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- All Subjects: Biochemistry
- Creators: Chiu, Po-Lin
- Creators: Stephanopoulos, Nicholas
Description
The field of biomedical research relies on the knowledge of binding interactions between various proteins of interest to create novel molecular targets for therapeutic purposes. While many of these interactions remain a mystery, knowledge of these properties and interactions could have significant medical applications in terms of understanding cell signaling and immunological defenses. Furthermore, there is evidence that machine learning and peptide microarrays can be used to make reliable predictions of where proteins could interact with each other without the definitive knowledge of the interactions. In this case, a neural network was used to predict the unknown binding interactions of TNFR2 onto LT-ɑ and TRAF2, and PD-L1 onto CD80, based off of the binding data from a sampling of protein-peptide interactions on a microarray. The accuracy and reliability of these predictions would rely on future research to confirm the interactions of these proteins, but the knowledge from these methods and predictions could have a future impact with regards to rational and structure-based drug design.
ContributorsPoweleit, Andrew Michael (Author) / Woodbury, Neal (Thesis director) / Diehnelt, Chris (Committee member) / Chiu, Po-Lin (Committee member) / School of Molecular Sciences (Contributor, Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
Description
The goal of this thesis was to simplify the sample preparation process for cryogenic electron microscopy (cryo-EM), clearing the way for the imaging of larger biomolecules and further expansion of the field. Various protic ionic liquids (PILs) were chosen for synthesis according to their pH and other physical properties. After several failed synthesizes, one PIL, cholinium dihydrogen phosphate, was chosen for further testing. This solution was put through a series of vitrification tests in order to understand its crystallization limits. Once limits were understood, cholinium dihydrogen phosphate was combined with ribosomal proteins and viewed under a transmission electron microscope to collect negative stain images. After adjusting the ratio of PIL to buffer and the concentration of ribosomes, images of whole intact ribosomes were captured. Samples were then placed in an EM grid, manually dipped in liquid nitrogen, and viewed using the the cryo-EM. These grids revealed ice too thick to properly image, an issue that was not solved by using a more aggressive blotting technique. Although the sample preparation process was not simplified, progress was made towards doing so and further testing using different techniques may result in success.
ContributorsStreet, Maya Ann (Author) / Angell, Charles Austen (Thesis director) / Chiu, Po-Lin (Committee member) / Materials Science and Engineering Program (Contributor) / School of Molecular Sciences (Contributor) / School of Human Evolution & Social Change (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
DNA nanotechnology uses the reliability of Watson-Crick base pairing to program and generate two-dimensional and three-dimensional nanostructures using single-stranded DNA as the structural material. DNA nanostructures show great promise for the future of bioengineering, as there are a myriad of potential applications that utilize DNA’s chemical interactivity and ability to bind other macromolecules and metals. DNA origami is a method of constructing nanostructures, which consists of a long “scaffold” strand folded into a shape by shorter “staple” oligonucleotides. Due to the negative charge of DNA molecules, divalent cations, most commonly magnesium, are required for origami to form and maintain structural integrity. The experiments in this paper address the discrepancy between salt concentrations required for origami stability and the salt concentrations present in living systems. The stability of three structures, a two-dimensional triangle, a three-dimensional solid cuboid and a three-dimensional wireframe icosahedron were examined in buffer solutions containing various concentrations of salts. In these experiments, DNA origami structures remained intact in low-magnesium conditions that emulate living cells, supporting their potential for widespread biological application in the future.
ContributorsSeverson, Grant William (Author) / Stephanopoulos, Nicholas (Thesis director) / Mills, Jeremy (Committee member) / School of Molecular Sciences (Contributor) / School of Life Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2020-05
Description
Extensive efforts have been made to develop efficient and low-cost methods for diagnostics to identify molecular biomarkers that are linked to a wide array of conditions, including cancer. A highly developed method includes utilizing the gene-editing enzyme CRISPR-Cas12a (Cpf1), which demonstrates double-stranded DNase activity with RuvC catalytic domain with high sensitivity and specificity. This DNase activity is RNA-guided and requires a T-rich PAM site on the target sequence for functional cleavage. There have been recent efforts to utilize this DNase activity of Cas12a by combining it with isothermal amplification and analysis by lateral strip tests. This project examined CRISPR-based early detection of microRNA biomarkers. MicroRNA are short RNA molecules that have large roles in post-transcriptional gene regulation. However, due the short length of microRNA and its single-stranded nature, it is challenging to use Cas12a for microRNA detection using existing methods. Thus, this project investigated the potential of two microRNA detection strategies for recognition by CRISPR-Cas12a. These methods were microRNA-splinted ligation with polymerase chain reaction (PCR) and MicroRNA-specific reverse transcriptase PCR (RT-PCR). Gel imaging demonstrated effective amplification of ligated DNA through microRNA-splinted ligation with PCR/RPA. In addition, lateral strips tests showed effective cleavage of the target sequences by Cas12a. However, RT-PCR method demonstrated low amplification by PCR and inefficient poly(A) elongation. This project paves the way for the detection of an extensive range of microRNA biomarkers that are linked to an array of diseases. Future directions include analysis and modifications of RT-PCR method to improve experimental results, extending these detection methods to a larger range of microRNA sequences, and eventually utilizing them for detection in human samples.
ContributorsStaren, Michael Steven (Author) / Green, Alexander (Thesis director) / Stephanopoulos, Nicholas (Committee member) / Diehnelt, Chris (Committee member) / School of Life Sciences (Contributor) / College of Health Solutions (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
The main objective of this project is to create a hydrogel based material system to capture and release CCRF-CEM Leukemia cancer cells via chemo-mechanical modulation. This system is composed of an aptamer-functionalized hydrogel thin film at the bottom of a microfluidic channel, which changes its film thickness as the temperature of the fluid in the system changes. The functionalized hydrogel film has been created as the primary steps to creating the microfluidic device that could capture and release leukemia cells by turning the temperature of the fluid and length of exposure. Circulating tumor cells have recently become a highly studied area since they have become associated with the likelihood of patient survival. Further, circulating tumor cells can be used to determine changes in the genome of the cancer leading to targeted treatment. First, the aptamers were attached onto the hydrogel through an EDC/NHS reaction. The aptamers were verified to be attached onto the hydrogel through FTIR spectroscopy. The cell capture experiments were completed by exposing the hydrogel to a solution of leukemia cells for 10 minutes at room temperature. The cell release experiments were completed by exposing the hydrogel to a 40°C solution. Several capture and release experiments were completed to measure how many cells could be captured, how quickly, and how many cells captured were released. The aptamers were chemically attached to the hydrogel. 300 cells per square millimeter could be captured at a time in a 10 minute time period and released in a 5 minute period. Of the cells captured, 96% of them were alive once caught. 99% of cells caught were released once exposed to elevated temperature. The project opens the possibility to quickly and efficiently capture and release tumor cells using only changes in temperature. Further, most of the cells that were captured were alive and nearly all of those were released leading to high survival and capture efficiency.
ContributorsPaxton, Rebecca Joanne (Author) / Stephanopoulos, Nicholas (Thesis director) / He, Ximin (Committee member) / Gould, Ian (Committee member) / Materials Science and Engineering Program (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2016-12
Description
Membrane proteins act as sensors, gatekeepers and information carriers in the cell membranes. Functional engineering of these proteins is important for the development of molecular tools for biosensing, therapeutics and as components of artificial cells. However, using protein engineering to modify existing protein structures is challenging due to the limitations of structural changes and difficulty in folding polypeptides into defined protein structures. Recent studies have shown that nanoscale architectures created by DNA nanotechnology can be used to mimic various protein functions, including some membrane proteins. However, mimicking the highly sophisticated structural dynamics of membrane proteins by DNA nanostructures is still in its infancy, mainly due to lack of transmembrane DNA nanostructures that can mimic the dynamic behavior, ubiquitous to membrane proteins. Here, I demonstrate design of dynamic DNA nanostructures to mimic two important class of membrane proteins. First, I describe a DNA nanostructure that inserts through lipid membrane and dynamically reconfigures upon sensing a membrane-enclosed DNA or RNA target, thereby transducing biomolecular information across the lipid membrane similar to G-protein coupled receptors (GPCR’s). I use the non-destructive sensing property of our GPCR-mimetic nanodevice to sense cancer associated micro-RNA biomarkers inside exosomes without the need of RNA extraction and amplification. Second, I demonstrate a fully reversibly gated DNA nanopore that mimics the ligand mediated gating of ion channel proteins. The 20.4 X 20.4 nm-wide channel of the DNA nanopore allows timed delivery of folded proteins across synthetic and biological membranes. These studies represent early examples of dynamic DNA nanostructures in mimicking membrane protein functions. I envision that they will be used in synthetic biology to create artificial cells containing GPCR-like and ion channel-like receptors, in site-specific drug or vaccine delivery and highly sensitive biosensing applications.
ContributorsDey, Swarup (Author) / Yan, Hao (Thesis advisor) / Hariadi, Rizal F (Thesis advisor) / Liu, Yan (Committee member) / Stephanopoulos, Nicholas (Committee member) / Arizona State University (Publisher)
Created2021
Description
The understanding of protein functions in vivo is very important since the protein is the building block of a cell. Cryogenic electron microscopy (cryo-EM) is capable of visualizing protein samples in their near-native states in high-resolution details. Cryo-EM enables the visualization of biomolecular structures at multiscale ranging from a cellular structure to an atomic structure of protein subunit.Neurodegenerative diseases, like Alzheimer’s disease and frontotemporal dementia, have multiple dysregulated signaling pathways. In my doctoral studies, I investigated two protein complexes relevant to these disorders: one is the proNGF- p75 neurotrophin receptor (p75NTR)- sortilin neurotrophin complex and the other is the p97R155H mutant complex. The neurotrophins are a family of soluble basic growth factors involved in the development, maintenance, and proliferation of neurons in the central nervous system (CNS) and peripheral nervous system (PNS). The ligand for the neuronal receptors dictates the fate of the neuronal cells. My studies focused on understanding the binding interfaces between the proteins in the proNGF-p75NTR-sortilin neuronal apoptotic complex. I have performed the biochemical characterization of the complex to understand how the complex formation occurs.
Single amino-acid mutation of R155H on the N-domain of p97 is known to be the prevalent mutation in 40% patients suffering from neurodegenerative disease. The p97R155H mutant exhibits abnormal ATPase activity and cofactor dysregulation. I pursued biochemical characterization in combination with single-particle cryo-EM to explore the interaction of p97R155H mutant with its cofactor p47 and determined the full-length structures of the p97R155H-p47 assemblies for the first time. About 40% p97R155H organizes into higher order dodecamers, which lacks nucleotide binding, does not bind to p47, and closely resembles the structure of p97 bound with an adenosine triphosphate (ATP)-competitive inhibitor, CB-5083, suggesting an inactive state of the p97R155H mutant. The structures also revealed conformational changes of the arginine fingers which might contribute to the elevated p97R155H ATPase activity. Because the D1-D2 domain communication is important in regulating the ATPase function, I further studied the functions of the conserved L464 residue on the D1-D2 linker using mutagenesis and single-particle cryo-EM. The biochemical and structural results suggested the torsional constraint of the D1-D2 linker likely modulates the D2 ATPase activity. Our studies thus contributed to develop deeper knowledge of the intricate cellular mechanisms and the proteins affected in disease pathways.
ContributorsNandi, Purbasha (Author) / Chiu, Po-Lin (Thesis advisor) / Mazor, Yuval (Committee member) / Hansen, Debra T (Committee member) / Arizona State University (Publisher)
Created2022
Description
In recent years, researchers have employed DNA and protein nanotechnology to develop nanomaterials for applications in the fields of regenerative medicine, gene therapeutic, and materials science. In the current state of research, developing a biomimetic approach to fabricate an extracellular matrix (ECM)-like material has faced key challenges. The difficulty arises due to achieving spatiotemporal complexity that rivals the native ECM. Attempts to replicate the ECM using hydrogels have been limited in their ability to recapitulate its structural and functional properties. Moreover, the biological activities of the ECM, such as cell adhesion, proliferation, and differentiation, are mediated by ECM proteins and their interactions with cells, making it difficult to reproduce these activities in vitro.Thus, the work presented in my dissertation represents efforts to develop DNA and protein-based materials that mimic the biological properties of the ECM. The research involves the design, synthesis, and characterization of nanomaterials that exhibit unique physical, chemical, and mechanical properties. Two specific aspects of the biomimetic system have been to include (1) a modular protein building block to change the bioactivity of the system and (2) to temporally control the self-assembly of the protein nanofiber using different coiled coil mechanisms. The protein nanofibers were characterized using atomic force microscopy, transmission electron microscopy, and super-resolution DNA Point Accumulation for Imaging in Nanoscale Topology. The domains chosen are the fibronectin domains, Fn-III10, Fn-III9-10, and Fn-III12-14, with bioactivity such as cell adhesion and growth factor binding.
To extend this approach, these cys-nanofibers have been embedded in a hyaluronic acid scaffold to enable bioactivity and fibrous morphologies. Nanofiber integration within the HA gel has been shown to promote tunable mechanical properties and architectures, in addition to promoting a temporal display of the protein nanofibers. The hydrogels were characterized using scanning electron microscopy, mechanical compression testing, and fluorescence microscopy. The findings in this dissertation highlight the promise of biomimetic DNA and protein nanomaterials as a versatile approach for developing next-generation materials with unprecedented properties and functions. These findings continue to push the boundaries of what is possible in nanotechnology, leading to new discoveries that will have a significant impact on society.
ContributorsBernal-Chanchavac, Julio (Author) / Stephanopoulos, Nicholas (Thesis advisor) / Jones, Anne (Committee member) / Mills, Jeremy (Committee member) / Arizona State University (Publisher)
Created2023
Description
Exoelectrogenic organisms transfer electrons from their quinone pool to extracellular acceptors over m-scale distances through appendages known as “biological nanowires”. These structures have been described as cytochrome-rich membrane extensions or pili. However, the components and mechanisms of this long-range electron transfer remain largely unknown. This dissertation describes supramolecular assembly of a tetraheme cytochrome into well-defined models of microbial nanowires and uses those structures to explore the mechanisms of ultra-long-range electron transfer. Chiral-induced-spin-selectivity through the cytochrome is also demonstrated. Nanowire extensions in Shewanella oneidensis have been hypothesized to transfer electrons via electron tunneling through proteinaceous structures that reinforce π-π stacking or through electron hopping via redox cofactors found along their lengths. To provide a model to evaluate the possibility of electron hopping along micron-scale distances, the first part of this dissertation describes the construction of a two-component, supramolecular nanostructure comprised of a small tetraheme cytochrome (STC) from Shewanella oneidensis fused to a peptide domain that self-assembles with a β-fibrillizing peptide. Structural and electrical characterization shows that the self-assembled protein fibers have dimensions relevant to understanding ultralong-range electron transfer and conduct electrons along their length via a cytochrome-mediated mechanism of electron transfer. The second part of this dissertations shows that a model three-component fiber construct based on charge complementary peptides and the redox protein can also be assembled. Structural and electrical characterization of the three-component structure also demonstrates desirable dimensions and electron conductivity along the length via a cytochrome-mediated mechanism.
In vivo, it has been hypothesized that cytochromes in the outer surface conduit are spin-selective. However, cytochromes in the periplasm of Shewanella oneidensis have not been shown to be spin selective, and the physiological impact of the chiral-induced-spin-selectivity (CISS) effect on microbial electron transport remains unclear. In the third part of this dissertation, investigations via spin polarization and a spin-dependent conduction study show that STC is spin selective, suggesting that spin selectivity may be an important factor in the electron transport efficiency of exoelectrogens.
In conclusion, this dissertation enables a better understanding of long-range electron transfer in bacterial nanowires and bioelectronic circuitry and offers suggestions for how to construct enhanced biosensors.
ContributorsNWACHUKWU, JUSTUS NMADUKA (Author) / Jones, Anne K. (Thesis advisor) / Mills, Jeremy (Committee member) / Stephanopoulos, Nicholas (Committee member) / Arizona State University (Publisher)
Created2023
Description
Receiving signals and responding to the environment is crucial for survival for every living organism. One of those signals is being able to detect environmental and visceral temperatures. Transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential melastatin 8 (TRPM8) are ion channels within cells that allow higher organisms to detect hot and cold temperatures, respectively. These TRP channels are also implicated in diverse physiological roles including pain, obesity, and cancer. As a result, these channels have garnered interest as potential targets for therapeutic interventions. However, the entanglement of TRPV1 and TRPM8 polymodal activation where it responds to a variety of different stimuli has caused adverse side effects of body thermal dysregulation and misregulation when antagonizing these channels as drug targets. This dissertation will dissect the molecular mechanism and regulation of TRPV1 and TRPM8. An in-depth look into the complex and conflicting results in trying to find the key area for thermosensation as well as looking into disentangling the polymodal activation modes in TRPV1. The regulatory mechanism between TRPM8 with phosphoinositide interacting regulator of TRPs (PIRT) and calmodulin will be examined using nuclear magnetic resonance (NMR). A computational, experimental, and methodical approach into ancestral TRPM8 orthologs using whole-cell patch-clamp electrophysiology, calcium mobilization assay, and cellular thermal shift assay (CETSA) to determine whether these modes of activation can be decoupled. Lastly, smaller studies are covered like developing a way to delivery full-length and truncated protein using amphipols to artificial and live cells without the biological regulatory processes and the purification of the TRPM8 transmembrane domain (TMD). In the end, two successful methods were developed to study the polymodal activation of proteins.
ContributorsLuu, Dustin Dean (Author) / Van Horn, Wade D (Thesis advisor) / Redding, Kevin E (Committee member) / Chiu, Po-Lin (Committee member) / Arizona State University (Publisher)
Created2023