Matching Items (17)
Description
ABSTRACT
X-Ray crystallography and NMR are two major ways of achieving atomic
resolution of structure determination for macro biomolecules such as proteins. Recently, new developments of hard X-ray pulsed free electron laser XFEL opened up new possibilities to break the dilemma of radiation dose and spatial resolution in diffraction imaging by outrunning radiation damage with ultra high brightness femtosecond X-ray pulses, which is so short in time that the pulse terminates before atomic motion starts. A variety of experimental techniques for structure determination of macro biomolecules is now available including imaging of protein nanocrystals, single particles such as viruses, pump-probe experiments for time-resolved nanocrystallography, and snapshot wide- angle x-ray scattering (WAXS) from molecules in solution. However, due to the nature of the "diffract-then-destroy" process, each protein crystal would be destroyed once
probed. Hence a new sample delivery system is required to replenish the target crystal at a high rate. In this dissertation, the sample delivery systems for the application of XFELs to biomolecular imaging will be discussed and the severe challenges related to the delivering of macroscopic protein crystal in a stable controllable way with minimum waste of sample and maximum hit rate will be tackled with several different development of injector designs and approaches. New developments of the sample delivery system such as liquid mixing jet also opens up new experimental methods which gives opportunities to study of the chemical dynamics in biomolecules in a molecular structural level. The design and characterization of the system will be discussed along with future possible developments and applications. Finally, LCP injector will be discussed which is critical for the success in various applications.
X-Ray crystallography and NMR are two major ways of achieving atomic
resolution of structure determination for macro biomolecules such as proteins. Recently, new developments of hard X-ray pulsed free electron laser XFEL opened up new possibilities to break the dilemma of radiation dose and spatial resolution in diffraction imaging by outrunning radiation damage with ultra high brightness femtosecond X-ray pulses, which is so short in time that the pulse terminates before atomic motion starts. A variety of experimental techniques for structure determination of macro biomolecules is now available including imaging of protein nanocrystals, single particles such as viruses, pump-probe experiments for time-resolved nanocrystallography, and snapshot wide- angle x-ray scattering (WAXS) from molecules in solution. However, due to the nature of the "diffract-then-destroy" process, each protein crystal would be destroyed once
probed. Hence a new sample delivery system is required to replenish the target crystal at a high rate. In this dissertation, the sample delivery systems for the application of XFELs to biomolecular imaging will be discussed and the severe challenges related to the delivering of macroscopic protein crystal in a stable controllable way with minimum waste of sample and maximum hit rate will be tackled with several different development of injector designs and approaches. New developments of the sample delivery system such as liquid mixing jet also opens up new experimental methods which gives opportunities to study of the chemical dynamics in biomolecules in a molecular structural level. The design and characterization of the system will be discussed along with future possible developments and applications. Finally, LCP injector will be discussed which is critical for the success in various applications.
ContributorsWang, Dingjie (Author) / Spence, John CH (Thesis advisor) / Weierstall, Uwe (Committee member) / Schmidt, Kevin (Committee member) / Fromme, Petra (Committee member) / Ozkan, Banu (Committee member) / Arizona State University (Publisher)
Created2014
Description
The goal of this theoretical study of infrared spectra was to ascertain to what degree molecules may be identified from their IR spectra and which spectral regions are best suited for this purpose. The frequencies considered range from the lowest frequency molecular vibrations in the far-IR, terahertz region (below ~3 THz or 100 cm-1) up to the highest frequency vibrations (~120 THz or 4000 cm-1). An emphasis was placed on the IR spectra of chemical and biological threat molecules in the interest of detection and prevention. To calculate IR spectra, the technique of normal mode analysis was applied to organic molecules ranging in size from 8 to 11,352 atoms. The IR intensities of the vibrational modes were calculated in terms of the derivative of the molecular dipole moment with respect to each normal coordinate. Three sets of molecules were studied: the organophosphorus G- and V-type nerve agents and chemically related simulants (15 molecules ranging in size from 11 to 40 atoms); 21 other small molecules ranging in size from 8 to 24 atoms; and 13 proteins ranging in size from 304 to 11,352 atoms. Spectra for the first two sets of molecules were calculated using quantum chemistry software, the last two sets using force fields. The "middle" set used both methods, allowing for comparison between them and with experimental spectra from the NIST/EPA Gas-Phase Infrared Library. The calculated spectra of proteins, for which only force field calculations are practical, reproduced the experimentally observed amide I and II bands, but they were shifted by approximately +40 cm-1 relative to experiment. Considering the entire spectrum of protein vibrations, the most promising frequency range for differentiating between proteins was approximately 600-1300 cm-1 where water has low absorption and the proteins show some differences.
ContributorsMott, Adam J (Author) / Rez, Peter (Thesis advisor) / Ozkan, Banu (Committee member) / Shumway, John (Committee member) / Thorpe, Michael (Committee member) / Vaiana, Sara (Committee member) / Arizona State University (Publisher)
Created2012
Description
A form of nanoscale steganography exists described as DNA origami cryptography which is a technique of secure information encryption through scaffold, staple, and varying docking strand self- assembling mixtures. The all-DNA steganography based origami was imaged through high-speed DNA-PAINT super-resolution imaging which uses periodic docking sequences to eliminate the need for protein binding. The purpose of this research was to improve upon the DNA origami cryptography protocol by encrypting information in 2D Rothemund Rectangular DNA Origami (RRO) and 3D cuboctahedron DNA origami as a platform of self-assembling DNA nanostructures to increase the routing possibilities of the scaffold. The initial focus of the work was increasing the incorporation efficiency of all individual docking spots for full 20nm grid RRO pattern readout. Due to this procedural optimization was pursued by altering annealing cycle length, centrifugal spin rates for purification, and lengthening docking strands vs. imager poly T linkers. A 14nm grid was explored as an intermediate prior to the 10nm grid for comparison of optimized experimental procedure for a higher density encryption pattern option. Imager concentration was discovered to be a vital determining factor in effectively resolving the 10nm grids due to high concentrations of imager strands inducing simultaneous blinking of adjacent docking strands to be more likely causing the 10nm grids to not be resolved. A 2 redundancy and 3 redundancy encryption scheme was developed for the 10nm grid RRO to be encrypted with. Further experimentation was completed to resolve full 10nm DNA-origami grids and encrypt with the message ”ASU”. The message was successfully encrypted and resolved through the high density 10nm grid with 2 and 3 redundancy patterns. A cuboctahedron 3D origami was explored with DNA-PAINT techniques as well resulting in successful resolution of the z-axis through variation of biotin linker length and calibration file. Positive results for short message ”0407” encryption of the cuboctahedron were achieved. Data encryption in DNA origami is further being explored and could be an optimal solution for higher density data storage with greater longevity of media.
ContributorsSukhareva, Daria (Author) / Hariadi, Rizal (Thesis director) / Sulc, Petr (Committee member) / Matthies, Michael (Committee member) / Barrett, The Honors College (Contributor) / School of Molecular Sciences (Contributor)
Created2023-05
Description
Cyclodextrins are known for their pharmaceutical applications in a range of pathologies. Beta(ꞵ)-cyclodextrins have been suggested to be effective scaffolds that can ligate to peptides when chemically modified, which has the potential to be cost-effective in comparison to other available treatments for antiviral therapeutics. It is hypothesized that a ꞵ-cyclodextrin platform can be modified through a few-step reaction process to develop a ꞵ-cyclodextrin-DBCO-GFP nanobody. The findings of this few-step reaction support the general approach of conjugating the ꞵ-cyclodextrin derivative to GPF nanobody for developing a cyclodextrin antiviral scaffold.
ContributorsTaniguchi, Tohma (Author) / Hariadi, Rizal (Thesis director) / Stephanopoulos, Nicholas (Committee member) / Sasmal, Ranjan (Committee member) / Barrett, The Honors College (Contributor) / School of Molecular Sciences (Contributor)
Created2023-05
Description
With climate change threatening to increase the frequency of global pandemics, the need for quick and adaptable responses to novel viruses will become paramount. DNA nanotechnology offers a highly customizable, biocompatible approach to combating novel outbreaks. For any DNA nanotechnology-based therapeutic to have future success in vivo, the structure must be able to withstand serological conditions for an extended time period. In this study, the stability of a wireframe DNA snub cube with attached nbGFP used to bind a nonessential viral epitope on Pseudorabies virus is evaluated in vitro both with and without one of two modifications designed to enhance stability: 1) the use of trivalent spermidine cations during thermal annealing of the nanostructure, and 2) the introduction of a polylysine-polyethylene glycol coating to the conjugated nanostructure. The design, synthesis, and purification of the multivalent inhibitor were also evaluated and optimized. Without modification, the snub cube nanostructure was stable for up to 8 hours in culture media supplemented with 10% FBS. The spermidine-annealed nanostructures demonstrated lesser degrees of stability and greater degradation than the unmodified structures, whereas the polylysine-coated structures demonstrated equivalent stability at lower valencies and enhanced stability at the highest valency of the snub cube inhibitor. These results support the potential for the polylysine-polyethylene glycol coating as a potential method for enhancing the stability of the snub cube for future in vivo applications.
ContributorsLeff, Chloe (Author) / Hariadi, Rizal (Thesis director) / Stephanopoulos, Nicholas (Committee member) / Pradhan, Swechchha (Committee member) / Barrett, The Honors College (Contributor) / School of Life Sciences (Contributor) / School of Molecular Sciences (Contributor)
Created2023-05
Description
The propulsion matrix provides a compact description of the locomotion of a single flagella molecular motor in a low Reynolds number environment. The locomotion properties of individual flagellar motors are central to bacterial behavior, including chemotaxis, pathogenesis, and biofilm formation. However, because conventional hydrodynamic measurement approaches require applied forces, torques, or fluid flows, it is not possible to directly measure the propulsion matrix for an individual microscale helical filament. Here, the limitations inherent to conventional measurement approaches are overcome using a combination of theoretical, experimental, and computational advancements. First, the relationship between the elements of the propulsion matrix with translational and rotational Brownian motion is derived using the fluctuation-dissipation theorem. Next, a volumetric fluorescent imaging using high resolution oblique plane microscopy with sufficient spatio-temporal resolution is conducted to resolve both translation and rotation of individual helical filaments isolated from E.coli's flagellar motor. Finally, a computational framework is developed to track individual helical filaments across six degrees of freedom, extract diffusion coefficients, and quantify the temporal correlation between translation and rotation. This study computed the maximum propulsion efficiency to be around 1.7%. Direct measurement of propulsion efficiency generally agrees with the ensemble and large-scale measurements previously performed using conventional hydrodynamic measurements. The findings suggest that the approach described here can be extended to more complex in-vitro experiments that evaluate microscale molecular motors. For example, evaluating sperm motility without inducing chemotaxis or utilizing a microfluidic setup.
ContributorsDjutanta, Franky (Author) / Hariadi, Rizal (Thesis advisor) / Wang, Robert (Thesis advisor) / Yurke, Bernard (Committee member) / Herrmann, Marcus (Committee member) / Huang, Huei-Ping (Committee member) / Arizona State University (Publisher)
Created2022
Description
Transition metal ions such as Zn2+, Mn2+, Co2+, and Fe2+ play crucial roles in organisms from all kingdoms of life. The homeostasis of these ions is mainly regulated by a group of secondary transporters from the cation diffusion facilitator (CDF) family. The mammalian zinc transporters (ZnTs), a subfamily of CDF, have been an important target for study as they are associated with several diseases, such as diabetes, delayed growth and osteopenia, Alzheimer’s disease, and Parkinsonism. The bacterial homolog of ZnTs, YiiP, is the first CDF transporter with a determined structure and is used as a model for studying the structural and mechanistic properties of CDF transporters. On the other hand, Molecular dynamics simulation has emerged as a valuable computational tool for exploring the physical basis of biological macromolecules' structure and function with atomic precision at femtosecond resolution. This work aims to elucidate the roles of the three Zn$2+ binding sites found on each YiiP protomer and the role of protons in the transport process of CDFs, which remain under debate despite previous thermodynamic and structural studies on YiiP. Cryo-EM, microscale thermophoresis (MST) and molecular dynamics (MD) simulations were used to address these questions. With a Zn2+ model that accurately reproduces experimental structures of the binding clusters, the dynamical influence of zinc binding on the transporter was accessed through MD simulations, which was consistent with the new cryo-EM structures. Zinc binding affinities obtained through MST were used to infer the stoichiometry of Zn2+/H+ antiport in combination with a microscopic thermodynamic model and constant pH simulations. The most likely microstates of H$^+$ and Zn2+ binding indicated a transport stoichiometry of 1 Zn2+ to 2-3 H+ depending on the external pH. A model describing the entire transport cycle of YiiP was finally built on these findings, providing insight into the structural and mechanistic properties of CDF transporters.
ContributorsFan, Shujie (Author) / Beckstein, Oliver (Thesis advisor) / Ozkan, Banu (Committee member) / Heyden, Matthias (Committee member) / Van Horn, Wade (Committee member) / Arizona State University (Publisher)
Created2023
Description
The Bayesian paradigm provides a flexible and versatile framework for modeling complex biological systems without assuming a fixed functional form or other constraints on the underlying data. This dissertation explores the use of Bayesian nonparametric methods for analyzing fluorescence microscopy data in biophysics, with a focus on enumerating diffraction-limited particles, reconstructing potentials from trajectories corrupted by measurement noise, and inferring potential energy landscapes from fluorescence intensity experiments. This research demonstrates the power and potential of Bayesian methods for solving a variety of problems in fluorescence microscopy and biophysics more broadly.
ContributorsBryan IV, J Shepard (Author) / Presse, Steve (Thesis advisor) / Ozkan, Banu (Committee member) / Wadhwa, Navish (Committee member) / Shepherd, Doug (Committee member) / Arizona State University (Publisher)
Created2023
Description
Secondary active transporters play significant roles in maintaining living cells' homeostasis by utilizing the electrochemical gradient in driving ions or protons as the source of free energy to transport substrate through biological membranes.A broadly recognized molecular framework, the alternating access model, describes the transport mechanism as the transporter undergoes conformational changes between different conformations and alternatingly exposes its binding site to intracellular and extracellular sides and, thus, exchanges ion and substrate in a cyclical manner.
Recent progress in structural biology brought the first-ever structural insights into the mammalian Cation-Proton Antiporters (CPA) family of proteins.
However, the dynamic atomic-level information about the interactions between the newly discovered structures and the bound ion or the corresponding substrate remains unknown.
With Molecular Dynamics (MD), multiple spontaneous ion binding events were observed in the equilibrium simulations, revealing the binding site topology of Horse Sodium-Proton Exchanger 9 (NHE9) and Bison Sodium-Proton Antiporter 2 (NHA2) in their preferred protonation state.
Further investigation into more CPA homologs compared various aspects, including sequence identity, binding site topology, and energetic properties, and obtained general insights into the similarities shared by the binding process of CPA members.
The putative binding site and other conserved residues in their actively ion-bound poses were identified for each model, and their similarities were compared.
The energetic properties accessed by the three-dimensional free energy profile, initially found to be binding unfavorable for the experimental structures, were recalculated based on the simulation data. The updated results show consistency with the correct binding affinity as indicated by the experimental methods.
This work provided a general picture of the structures and the ion-protein interaction of CPA proteins and serves as comprehensive guidance for any related future structural and computational work.
ContributorsZhang, Chenou (Author) / Beckstein, Oliver (Thesis advisor) / Ozkan, Banu (Committee member) / Ros, Robert (Committee member) / Singharoy, Abhishek (Committee member) / Arizona State University (Publisher)
Created2023
Description
Scientists are entrusted with developing novel molecular strategies for effective prophylactic and therapeutic interventions. Antivirals are indispensable tools that can be targeted at viral domains directly or at cellular domains indirectly to obstruct viral infections and reduce pathogenicity. Despite their transformative potential in healthcare, to date, antivirals have been clinically approved to treat only 10 out of the greater than 200 known pathogenic human viruses. Additionally, as obligate intracellular parasites, many virus functions are intimately coupled with host cellular processes. As such, the development of a clinically relevant antiviral is challenged by the limited number of clear targets per virus and necessitates an extensive insight into these molecular processes. Compounding this challenge, many viral pathogens have evolved to evade effective antivirals. Therefore, a means to develop virus- or strain-specific antivirals without detailed insight into each idiosyncratic biochemical mechanism may aid in the development of antivirals against a larger swath of pathogens. Such an approach will tremendously benefit from having the specific molecular recognition of viral species as the lowest barrier. Here, I modify a nanobody (anti-green fluorescent protein) that specifically recognizes non-essential epitopes (glycoprotein M-pHluorin chimera) presented on the extra virion surface of a virus (Pseudorabies virus strain 486). The nanobody switches from having no inhibitory properties (tested up to 50 μM) to ∼3 nM IC50 in in vitro infectivity assays using porcine kidney (PK15) cells. The nanobody modifications use highly reliable bioconjugation to a three-dimensional wireframe deoxyribonucleic acid (DNA) origami scaffold. Mechanistic studies suggest that inhibition is mediated by the DNA origami scaffold bound to the virus particle, which obstructs the internalization of the viruses into cells, and that inhibition is enhanced by avidity resulting from multivalent virus and scaffold interactions. The assembled nanostructures demonstrate negligible cytotoxicity (<10 nM) and sufficient stability, further supporting their therapeutic potential. If translatable to other viral species and epitopes, this approach may open a new strategy that leverages existing infrastructures – monoclonal antibody development, phage display, and in vitro evolution - for rapidly developing novel antivirals in vivo.
ContributorsPradhan, Swechchha (Author) / Hariadi, Rizal (Thesis advisor) / Hogue, Ian (Committee member) / Varsani, Arvind (Committee member) / Chen, Qiang (Committee member) / Arizona State University (Publisher)
Created2022