) is capable of forming stable helical structures with complementary strands of itself and RNA. This provides a plausible mechanism for genetic information transfer between TNA and RNA. Therefore TNA has been proposed as a potential RNA progenitor. Using molecular evolution, functional sequences …

Contributors
Zhang, Su, Chaut, John C, Ghirlanda, Giovanna, et al.
Created Date
2011

Telomerase ribonucleoprotein is a unique reverse transcriptase that adds telomeric DNA repeats to chromosome ends. Telomerase RNA (TER) is extremely divergent in size, sequence and has to date only been identified in vertebrate, yeast, ciliate and plant species. Herein, the identification and characterization of TERs from an evolutionarily distinct group, filamentous fungi, is presented. Based on phylogenetic analysis of 69 TER sequences and mutagenesis analysis of in vitro reconstituted Neurospora telomerase, we discovered a conserved functional core in filamentous fungal TERs sharing homologous structural features with vertebrate TERs. This core contains the template-pseudoknot and P6/P6.1 domains, essential for enzymatic activity, …

Contributors
Qi, Xiaodong, Chen, Julian, Ghirlanda, Giovanna, et al.
Created Date
2011

The cyanobacterium Synechocystis sp. PCC 6803 performs oxygenic photosynthesis. Light energy conversion in photosynthesis takes place in photosystem I (PSI) and photosystem II (PSII) that contain chlorophyll, which absorbs light energy that is utilized as a driving force for photosynthesis. However, excess light energy may lead to formation of reactive oxygen species that cause damage to photosynthetic complexes, which subsequently need repair or replacement. To gain insight in the degradation/biogenesis dynamics of the photosystems, the lifetimes of photosynthetic proteins and chlorophyll were determined by a combined stable-isotope (15N) and mass spectrometry method. The lifetimes of PSII and PSI proteins ranged …

Contributors
Yao, Cheng I Daniel, Vermaas, Wim, Fromme, Petra, et al.
Created Date
2011

Telomerase is a specialized enzyme that adds telomeric DNA repeats to the chromosome ends to counterbalance the progressive telomere shortening over cell divisions. It has two essential core components, a catalytic telomerase reverse transcriptase protein (TERT), and a telomerase RNA (TR). TERT synthesizes telomeric DNA by reverse transcribing a short template sequence in TR. Unlike TERT, TR is extremely divergent in size, sequence and structure and has only been identified in three evolutionarily distant groups. The lack of knowledge on TR from important model organisms has been a roadblock for vigorous studies on telomerase regulation. To address this issue, a …

Contributors
Li, Yang, Chen, Julian Jl, Yan, Hao, et al.
Created Date
2011

ATP synthase is a large multimeric protein complex responsible for generating the energy molecule adenosine triphosphate (ATP) in most organisms. The catalysis involves the rotation of a ring of c-subunits, which is driven by the transmembrane electrochemical gradient. This dissertation reports how the eukaryotic c-subunit from spinach chloroplast ATP synthase has successfully been expressed in Escherichia coli and purified in mg quantities by incorporating a unique combination of methods. Expression was accomplished using a codon optimized gene for the c-subunit, and it was expressed as an attachment to the larger, more soluble, native maltose binding protein (MBP-c1). The fusion protein …

Contributors
Lawrence, Robert Michael, Fromme, Petra, Chen, Julian J.L., et al.
Created Date
2011

In the 1970s James Watson recognized the inability of conventional DNA replication machinery to replicate the extreme termini of chromosomes known as telomeres. This inability is due to the requirement of a building block primer and was termed the end replication problem. Telomerase is nature's answer to the end replication problem. Telomerase is a ribonucleoprotein which extends telomeres through reverse transcriptase activity by reiteratively copying a short intrinsic RNA sequence to generate 3' telomeric extensions. Telomeres protect chromosomes from erosion of coding genes during replication, as well as differentiate native chromosome ends from double stranded breaks. However, controlled erosion of …

Contributors
Bley, Christopher James, Chen, Julian, Allen, James, et al.
Created Date
2011

Like most other phototrophic organisms the cyanobacterium Synechocystis sp. PCC 6803 produces carotenoids. These pigments often bind to proteins and assume various functions in light harvesting, protection from reactive oxygen species (ROS) and protein stabilization. One hypothesis was that carotenoids bind to the surface (S-)layer protein. In this work the Synechocystis S-layer protein was identified as Sll1951 and the effect on the carotenoid composition of this prokaryote by disruption of sll1951 was studied. Loss of the S-layer, which was demonstrated by electron microscopy, did not result in loss of carotenoids or changes in the carotenoid profile of the mutant, which …

Contributors
Trautner, Christoph, Vermaas, Willem Fj, Chandler, Douglas E, et al.
Created Date
2011

Conformational changes in biomolecules often take place on longer timescales than are easily accessible with unbiased molecular dynamics simulations, necessitating the use of enhanced sampling techniques, such as adaptive umbrella sampling. In this technique, the conformational free energy is calculated in terms of a designated set of reaction coordinates. At the same time, estimates of this free energy are subtracted from the potential energy in order to remove free energy barriers and cause conformational changes to take place more rapidly. This dissertation presents applications of adaptive umbrella sampling to a variety of biomolecular systems. The first study investigated the effects …

Contributors
Spiriti, Justin Matthew, Van Der Vaart, Arjan, Chizmeshya, Andrew, et al.
Created Date
2011

Molecular dynamics (MD) simulations provide a particularly useful approach to understanding conformational change in biomolecular systems. MD simulations provide an atomistic, physics-based description of the motions accessible to biomolecular systems on the pico- to micro-second timescale, yielding important insight into the free energy of the system, the dynamical stability of contacts and the role of correlated motions in directing the motions of the system. In this thesis, I use molecular dynamics simulations to provide molecular mechanisms that rationalize structural, thermodynamic, and mutation data on the interactions between the lac repressor headpiece and its O1 operator DNA as well as the …

Contributors
Barr, Daniel, Van Der Vaart, Arjan, Matyushov, Dmitry, et al.
Created Date
2011

The elaborate signals of animals are often costly to produce and maintain, thus communicating reliable information about the quality of an individual to potential mates or competitors. The properties of the sensory systems that receive signals can drive the evolution of these signals and shape their form and function. However, relatively little is known about the ecological and physiological constraints that may influence the development and maintenance of sensory systems. In the house finch (Carpodacus mexicanus) and many other bird species, carotenoid pigments are used to create colorful sexually selected displays, and their expression is limited by health and dietary …

Contributors
Toomey, Matthew B, Mcgraw, Kevin J, Deviche, Pierre, et al.
Created Date
2011

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, …

Contributors
Martin Ii, James Leo, Frasch, Wayne D, Chandler, Douglas, et al.
Created Date
2012

The need for a renewable and sustainable light-driven energy source is the motivation for this work, which utilizes a challenging, yet practical and attainable bio-inspired approach to develop an artificial oxygen evolving complex, which builds upon the principles of the natural water splitting mechanism in oxygenic photosynthesis. In this work, a stable framework consisting of a three-dimensional DNA tetrahedron has been used for the design of a bio-mimic of the Oxygen-Evolving Complex (OEC) found in natural Photosystem II (PSII). PSII is a large protein complex that evolves all the oxygen in the atmosphere, but it cannot be used directly in …

Contributors
Rendek, Kim, Fromme, Petra, Chen, Julian, et al.
Created Date
2012

DNA nanotechnology has been a rapidly growing research field in the recent decades, and there have been extensive efforts to construct various types of highly programmable and robust DNA nanostructures. Due to the advantage that DNA nanostructure can be used to organize biochemical molecules with precisely controlled spatial resolution, herein we used DNA nanostructure as a scaffold for biological applications. Targeted cell-cell interaction was reconstituted through a DNA scaffolded multivalent bispecific aptamer, which may lead to promising potentials in tumor therapeutics. In addition a synthetic vaccine was constructed using DNA nanostructure as a platform to assemble both model antigen and …

Contributors
Liu, Xiaowei, Liu, Yan, Chang, Yung, et al.
Created Date
2012

Deoxyribonucleic acid (DNA), a biopolymer well known for its role in preserving genetic information in biology, is now drawing great deal of interest from material scientists. Ease of synthesis, predictable molecular recognition via Watson-Crick base pairing, vast numbers of available chemical modifications, and intrinsic nanoscale size makes DNA a suitable material for the construction of a plethora of nanostructures that can be used as scaffold to organize functional molecules with nanometer precision. This dissertation focuses on DNA-directed organization of metallic nanoparticles into well-defined, discrete structures and using them to study photonic interaction between fluorophore and metal particle. Presented here are …

Contributors
Pal, Suchetan, Liu, Yan, Yan, Hao, et al.
Created Date
2012

Spinal muscular atrophy (SMA) is a neurodegenerative disease that results in the loss of lower body muscle function. SMA is the second leading genetic cause of death in infants and arises from the loss of the Survival of Motor Neuron (SMN) protein. SMN is produced by two genes, smn1 and smn2, that are identical with the exception of a C to T conversion in exon 7 of the smn2 gene. SMA patients lacking the smn1 gene, rely on smn2 for production of SMN. Due to an alternative splicing event, smn2 primarily encodes a non-functional SMN lacking exon 7 (SMN D7) …

Contributors
Niday, Tracy Christina, Allen, James P, Wachter, Rebekka, et al.
Created Date
2012

In somatic cells, the mitotic spindle apparatus is centrosomal and several isoforms of Protein Kinase C (PKC) have been associated with the mitotic spindle, but their role in stabilizing the mitotic spindle is unclear. Other protein kinases such as, Glycogen Synthase Kinase 3â (GSK3â) also have been shown to be associated with the mitotic spindle. In the study in chapter 2, we show the enrichment of active (phosphorylated) PKCæ at the centrosomal region of the spindle apparatus in metaphase stage of 3T3 cells. In order to understand whether the two kinases, PKC and GSK3â are associated with the mitotic spindle, …

Contributors
Chakravadhanula, Madhavi, Capco, David G., Chandler, Douglas, et al.
Created Date
2012

Membrane protein structure is continuing to be a topic of interest across the scientific community. However, high resolution structural data of these proteins is difficult to obtain. The amino acid transport protein, Outer Envelope Protein, 16kDa (OEP16) is a transmembrane protein channel that allows the passive diffusion of amino acids across the outer chloroplast membrane, and is used as a model protein in order to establish methods that ultimately reveal structural details about membrane proteins using nuclear magnetic resonance (NMR) spectroscopy. Methods include recombinant expression of isotope enriched inclusion bodies, purification and reconstitution in detergent micelles, and pre-characterization techniques including …

Contributors
Zook, James Duncan, Fromme, Petra, Chen, Julian, et al.
Created Date
2012

Hydrogenases, the enzymes that reversibly convert protons and electrons to hydrogen, are used in all three domains of life. [NiFe]-hydrogenases are considered best suited for biotechnological applications because of their reversible inactivation with oxygen. Phylogenetically, there are four groups of [NiFe]-hydrogenases. The best characterized group, "uptake" hydrogenases, are membrane-bound and catalyze hydrogen oxidation in vivo. In contrast, the group 3 [NiFe]-hydrogenases are heteromultimeric, bifunctional enzymes that fulfill various cellular roles. In this dissertation, protein film electrochemistry (PFE) is used to characterize the catalytic properties of two group 3 [NiFe]-hydrogenases: HoxEFUYH from Synechocystsis sp. PCC 6803 and SHI from Pyrococcus furiosus. …

Contributors
Mcintosh, Chelsea Lee, Jones, Anne K, Ghirlanda, Giovanna, et al.
Created Date
2012

While exercising mammalian muscle increasingly relies on carbohydrates for fuel as aerobic exercise intensity rises above the moderate range, flying birds are extraordinary endurance athletes and fuel flight, a moderate-high intensity exercise, almost exclusively with lipid. In addition, Aves have long lifespans compared to weight-matched mammals. As skeletal muscle mitochondria account for the majority of oxygen consumption during aerobic exercise, the primary goal was to investigate differences in isolated muscle mitochondria between these species and to examine to what extent factors intrinsic to mitochondria may account for the behavior observed in the intact tissue and whole organism. First, maximal enzyme …

Contributors
Kuzmiak, Sarah, Willis, Wayne T, Mandarino, Lawrence, et al.
Created Date
2012

Acquisition of fluorescence via autocatalytic processes is unique to few proteins in the natural world. Fluorescent proteins (FPs) have been integral to live-cell imaging techniques for decades; however, mechanistic information is still emerging fifty years after the discovery of the original green fluorescent protein (GFP). Modification of the fluorescence properties of the proteins derived from GFP allows increased complexity of experiments and consequently, information content of the data acquired. The importance of arginine-96 in GFP has been widely discussed. It has been established as vital to the kinetics of chromophore maturation and to the overall fold of GFP before post-translational …

Contributors
Watkins, Jennifer L., Wachter, Rebekka M, Ghirlanda, Giovanna, et al.
Created Date
2012

The green fluorescent protein (GFP)-like fluorescent proteins play an important role for the color of reef-building corals. Different colors of extant coral fluorescent proteins (FPs) have evolved from a green ancestral protein. Interestingly, green-to-red photoconversion FPs (Kaede-type Red FPs) are only found in clade D from Scleractinia (Faviina suborder). Therefore, I focus on the evolution of Kaede-type FPs from Faviina suborder ancestral FP. A total of 13 mutations have been identified previously that recapitulate the evolution of Kaede-type red FPs from the ancestral green FP. To examine the effect of each mutation, total ten reconstructed FPs were analyzed and six …

Contributors
Kim, Hanseong, Wachter, Rebekka M, Fromme, Petra, et al.
Created Date
2012

The sun provides Earth with a virtually limitless source of energy capable of sustaining all of humanity's needs. Photosynthetic organisms have exploited this energy for eons. However, efficiently converting solar radiation into a readily available and easily transportable form is complex. New materials with optimized physical, electrochemical, and photophysical properties are at the forefront of organic solar energy conversion research. In the work presented herein, porphyrin and organometallic dyes with widely-varied properties were studied for solar energy applications. In one project, porphyrins and porphyrin-fullerene dyads with aniline-like features were polymerized via electrochemical methods into semiconductive thin films. These were shown …

Contributors
Brennan, Bradley, Gust, Devens, Moore, Thomas A, et al.
Created Date
2012

V(D)J recombination is responsible for generating an enormous repertoire of immunoglobulins and T cell receptors, therefore it is a centerpiece to the formation of the adaptive immune system. The V(D)J recombination process proceeds through two steps, site-specific cleavage at RSS (Recombination Signal Sequence) site mediated by the RAG recombinase (RAG1/2) and the subsequent imprecise resolution of the DNA ends, which is carried out by the ubiquitous non-homologous end joining pathway (NHEJ). The V(D)J recombination reaction is obliged to be tightly controlled under all circumstances, as it involves generations of DNA double strand breaks, which are considered the most dangerous lesion …

Contributors
Wang, Guannan, Chang, Yung, Levitus, Marcia, et al.
Created Date
2012

The ribosome is a ribozyme and central to the biosynthesis of proteins in all organisms. It has a strong bias against non-alpha-L-amino acids, such as alpha-D-amino acids and beta-amino acids. Additionally, the ribosome is only able to incorporate one amino acid in response to one codon. It has been demonstrated that reengineering of the peptidyltransferase center (PTC) of the ribosome enabled the incorporation of both alpha-D-amino acids and beta-amino acids into full length protein. Described in Chapter 2 are five modified ribosomes having modifications in the peptidyltrasnferase center in the 23S rRNA. These modified ribosomes successfully incorporated five different beta-amino …

Contributors
Maini, Rumit, Hecht, Sidney M, Gould, Ian, et al.
Created Date
2013

The bleomycins are a family of glycopeptide-derived antibiotics isolated from various Streptomyces species and have been the subject of much attention from the scientific community as a consequence of their antitumor activity. Bleomycin clinically and is an integral part of a number of combination chemotherapy regimens. It has previously been shown that bleomycin has the ability to selectively target tumor cells over their non-malignant counterparts. Pyrimidoblamic acid, the N-terminal metal ion binding domain of bleomycin is known to be the moiety that is responsible for O2 activation and the subsequent chemistry leading to DNA strand scission and overall antitumor activity. …

Contributors
Bozeman, Trevor, Hecht, Sidney M, Chaput, John, et al.
Created Date
2013

X-ray diffraction is the technique of choice to determine the three-dimensional structures of proteins. In this study it has been applied to solve the structure of the survival motor neuron (SMN) proteins, the Fenna-Mathews-Olson (FMO) from Pelodictyon phaeum (Pld. phaeum) protein, and the synthetic ATP binding protein DX. Spinal muscular atrophy (SMA) is an autosomal recessive genetic disease resulting in muscle atrophy and paralysis via degeneration of motor neurons in the spinal cord. In this work, we used X-ray diffraction technique to solve the structures of the three variant of the of SMN protein, namely SMN 1-4, SMN-WT, and SMN-Δ7. …

Contributors
Seng, Chenda Ouk, Allen, James P., Wachter, Rebekka, et al.
Created Date
2013

The biological and chemical diversity of protein structure and function can be greatly expanded by position-specific incorporation of non-natural amino acids bearing a variety of functional groups. Non-cognate amino acids can be incorporated into proteins at specific sites by using orthogonal aminoacyl-tRNA synthetase/tRNA pairs in conjunction with nonsense, rare, or 4-bp codons. There has been considerable progress in developing new types of amino acids, in identifying novel methods of tRNA aminoacylation, and in expanding the genetic code to direct their position. Chemical aminoacylation of tRNAs is accomplished by acylation and ligation of a dinucleotide (pdCpA) to the 3'-terminus of truncated …

Contributors
Nangreave, Ryan Christopher, Hecht, Sidney M, Yan, Hao, et al.
Created Date
2013

There is a critical need for the development of clean and efficient energy sources. Hydrogen is being explored as a viable alternative to fuels in current use, many of which have limited availability and detrimental byproducts. Biological photo-production of H2 could provide a potential energy source directly manufactured from water and sunlight. As a part of the photosynthetic electron transport chain (PETC) of the green algae Chlamydomonas reinhardtii, water is split via Photosystem II (PSII) and the electrons flow through a series of electron transfer cofactors in cytochrome b6f, plastocyanin and Photosystem I (PSI). The terminal electron acceptor of PSI …

Contributors
Reifschneider, Kiera T., Redding, Kevin, Fromme, Petra, et al.
Created Date
2013

Since Darwin popularized the evolution theory in 1895, it has been completed and studied through the years. Starting in 1990s, evolution at molecular level has been used to discover functional molecules while studying the origin of functional molecules in nature by mimicing the natural selection process in laboratory. Along this line, my Ph.D. dissertation focuses on the in vitro selection of two important biomolecules, deoxynucleotide acid (DNA) and protein with binding properties. Chapter two focuses on in vitro selection of DNA. Aptamers are single-stranded nucleic acids that generated from a random pool and fold into stable three-dimensional structures with ligand …

Contributors
Jiang, Bing, Chaput, John C, Chen, Julian, et al.
Created Date
2013

The discovery of DNA helical structure opened the door of modern molecular biology. Ned Seeman utilized DNA as building block to construct different nanoscale materials, and introduced a new field, know as DNA nanotechnology. After several decades of development, different DNA structures had been created, with different dimension, different morphology and even with complex curvatures. In addition, after construction of enough amounts DNA structure candidates, DNA structure template, with excellent spatial addressability, had been used to direct the assembly of different nanomaterials, including nanoparticles and proteins, to produce different functional nanomaterials. However there are still many challenges to fabricate functional …

Contributors
Zhao, Zhao, Yan, Hao, Liu, Yan, et al.
Created Date
2013

Cyanovirin-N (CV-N) is a naturally occurring lectin originally isolated from the cyanobacteria Nostoc ellipsosporum. This 11 kDa lectin is 101 amino acids long with two binding sites, one at each end of the protein. CV-N specifically binds to terminal Manα1-2Manα motifs on the branched, high mannose Man9 and Man8 glycosylations found on enveloped viruses including Ebola, Influenza, and HIV. wt-CVN has micromolar binding to soluble Manα1-2Manα and also inhibits HIV entry at low nanomolar concentrations. CV-N's high affinity and specificity for Manα1-2Manα makes it an excellent lectin to study for its glycan-specific properties. The long-term aim of this project is …

Contributors
Ruben, Melissa, Ghirlanda, Giovanna, Allen, James, et al.
Created Date
2013

The principle of Darwinian evolution has been applied in the laboratory to nucleic acid molecules since 1990, and led to the emergence of in vitro evolution technique. The methodology of in vitro evolution surveys a large number of different molecules simultaneously for a pre-defined chemical property, and enrich for molecules with the particular property. DNA and RNA sequences with versatile functions have been identified by in vitro selection experiments, but many basic questions remain to be answered about how these molecules achieve their functions. This dissertation first focuses on addressing a fundamental question regarding the molecular recognition properties of in …

Contributors
Yu, Hanyang, Chaput, John C, Chen, Julian, et al.
Created Date
2013

DNA has recently emerged as an extremely promising material to organize molecules on nanoscale. The reliability of base recognition, self-assembling behavior, and attractive structural properties of DNA are of unparalleled value in systems of this size. DNA scaffolds have already been used to organize a variety of molecules including nanoparticles and proteins. New protein-DNA bio-conjugation chemistries make it possible to precisely position proteins and other biomolecules on underlying DNA scaffolds, generating multi-biomolecule pathways with the ability to modulate inter-molecular interactions and the local environment. This dissertation focuses on studying the application of using DNA nanostructure to direct the self-assembly of …

Contributors
Liu, Minghui, Yan, Hao, Liu, Yan, et al.
Created Date
2013

Photosystem I (PSI) is a multi-subunit, pigment-protein complex that catalyzes light-driven electron transfer (ET) in its bi-branched reaction center (RC). Recently it was suggested that the initial charge separation (CS) event can take place independently within each ec2/ec3 chlorophyll pair. In order to improve our understanding of this phenomenon, we have generated new mutations in the PsaA and PsaB subunits near the electron transfer cofactor 2 (ec2 chlorophyll). PsaA-Asn604 accepts a hydrogen bond from the water molecule that is the axial ligand of ec2B and the case is similar for PsaB-Asn591 and ec2A. The second set of targeted sites was …

Contributors
BADSHAH, SYED LAL, REDDING, KEVIN E, FROMME, PETRA, et al.
Created Date
2014

The AAA+ ATPase Rubisco activase (Rca) regulates the activity of Rubisco, the photosynthetic enzyme responsible for catalyzing biological carbon fixation. However, the detailed mechanism by which Rca self-association controls Rubisco reactivation activity remains poorly understood. In this work, we are using fluorescence correlation spectroscopy (FCS) to better characterize the thermodynamics of the assembly process of cotton Rca. We present FCS data for Rca in the presence of Mg*ATPgS and Mg*ADP and for the D173N Walker B motif mutant in the presence of Mg*ATP. Our data are consistent with promotion and stabilization of hexamers by Mg*ATPgS and Mg*ATP, whereas Mg*ADP facilitates …

Contributors
Kuriata, Agnieszka Magdalena, Wachter, Rebekka, Redding, Kevin, et al.
Created Date
2014

An animal's ability to produce protein-based silk materials has evolved independently in many different arthropod lineages, satisfying various ecological necessities. However, regardless of their wide range of uses and their potential industrial and biomedical applications, advanced knowledge on the molecular structure of silk biopolymers is largely limited to those produced by spiders (order Araneae) and silkworms (order Lepidoptera). This thesis provides an in-depth molecular-level characterization of silk fibers produced by two vastly different insects: the caddisfly larvae (order Trichoptera) and the webspinner (order Embioptera). The molecular structure of caddisfly larval silk from the species <italic>Hesperophylax consimilis</italic> was characterized using solid-state …

Contributors
Addison, John Bennett, Yarger, Jeffery L, Holland, Gregory P, et al.
Created Date
2014

A vast amount of energy emanates from the sun, and at the distance of Earth, approximately 172,500 TW reaches the atmosphere. Of that, 80,600 TW reaches the surface with 15,600 TW falling on land. Photosynthesis converts 156 TW in the form of biomass, which represents all food/fuel for the biosphere with about 20 TW of the total product used by humans. Additionally, our society uses approximately 20 more TW of energy from ancient photosynthetic products i.e. fossil fuels. In order to mitigate climate problems, the carbon dioxide must be removed from the human energy usage by replacement or recycling as …

Contributors
Vaughn, Michael David, Moore, Thomas, Fromme, Petra, et al.
Created Date
2014

Cyanovirin-N (CVN) is a cyanobacterial lectin with potent anti-HIV activity, mediated by binding to the N-linked oligosaccharide moiety of the envelope protein gp120. CVN offers a scaffold to develop multivalent carbohydrate-binding proteins with tunable specificities and affinities. I present here biophysical calculations completed on a monomeric-stabilized mutant of cyanovirin-N, P51G-m4-CVN, in which domain A binding activity is abolished by four mutations; with comparisons made to CVN<super>mutDB</super>, in which domain B binding activity is abolished. Using Monte Carlo calculations and docking simulations, mutations in CVN<super>mutDB</super> were considered singularly, and the mutations E41A/G and T57A were found to impact the affinity towards …

Contributors
Woodrum, Brian William, Ghirlanda, Giovanna, Redding, Kevin, et al.
Created Date
2014

Membrane proteins are a vital part of cellular structure. They are directly involved in many important cellular functions, such as uptake, signaling, respiration, and photosynthesis, among others. Despite their importance, however, less than 500 unique membrane protein structures have been determined to date. This is due to several difficulties with macromolecular crystallography, primarily the difficulty of growing large, well-ordered protein crystals. Since the first proof of concept for femtosecond nanocrystallography showing that diffraction patterns can be collected on extremely small crystals, thus negating the need to grow larger crystals, there have been many exciting advancements in the field. The technique …

Contributors
Kupitz, Christopher, Fromme, Petra, Spence, John C., et al.
Created Date
2014

ABSTRACT Post Translational Modifications (PTMs) are a series of chemical modifications with the capacity to expand the structural and functional repertoire of proteins. PTMs can regulate protein-protein interaction, localization, protein turn-over, the active state of the protein, and much more. This can dramatically affect cell processes as relevant as gene expression, cell-cell recognition, and cell signaling. Along these lines, this Ph.D. thesis examines the role of two of the most important PTMs: glycosylation and phosphorylation. In chapters 2, 3 and 4, a 10,000 peptide microarray is used to analyze the glycan variations in a series lipopolysaccharides (LPS) from Gram negative …

Contributors
Morales Betanzos, Carlos Alberto, LaBaer, Joshua, Allen, James, et al.
Created Date
2014

Protein-surface interactions, no matter structured or unstructured, are important in both biological and man-made systems. Unstructured interactions are more difficult to study with conventional techniques due to the lack of a specific binding structure. In this dissertation, a novel approach is employed to study the unstructured interactions between proteins and heterogonous surfaces, by looking at a large number of different binding partners at surfaces and using the binding information to understand the chemistry of binding. In this regard, surface-bound peptide arrays are used as a model for the study. Specifically, in Chapter 2, the effects of charge, hydrophobicity and length …

Contributors
Wang, Wei, Woodbury, Neal W, Liu, Yan, et al.
Created Date
2014

Telomerase is a unique reverse transcriptase that has evolved specifically to extend the single stranded DNA at the 3' ends of chromosomes. To achieve this, telomerase uses a small section of its integral RNA subunit (TR) to reiteratively copy a short, canonically 6-nt, sequence repeatedly in a processive manner using a complex and currently poorly understood mechanism of template translocation to stop nucleotide addition, regenerate its template, and then synthesize a new repeat. In this study, several novel interactions between the telomerase protein and RNA components along with the DNA substrate are identified and characterized which come together to allow …

Contributors
Brown, Andrew, Chen, Julian J. L., Jones, Anne, et al.
Created Date
2014

DNA nanotechnology is one of the most flourishing interdisciplinary research fields. Through the features of programmability and predictability, DNA nanostructures can be designed to self-assemble into a variety of periodic or aperiodic patterns of different shapes and length scales, and more importantly, they can be used as scaffolds for organizing other nanoparticles, proteins and chemical groups. By leveraging these molecules, DNA nanostructures can be used to direct the organization of complex bio-inspired materials that may serve as smart drug delivery systems and in vitro or in vivo bio-molecular computing and diagnostic devices. In this dissertation I describe a systematic study …

Contributors
Wei, Xixi, Liu, Yan, Yan, Hao, et al.
Created Date
2014

Photosynthesis is the primary source of energy for most living organisms. Light harvesting complexes (LHC) play a vital role in harvesting sunlight and passing it on to the protein complexes of the electron transfer chain which create the electrochemical potential across the membrane which drives ATP synthesis. phycobilisomes (PBS) are the most important LHCs in cyanobacteria. PBS is a complex of three light harvesting proteins: phycoerythrin (PE), phycocyanin (PC) and allophycocyanin (APC). This work has been done on a newly discovered cyanobacterium called Leptolyngbya Heron Island (L.HI). This study has three important goals: 1) Sequencing, assembly and annotation of the …

Contributors
Paul, Robin, Fromme, Petra, Ros, Alexandra, et al.
Created Date
2014

Rapid and reliable separation and analysis of proteins require powerful analytical methods. The analysis of proteins becomes especially challenging when only small sample volumes are available, concomitantly with low concentrations of proteins. Time critical situations pose additional challenges. Due to these challenges, conventional macro-scale separation techniques reach their limitations. While microfluidic devices require only pL-nL sample volumes, they offer several advantages such as speed, efficiency, and high throughput. This work elucidates the capability to manipulate proteins in a rapid and reliable manner with a novel migration technique, namely dielectrophoresis (DEP). Since protein analysis can often be achieved through a combination …

Contributors
Nakano, Asuka, Ros, Alexandra, Hayes, Mark, et al.
Created Date
2014

Proteins and peptides fold into dynamic structures that access a broad functional landscape, however, designing artificial polypeptide systems continues to be a great chal-lenge. Conversely, deoxyribonucleic acid (DNA) engineering is now routinely used to build a wide variety of two dimensional and three dimensional (3D) nanostructures from simple hybridization based rules, and their functional diversity can be significantly ex-panded through site specific incorporation of the appropriate guest molecules. This dis-sertation describes a gentle methodology for using short (8 nucleotide) peptide nucleic acid (PNA) linkers to assemble polypeptides within a 3D DNA nanocage, as a proof of concept for constructing artificial …

Contributors
Flory, Justin, Fromme, Petra, Yan, Hao, et al.
Created Date
2014

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 …

Contributors
Shi, Xiangyan, Yarger, Jeffery L, Holland, Gregory P, et al.
Created Date
2014

DNA is a unique, highly programmable and addressable biomolecule. Due to its reliable and predictable base recognition behavior, uniform structural properties, and extraordinary stability, DNA molecules are desirable substrates for biological computation and nanotechnology. The field of DNA computation has gained considerable attention due to the possibility of exploiting the massive parallelism that is inherent in natural systems to solve computational problems. This dissertation focuses on building novel types of computational DNA systems based on both DNA reaction networks and DNA nanotechnology. A series of related research projects are presented here. First, a novel, three-input majority logic gate based on …

Contributors
Li, Wei, Yan, Hao, Liu, Yan, et al.
Created Date
2014

The communication of genetic material with biomolecules has been a major interest in cancer biology research for decades. Among its different levels of involvement, DNA is known to be a target of several antitumor agents. Additionally, tissue specific interaction between macromolecules such as proteins and structurally important regions of DNA has been reported to define the onset of certain types of cancers. Illustrated in Chapter 1 is the general history of research on the interaction of DNA and anticancer drugs, most importantly different congener of bleomycin (BLM). Additionally, several synthetic analogues of bleomycin, including the structural components and functionalities, are …

Contributors
Roy, Basab, Hecht, Sidney M, Jones, Anne, et al.
Created Date
2014

Redox reactions are crucial to energy transduction in biology. Protein film electrochemistry (PFE) is a technique for studying redox proteins in which the protein is immobilized at an electrode surface so as to allow direct exchange of electrons. Establishing a direct electronic connection eliminates the need for redox­active mediators, thus allowing for interrogation of the redox protein of interest. PFE has proven a versatile tool that has been used to elucidate the properties of many technologically relevant redox proteins including hydrogenases, laccases, and glucose oxidase. This dissertation is comprised of two parts: extension of PFE to a novel electrode material …

Contributors
Kwan, Patrick, Jones, Anne K, Francisco, Wilson, et al.
Created Date
2014

Quiescin sulfhydryl oxidase 1 (QSOX1) is a highly conserved disulfide bond-generating enzyme that represents the ancient fusion of two major thiol-disulfide oxidoreductase gene families: thioredoxin and ERV. QSOX1 was first linked with cancer after being identified as overexpressed in pancreatic ductal adenocarcinoma (but not in adjacent normal ductal epithelia, infiltrating lymphocytes, or chronic pancreatitis). QSOX1 overexpression has been confirmed in a number of other histological tumor types, such as breast, lung, kidney, prostate, and others. Expression of QSOX1 supports a proliferative and invasive phenotype in tumor cells, and its enzymatic activity is critical for promoting an invasive phenotype. An in …

Contributors
Hanavan, Paul Daniel, Lake, Douglas, LaBaer, Joshua, et al.
Created Date
2015

Efficient separation techniques for organelles and bacteria in the micron- and sub-micron range are required for various analytical challenges. Mitochondria have a wide size range resulting from the sub-populations, some of which may be associated with diseases or aging. However, traditional methods can often not resolve within-species size variations. Strategies to separate mitochondrial sub-populations by size are thus needed to study the importance of this organelle in cellular functions. Additionally, challenges also exist in distinguishing the sub-populations of bio-species which differ in the surface charge while possessing similar size, such as Salmonella typhimurium (Salmonella). The surface charge of Salmonella wild-type …

Contributors
Luo, Jinghui, Ros, Alexandra, Hayes, Mark, et al.
Created Date
2015

Advances in chemical synthesis have enabled new lines of research with unnatural genetic polymers whose modified bases or sugar-phosphate backbones have potential therapeutic and biotechnological applications. Maximizing the potential of these synthetic genetic systems requires inventing new molecular biology tools that can both generate and faithfully replicate unnatural polymers of significant length. Threose nucleic acid (TNA) has received significant attention as a complete replication system has been developed by engineering natural polymerases to broaden their substrate specificity. The system, however, suffers from a high mutational load reducing its utility. This thesis will cover the development of two new polymerases capable …

Contributors
Dunn, Matthew Ryan, Chaput, John C, LaBaer, Joshua, et al.
Created Date
2015

Molecular docking serves as an important tool in modeling protein-ligand interactions. Most of the docking approaches treat the protein receptor as rigid and move the ligand in the binding pocket through an energy minimization, which is an incorrect approach as proteins are flexible and undergo conformational changes upon ligand binding. However, modeling receptor backbone flexibility in docking is challenging and computationally expensive due to the large conformational space that needs to be sampled. A novel flexible docking approach called BP-Dock (Backbone Perturbation docking) was developed to overcome this challenge. BP-Dock integrates both backbone and side chain conformational changes of a …

Contributors
Bolia, Ashini, Ozkan, Sefika Banu, Ghirlanda, Giovanna, et al.
Created Date
2015

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 …

Contributors
Morgan, Ashli, Wang, Xu, Allen, James, et al.
Created Date
2015

Atomic force microscopy (AFM) has become an important tool to characterize and image surfaces with nanoscale resolution. AFM imaging technique has been utilized to study a wide range of substances such as DNA, proteins, cells, silicon surfaces, nanowires etc. Hence AFM has become extremely important in the field of biochemistry, cell biology and material science. Functionalizing the AFM tip made it possible to detect molecules and their interaction using recognition imaging at single molecule level. Also the unbinding force of two molecules can be investigated based on AFM based single molecule force spectroscopy. In the first study, a new chemical …

Contributors
SENAPATI, SUBHADIP, Lindsay, Stuart, Zhang, Peiming, et al.
Created Date
2015

The Heliobacterial reaction center (HbRC) is generally regarded as the most primitive photosynthetic reaction center (RC) known. Even if the HbRC is structurally and functionally simple compared to higher plants, the mechanisms of energy transduction preceding, inside the core, and from the RC are not totally established. Elucidating these structures and mechanisms are paramount to determining where the HbRC is in the grand scheme of RC evolution. In this work, the function and properties of the solubilized cyt c553, PetJ, were investigated, as well as the role HbRC localized menaquinone plays in light-induced electron transfer, and the interaction of the …

Contributors
Kashey, Trevor Scott, Redding, Kevin E, Fromme, Petra, et al.
Created Date
2015

CTB-MPR649-684 is a translational fusion protein consisting of the cholera toxin B subunit (CTB) and the conserved residues 649-684 of gp41 membrane proximal region (MPR). It is a candidate vaccine component aimed at early steps of the HIV-1 infection by blocking viral mucosal transmission. Bacterially produced CTB-MPR was previously shown to induce HIV-1 transcytosis-blocking antibodies in mice and rabbits. However, the induction of high-titer MPR specific antibodies with HIV-1 transcytosis blocking ability remains a challenge as the immuno-dominance of CTB overshadows the response to MPR. X-ray crystallography was used to investigate the structure of CTB-MPR with the goal of identifying …

Contributors
Lee, Ho-Hsien, Fromme, Petra, Mor, Tsafrir, et al.
Created Date
2015

Adenosine triphosphate (ATP) is the universal chemical energy currency in most living cells, used to power many cellular reactions and generated by an enzyme supercomplex known as the ATP synthase, consisting of a hydrophilic F1 subcomplex and a membrane-bound FO subcomplex. Driven by the electrochemical gradient generated by the respiratory or photosynthetic electron transport chain, the rotation of the FO domain drives movements of the central stalk in response to conformational changes in the F1 domain, in which the physical energy is converted into chemical energy through the condensation of ADP and Pi to ATP. The exact mechanism how ATP …

Contributors
Yang, Jay-How, Fromme, Petra, Redding, Kevin, et al.
Created Date
2015

Biological fluids contain information-rich mixtures of biochemicals and particles such as cells, proteins, and viruses. Selective and sensitive analysis of these fluids can enable clinicians to accurately diagnose a wide range of pathologies. Fluid samples such as these present an intriguing challenge to researchers; they are packed with potentially vital information, but notoriously difficult to analyze. Rapid and inexpensive analysis of blood and other bodily fluids is a topic gaining substantial attention in both science and medicine. Current limitations to many analyses include long culture times, expensive reagents, and the need for specialized laboratory facilities and personnel. Improving these tests …

Contributors
Jones, Paul Vernon, Hayes, Mark, Ros, Alexandra, et al.
Created Date
2015

ABSTRACT The catalytic chaperone of Rubisco is AAA+ protein Rubisco activase (Rca), which hydrolyzes ATP and thus undergoes conformational change, helping in reactivating Rubisco. Rca reactivates Rubisco plausibly by removing its C- terminal tail from the opening of its active site thus releasing the inhibitor, a sugar phosphate molecule. Rubisco and Rca are regulated by the stromal environment, which includes the ATP/ADP ratio, Mg2+ concentration, redox potential etc. Here the mechanistic regulation of tobacco β-Rca was studied using steady state enzyme kinetics in terms of product inhibition, Mg2+ activation, cooperativity and asymmetry. A continuous Pi measurement assay was developed, and …

Contributors
Hazra, Suratna, Wachter, Rebekka M, Fromme, Petra, et al.
Created Date
2015

In vitro selection technologies allow for the identification of novel biomolecules endowed with desired functions. Successful selection methodologies share the same fundamental requirements. First, they must establish a strong link between the enzymatic function being selected (phenotype) and the genetic information responsible for the function (genotype). Second, they must enable partitioning of active from inactive variants, often capturing only a small number of positive hits from a large population of variants. These principles have been applied to the selection of natural, modified, and even unnatural nucleic acids, peptides, and proteins. The ability to select for and characterize new functional molecules …

Contributors
Larsen, Andrew, Chaput, John C, Jacobs, Bertram L, et al.
Created Date
2015

Telomerase enzyme is a truly remarkable enzyme specialized for the addition of short, highly repetitive DNA sequences onto linear eukaryotic chromosome ends. The telomerase enzyme functions as a ribonucleoprotein, minimally composed of the highly conserved catalytic telomerase reverse transcriptase and essential telomerase RNA component containing an internalized short template region within the vastly larger non-coding RNA. Even among closely related groups of species, telomerase RNA is astonishingly divergent in sequence, length, and secondary structure. This massive disparity is highly prohibitive for telomerase RNA identification from previously unexplored groups of species, which is fundamental for secondary structure determination. Combined biochemical enrichment …

Contributors
Podlevsky, Joshua David, Chen, Julian, Mangone, Marco, et al.
Created Date
2015

Biophysical techniques have been increasingly applied toward answering biological questions with more precision. Here, three different biological systems were studied with the goal of understanding their dynamic differences, either conformational dynamics within the system or oligomerization dynamics between monomers. With Cy3 on the 5' end of DNA, the effects of changing the terminal base pair were explored using temperature-dependent quantum yields. It was discovered, in combination with simulations, that a terminal thymine base has the weakest stacking interactions with the Cy3 dye compared to the other three bases. With ME1 heterodimers, the goal was to see if engineering a salt …

Contributors
Binder, Jennifer K., Levitus, Marcia, Wachter, Rebekka, et al.
Created Date
2015

Deoxyribonucleic acid (DNA) has emerged as an excellent molecular building block for nanoconstruction in addition to its biological role of preserving genetic information. Its unique features such as predictable conformation and programmable intra- and inter-molecular Watson-Crick base pairing interactions make it a remarkable engineering material. A variety of convenient design rules and reliable assembly methods have been developed to engineer DNA nanostructures. The ability to create designer DNA architectures with accurate spatial control has allowed researchers to explore novel applications in directed material assembly, structural biology, biocatalysis, DNA computing, nano-robotics, disease diagnosis, and drug delivery. This dissertation focuses on developing …

Contributors
Zhang, Fei, Yan, Hao, Liu, Yan, et al.
Created Date
2015

Biological fluids, in particular blood plasma, provide a vital source of information on the state of human health. While specific detection of biomarker species can aid in disease diagnostics, the complexity of plasma makes analysis challenging. Despite the challenge of complex sample analysis, biomarker quantification has become a primary interest in biomedical analysis. Due to the extremely specific interaction between antibody and analyte, immunoassays are attractive for the analysis of these samples and have gained popularity since their initial introduction several decades ago. Current limitations to diagnostics through blood testing include long incubation times, interference from non-specific binding, and the …

Contributors
Woolley, Christine F, Hayes, Mark A, Ros, Alexandra, et al.
Created Date
2015

The energy required in a eukaryotic cell is provided by mitochondria. Mitochondrial electron transport chain (ETC) coupled with oxidative phosphorylation generates ATP. During electron transport, electron leakage from the ETC produces reactive oxygen species (ROS). In healthy cells, there are preventive and defense mechanisms in place to manage ROS. Maintaining a steady balance of ROS is very important because overproduction of ROS can lead to several pathological conditions. There are several strategies to prevent ROS production. Addition of external antioxidants is widely used among them. Discussed in the first part of Chapter 1 is the mitochondrial ETC, ROS production and …

Contributors
Roy Chowdhury, Sandipan, Hecht, Sidney, Gould, Ian, et al.
Created Date
2016

Nature is a master at organizing biomolecules in all intracellular processes, and researchers have conducted extensive research to understand the way enzymes interact with each other through spatial and orientation positioning, substrate channeling, compartmentalization, and more. DNA nanostructures of high programmability and complexity provide excellent scaffolds to arrange multiple molecular/macromolecular components at nanometer scale to construct interactive biomolecular complexes and networks. Due to the sequence specificity at different positions of the DNA origami nanostructures, spatially addressable molecular pegboard with a resolution of several nm (less than 10 nm) can be achieved. So far, DNA nanostructures can be used to build …

Contributors
Yang, Yuhe Renee, Yan, Hao, Liu, Yan, et al.
Created Date
2016

One of the greatest problems facing society today is the development of a sustainable, carbon neutral energy source to curb the reliance on fossil fuel combustion as the primary source of energy. To overcome this challenge, research efforts have turned to biology for inspiration, as nature is adept at inter-converting low molecular weight precursors into complex molecules. A number of inorganic catalysts have been reported that mimic the active sites of energy-relevant enzymes such as hydrogenases and carbon monoxide dehydrogenase. However, these inorganic models fail to achieve the high activity of the enzymes, which function in aqueous systems, as they …

Contributors
Sommer, Dayn Joseph, Ghirlanda, Giovanna, Redding, Kevin, et al.
Created Date
2016

Viral protein U (Vpu) is a type-III integral membrane protein encoded by the Human Immunodeficiency Virus-1 (HIV- 1). It is expressed in infected host cells and plays vital roles in down-regulation of CD4 receptors in T cells and also in the budding of virions. But, there remain key structure/function questions regarding the mechanisms by which the Vpu protein contributes to HIV-1 pathogenesis and thus, it makes for an attractive target to study the structural attributes of this protein by elucidating a structural model with X-ray crystallography. This study describes a multi-pronged approach of heterologous over-expression of Vpu. The strategies of …

Contributors
Deb, Arpan, Leket-Mor, Tsafrir S, Fromme, Petra, et al.
Created Date
2016

Accurate virus detection is important for diagnosis in a timely manner to facilitate rapid interventions and treatments. RNA viruses affect an extensive amount of the world’s population, particularly in tropical countries where emerging infectious agents often arise. Current diagnostic methods have three main problems: they are time consuming, typically not field-portable, and expensive. My research goal is to develop rapid, field-portable and cost sensitive diagnostic methods for RNA viruses. Herein, two different approaches to detect RNA viruses were proposed: Conjugated gold nanoparticles for detection of viral particles or virus-specific antibodies by monitoring changes in their optical properties, and Tentacle Probes …

Contributors
Franco, Lina Stella, Mujica, Vladimiro, Blattman, Joseph N, et al.
Created Date
2016

The ability to manipulate the interaction between small molecules and biological macromolecules towards the study of disease pathogenesis has become a very important part of research towards treatment options for various diseases. The work described here shows both the use of DNA oligonucleotides as carriers for a nicotine hapten small molecule, and the use of microsomes to study the stability of compounds derived to treat mitochondrial diseases. Nicotine addiction is a worldwide epidemic because nicotine is one of the most widely used addictive substances. It is linked to early death, typically in the form of heart or lung disease. A …

Contributors
Schmierer, Margaret Louise, Hecht, Sidney M, Allen, James, et al.
Created Date
2016

The basic scheme for photosynthesis suggests the two photosystems existing in parity with one another. However, cyanobacteria typically maintain significantly more photosystem I (PSI) than photosystem II (PSII) complexes. I set out to evaluate this disparity through development and analysis of multiple mutants of the genetically tractable cyanobacterium Synechocystis sp. PCC 6803 that exhibit a range of expression levels of the main proteins present in PSI (Chapter 2). One hypothesis was that the higher abundance of PSI in this organism is used to enable more cyclic electron flow (CEF) around PSI to contribute to greater ATP synthesis. Results of this …

Contributors
Moore, Vicki, Vermaas, Willem, Wang, Xuan, et al.
Created Date
2017

The primary carbon fixing enzyme Rubisco maintains its activity through release of trapped inhibitors by Rubisco activase (Rca). Very little is known about the interaction, but binding has been proposed to be weak and transient. Extensive effort was made to develop Förster resonance energy transfer (FRET) based assays to understand the physical interaction between Rubisco and Rca, as well as understand subunit exchange in Rca. Preparations of labeled Rubisco and Rca were utilized in a FRET-based binding assay. Although initial data looked promising, this approach was not fruitful, as no true FRET signal was observed. One possibility is that under …

Contributors
Forbrook, Dayna, Wachter, Rebekka M, Allen, James, et al.
Created Date
2017

Development of efficient and renewable electrocatalytic systems is foundational to creation of effective means to produce solar fuels. Many redox enzymes are functional electrocatalysts when immobilized on an electrode, but long-term stability of isolated proteins limits use in applications. Thus there is interest in developing bio-inspired functional catalysts or electrocatalytic systems based on living organisms. This dissertation describes efforts to create both synthetic and biological electrochemical systems for electrocatalytic hydrogen production. The first part of this dissertation describes the preparation of three different types of proton reduction catalysts. First, four bioinspired diiron complexes of the form (μ-SRS)Fe(CO)3[Fe(CO)(N-N)] for SRS = …

Contributors
Laureanti, Joseph Anthony, Jones, Anne K., Moore, Thomas, et al.
Created Date
2017

The evolution of photosynthesis caused the oxygen-rich atmosphere in which we thrive today. Although the reaction centers involved in oxygenic photosynthesis probably evolved from a protein like the reaction centers in modern anoxygenic photosynthesis, modern anoxygenic reaction centers are poorly understood. One such anaerobic reaction center is found in Heliobacterium modesticaldum. Here, the photosynthetic properties of H. modesticaldum are investigated, especially as they pertain to its unique photochemical reaction center. The first part of this dissertation describes the optimization of the previously established protocol for the H. modesticaldum reaction center isolation. Subsequently, electron transfer is characterized by ultrafast spectroscopy; the …

Contributors
Gisriel, Christopher James, Redding, Kevin E, Jones, Anne K, et al.
Created Date
2017

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 …

Contributors
Coe, Jesse, Fromme, Petra, Sayres, Scott, et al.
Created Date
2018

Signal transduction networks comprising protein-protein interactions (PPIs) mediate homeostatic, diseased, and therapeutic cellular responses. Mapping these networks has primarily focused on identifying interactors, but less is known about the interaction affinity, rates of interaction or their regulation. To better understand the extent of the annotated human interactome, I first examined > 2500 protein interactions within the B cell receptor (BCR) signaling pathway using a current, cutting-edge bioluminescence-based platform called “NanoBRET” that is capable of analyzing transient and stable interactions in high throughput. Eighty-three percent (83%) of the detected interactions have not been previously reported, indicating that much of the BCR …

Contributors
Petritis, Brianne Ogata, LaBaer, Joshua, Lake, Douglas, et al.
Created Date
2018

The linear chromosomes ends in eukaryotes are protected by telomeres, a nucleoprotein structure that contains telomeric DNA with repetitive sequence and associated proteins. Telomerase is an RNA-dependent DNA polymerase that adds telomeric DNA repeats to the 3'-ends of chromosomes to offset the loss of terminal DNA repeats during DNA replication. It consists of two core components: a telomerase reverse transcriptase (TERT) and a telomerase RNA (TR). Telomerase uses a short sequence in its integral RNA component as template to add multiple DNA repeats in a processive manner. However, it remains unclear how the telomerase utilizes the short RNA template accurately …

Contributors
Chen, Yinnan, Chen, Julian J-L, Jones, Anne K, et al.
Created Date
2018

Lipids perform functions essential to life and have a variety of structures that are influenced by the organisms and environments that produced them. Lipids tend to resist degradation after cell death, leading to their widespread use as biomarkers in geobiology, though their interpretation is often tricky. Many lipid structures are shared among organisms and function in many geochemical conditions and extremes. I argue it is useful to interpret lipid distributions as a balance of functional necessity and energy cost. This work utilizes a quantitative thermodynamic framework for interpreting energetically driven adaptation in lipids. Yellowstone National Park is a prime location …

Contributors
Boyer, Grayson Maxwell, Shock, Everett, Hartnett, Hilairy, et al.
Created Date
2018

Glycans are monosaccharide-based heteropolymers that are found covalently attached to many different proteins and lipids and are ubiquitously displayed on the exterior surfaces of cells. Serum glycan composition and structure are well known to be altered in many different types of cancer. In fact, glycans represent a promising but only marginally accessed source of cancer markers. The approach used in this dissertation, which is referred to as “glycan node analysis”, is a molecularly bottom-up approach to plasma/serum (P/S) glycomics based on glycan linkage analysis that captures features such as α2-6 sialylation, β1-6 branching, and core fucosylation as single analytical signals. …

Contributors
Roshdiferdosi, Shadi, Borges, Chad R, Woodbury, Neal, et al.
Created Date
2018

Extracellular vesicles (EVs) represent a heterogeneous population of small vesicles, consisting of a phospholipidic bilayer surrounding a soluble interior cargo. These vesicles play an important role in cellular communication by virtue of their protein, RNA, and lipid content, which can be transferred among cells. Peripheral blood is a rich source of circulating EVs. An analysis of EVs in peripheral blood could provide access to unparalleled amounts of biomarkers of great diagnostic, prognostic as well as therapeutic value. In the current study, a plasma EV enrichment method based on pluronic co-polymer was first established and characterized. Plasma EVs from breast cancer …

Contributors
Zhong, Zhenyu, Spetzler, David, Yan, Hao, et al.
Created Date
2018

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 …

Contributors
Roy Chowdhury, Shatabdi, Fromme, Petra, Ros, Alexandra, et al.
Created Date
2018

Biological systems have long been known to utilize two processes for energy conservation: substrate-level phosphorylation and electron transport phosphorylation. Recently, a new bioenergetic process was discovered that increases ATP yields: flavin-based electron bifurcation (FBEB). This process couples an energetically favorable reaction with an energetically unfavorable one to conserve energy in the organism. Currently, the mechanisms of enzymes that perform FBEB are unknown. In this work, NADH-dependent reduced ferredoxin:NADP+ oxidoreductase (Nfn), a FBEB enzyme, is used as a model system to study this phenomenon. Nfn is a heterodimeric enzyme that reversibly couples the exergonic reduction of NADP+ by reduced ferredoxin with …

Contributors
Jennings, David, Jones, Anne K, Redding, Kevin E, et al.
Created Date
2018

Rubisco activase (Rca) from higher plants is a stromal ATPase essential for reactivating Rubiscos rendered catalytically inactive by endogenous inhibitors. Rca’s functional state is thought to consist of ring-like hexameric assemblies, similar to other members of the AAA+ protein superfamily. However, unlike other members, it does not form obligate hexamers and is quite polydisperse in solution, making elucidation of its self-association pathway challenging. This polydispersity also makes interpretation of traditional biochemical approaches difficult, prompting use of a fluorescence-based technique (Fluorescence Correlation Spectroscopy) to investigate the relationship between quaternary structure and function. Like cotton β Rca, tobacco β Rca appears to …

Contributors
Serban, Andrew J, Wachter, Rebekka M, Levitus, Marcia, et al.
Created Date
2018

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 …

Contributors
Sisco, Nicholas John, Van Horn, Wade D, Mills, Jeremy H, et al.
Created Date
2018

Measuring molecular interaction with membrane proteins is critical for understanding cellular functions, validating biomarkers and screening drugs. Despite the importance, developing such a capability has been a difficult challenge, especially for small molecules binding to membrane proteins in their native cellular environment. The current mainstream practice is to isolate membrane proteins from the cell membranes, which is difficult and often lead to the loss of their native structures and functions. In this thesis, novel detection methods for in situ quantification of molecular interactions with membrane proteins are described. First, a label-free surface plasmon resonance imaging (SPRi) platform is developed for …

Contributors
Zhang, Fenni, Tao, Nongjian, Chae, Junseok, et al.
Created Date
2018

Exposure of blood plasma/serum (P/S) to thawed conditions, greater than -30°C, can produce biomolecular changes that misleadingly impact measurements of clinical markers within archived samples. Reported here is a low sample-volume, dilute-and-shoot, intact protein mass spectrometric assay of albumin proteoforms called “ΔS-Cys-Albumin” that quantifies cumulative exposure of archived P/S samples to thawed conditions. The assay uses the fact that S-cysteinylation (oxidation) of albumin in P/S increases to a maximum value when exposed to temperatures greater than -30°C. The multi-reaction rate law that governs this albumin S-cysteinylation formation in P/S was determined and was shown to predict the rate of formation …

Contributors
Jeffs, Joshua W, Borges, Chad R, Van Horn, Wade, et al.
Created Date
2018

Biochemical reactions underlie all living processes. Their complex web of interactions is difficult to fully capture and quantify with simple mathematical objects. Applying network science to biology has advanced our understanding of the metabolisms of individual organisms and the organization of ecosystems, but has scarcely been applied to life at a planetary scale. To characterize planetary-scale biochemistry, I constructed biochemical networks using global databases of annotated genomes and metagenomes, and biochemical reactions. I uncover scaling laws governing biochemical diversity and network structure shared across levels of organization from individuals to ecosystems, to the biosphere as a whole. Comparing real biochemical …

Contributors
Smith, Harrison Brodsky, Walker, Sara I, Anbar, Ariel D, et al.
Created Date
2018

Single-cell proteomics and transcriptomics analysis are crucial to gain insights of healthy physiology and disease pathogenesis. The comprehensive profiling of biomolecules in individual cells of a heterogeneous system can provide deep insights into many important biological questions, such as the distinct cellular compositions or regulation of inter- and intracellular signaling pathways of healthy and diseased tissues. With multidimensional molecular imaging of many different biomarkers in patient biopsies, diseases can be accurately diagnosed to guide the selection of the ideal treatment. As an urgent need to advance single-cell analysis, imaging-based technologies have been developed to detect and quantify multiple DNA, RNA …

Contributors
Mondal, Manas, Guo, Jia, Gould, Ian, et al.
Created Date
2018

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 …

Contributors
Feng, Wei, Wang, Xu, Yarger, Jeff L, et al.
Created Date
2019

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 …

Contributors
Espiritu, Eduardo, Allen, James P, Jones, Anne K, et al.
Created Date
2019

The highly predictable structural and thermodynamic behavior of deoxynucleic acid (DNA) and ribonucleic acid (RNA) have made them versatile tools for creating artificial nanostructures over broad range. Moreover, DNA and RNA are able to interact with biological ligand as either synthetic aptamers or natural components, conferring direct biological functions to the nucleic acid devices. The applications of nucleic acids greatly relies on the bio-reactivity and specificity when applied to highly complexed biological systems. This dissertation aims to 1) develop new strategy to identify high affinity nucleic acid aptamers against biological ligand; and 2) explore highly orthogonal RNA riboregulators in vivo …

Contributors
Zhou, Yu, Yan, Hao, Green, Alexander, et al.
Created Date
2019