Matching Items (43)
Description
Non-alcoholic fatty liver disease occurs when triglycerides are stored in the liver leading to irreversible scarring and damage of liver tissue. Inside the liver, adipose triglyceride lipase is responsible for the breaking down of triglycerides and is regulated by the inhibitor g0/g1 switch gene 2 (G0S2). G0S2 is proposed to be one of the targets against drug design for non-alcoholic fatty liver disease, and more information is needed on the structure of this protein to aid in drug discovery. Here I describe the expression of G0S2 in an E. coli system as well as purification and biophysical characterization of a functional G0S2 in amounts viable for solution state Nuclear Magnetic Resonance (NMR) spectroscopy. Initial spectra of the isotopically labeled protein show well dispersed 15N resonance lines, clean 13C resonances, and dominant a-helices characteristics. These results show that a prepared G0S2 construct is suitable for solution NMR such that 20 amino acids are now assigned in the G0S2 portion of the protein, allowing for further NMR work with this protein for structural discovery. Further work with a large oligomeric complex of G0S2 with Maltose Binding Protein also shows promise for future cryo-EM work.
ContributorsMoran, Michael William (Author) / Fromma, Petra (Thesis advisor) / Guo, Jia (Committee member) / Liu, Wei (Committee member) / Arizona State University (Publisher)
Created2020
Description
G protein-coupled receptors (GPCRs) are known to be modulated by membrane cholesterol levels, but whether or not the effects are caused by specific receptor-cholesterol interactions or cholesterol’s general effects on the membrane is not well-understood. Results from coarse-grained molecular dynamics (CGMD) simulations coupled and structural bioinformatics offer new insights into how cholesterol modulates GPCR function by showing cholesterol interactions with β2AR that agree with previously published data. Additionally, differential and specific cholesterol binding in the CCK receptor subfamily was observed while revealing a previously unreported Cholesterol Recognition Amino-acid Consensus (CRAC) sequence that is also conserved across 38% of class A GPCRs. Mutation of this conserved CRAC sequence of the β2AR affects cholesterol stabilization of the receptor in a lipid bilayer. Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) has proven highly successful for structure determination of challenging membrane proteins crystallized in lipidic cubic phase, however, as most techniques, it has limitations. Using an optimized SFX experimental setup in a helium atmosphere we determined the room temperature structure of the adenosine A2A receptor (A2AAR) at 2.0 Å resolution and compared it with previous A2AAR structures determined in vacuum and/or at cryogenic temperatures. Specifically, we demonstrated the capability of utilizing high XFEL beam transmissions, in conjunction with a high dynamic range detector, to collect high-resolution SFX data while reducing crystalline material consumption and shortening the collection time required for a complete data set.
The results of these studies provide a better understanding of receptor-cholesterol interactions that can contribute to novel and improved therapeutics for a variety of diseases. Furthermore, the experimental setups presented herein can be applied to future molecular dynamics and SFX applications for protein nanocrystal samples to aid in structure-based discovery efforts of therapeutic targets that are difficult to crystallize.
The results of these studies provide a better understanding of receptor-cholesterol interactions that can contribute to novel and improved therapeutics for a variety of diseases. Furthermore, the experimental setups presented herein can be applied to future molecular dynamics and SFX applications for protein nanocrystal samples to aid in structure-based discovery efforts of therapeutic targets that are difficult to crystallize.
ContributorsGeiger, James (Author) / Liu, Wei (Thesis advisor) / Mills, Jeremy (Committee member) / Chiu, Po-Lin (Committee member) / Arizona State University (Publisher)
Created2020
Description
This thesis focuses on serial crystallography studies with X-ray free electron lasers
(XFEL) with a special emphasis on data analysis to investigate important processes
in bioenergy conversion and medicinal applications.
First, the work on photosynthesis focuses on time-resolved femtosecond crystallography
studies of Photosystem II (PSII). The structural-dynamic studies of the water
splitting reaction centering on PSII is a current hot topic of interest in the field, the
goal of which is to capture snapshots of the structural changes during the Kok cycle.
This thesis presents results from time-resolved serial femtosecond (fs) crystallography
experiments (TR-SFX) where data sets are collected at room temperature from a
stream of crystals that intersect with the ultrashort femtosecond X-ray pulses at an
XFEL with the goal to obtain structural information from the transient state (S4)
state of the cycle where the O=O bond is formed, and oxygen is released. The most
current techniques available in SFX/TR-SFX to handle hundreds of millions of raw
diffraction patterns are discussed, including selection of the best diffraction patterns,
allowing for their indexing and further data processing. The results include two 4.0 Å
resolution structures of the ground S1 state and triple excited S4 transient state.
Second, this thesis reports on the first international XFEL user experiments in
South Korea at the Pohang Accelerator Laboratory (PAL-XFEL). The usability of this
new XFEL in a proof-of-principle experiment for the study of microcrystals of human
taspase1 (an important cancer target) by SFX has been tested. The descriptions of
experiments and discussions of specific data evaluation challenges of this project in
light of the taspase1 crystals’ high anisotropy, which limited the resolution to 4.5 Å,
are included in this report
In summary, this thesis examines current techniques that are available in the
SFX/TR-SFX domain to study crystal structures from microcrystals damage-free,
with the future potential of making movies of biological processes.
(XFEL) with a special emphasis on data analysis to investigate important processes
in bioenergy conversion and medicinal applications.
First, the work on photosynthesis focuses on time-resolved femtosecond crystallography
studies of Photosystem II (PSII). The structural-dynamic studies of the water
splitting reaction centering on PSII is a current hot topic of interest in the field, the
goal of which is to capture snapshots of the structural changes during the Kok cycle.
This thesis presents results from time-resolved serial femtosecond (fs) crystallography
experiments (TR-SFX) where data sets are collected at room temperature from a
stream of crystals that intersect with the ultrashort femtosecond X-ray pulses at an
XFEL with the goal to obtain structural information from the transient state (S4)
state of the cycle where the O=O bond is formed, and oxygen is released. The most
current techniques available in SFX/TR-SFX to handle hundreds of millions of raw
diffraction patterns are discussed, including selection of the best diffraction patterns,
allowing for their indexing and further data processing. The results include two 4.0 Å
resolution structures of the ground S1 state and triple excited S4 transient state.
Second, this thesis reports on the first international XFEL user experiments in
South Korea at the Pohang Accelerator Laboratory (PAL-XFEL). The usability of this
new XFEL in a proof-of-principle experiment for the study of microcrystals of human
taspase1 (an important cancer target) by SFX has been tested. The descriptions of
experiments and discussions of specific data evaluation challenges of this project in
light of the taspase1 crystals’ high anisotropy, which limited the resolution to 4.5 Å,
are included in this report
In summary, this thesis examines current techniques that are available in the
SFX/TR-SFX domain to study crystal structures from microcrystals damage-free,
with the future potential of making movies of biological processes.
ContributorsKetawala, Gihan Kaushyal (Author) / Fromme, Petra (Thesis advisor) / Liu, Wei (Committee member) / Kirian, Richard (Committee member) / Arizona State University (Publisher)
Created2020
Description
G protein-coupled receptors (GPCRs) are a large family of proteins involved in the cell signaling and regulation of many biological and pathological processes in the human body. To fully understand their functions, various approaches are needed. This work combines several techniques to advance the study of GPCRs with the overarching goal of pursuing X-ray crystallization using lipidic cubic phase (LCP). In meso, or LCP crystallization method involves imbedding the GPCR into a lipid membrane-mimetic material which spontaneously forms when monoacylglycerols (MAGs) are mixed at the correct hydration level and temperature. It provides a stable environment for GPCRs and has been established as the most common method to resolve structural details of GPCRs (Chapter 2). Yet, before crystallization, GPCRs need to be put through several rounds of optimization of the construct design, including truncation of N- and C- termini, fusing different soluble proteins, and mutating the receptor (Chapter 3). Other methods were also used to gain structural insights into GPCR interactions, such as coarse-grained molecular dynamic simulations, which showed the specific regions of interactions with cholesterol molecules imbedded in the membranes (Chapter 4). This study demonstrated β2-adrenergic receptor (β2AR), a GPCR, as a model of a cholesterol-sensitive receptor. Mutations were made to test the effect of removing specific residues of interest on cholesterol stabilization through the LCP-Tm assay, producing results that align with the simulation data. Finally, the goal of the last study is to provide a guide to identify which host lipids form stable LCP phases for different applications (Chapter 5). Small angle X-ray scattering is used to identify phases in hundreds of different precipitant conditions in the search of suitable host lipid for LCP studies. The results present a systematic overview of the compatibility of common MAGs by screening them against different precipitant solutions including varying salts and polyethylene glycol (PEG) concentrations, different PEG sizes, the presence of detergent or protein in the sample, and the addition of cholesterol. Together, these studies present a variety of methods to advance the structural studies of GPCRs using LCP
ContributorsAL-SAHOURI, ZINA (Author) / Liu, Wei (Thesis advisor) / Stephanopoulos, Nicholas (Committee member) / Chiu, Po-Lin (Committee member) / Arizona State University (Publisher)
Created2021
Description
Crystal structure determination of biological macromolecules using the novel technique of serial femtosecond crystallography (SFX) is severely limited by the scarcity of X-ray free-electron laser (XFEL) sources. However, recent and future upgrades render microfocus beamlines at synchrotron-radiation sources suitable for room-temperature serial crystallography data collection also. Owing to the longer exposure times that are needed at synchrotrons, serial data collection is termed serial millisecond crystallography (SMX). As a result, the number of SMX experiments is growing rapidly, with a dozen experiments reported so far. Here, the first high-viscosity injector-based SMX experiments carried out at a US synchrotron source, the Advanced Photon Source (APS), are reported. Microcrystals (5–20 µm) of a wide variety of proteins, including lysozyme, thaumatin, phycocyanin, the human A[subscript 2A] adenosine receptor (A[subscript 2A]AR), the soluble fragment of the membrane lipoprotein Flpp3 and proteinase K, were screened. Crystals suspended in lipidic cubic phase (LCP) or a high-molecular-weight poly(ethylene oxide) (PEO; molecular weight 8 000 000) were delivered to the beam using a high-viscosity injector. In-house data-reduction (hit-finding) software developed at APS as well as the SFX data-reduction and analysis software suites Cheetah and CrystFEL enabled efficient on-site SMX data monitoring, reduction and processing. Complete data sets were collected for A[subscript 2A]AR, phycocyanin, Flpp3, proteinase K and lysozyme, and the structures of A[subscript 2A]AR, phycocyanin, proteinase K and lysozyme were determined at 3.2, 3.1, 2.65 and 2.05 Å resolution, respectively. The data demonstrate the feasibility of serial millisecond crystallography from 5–20 µm crystals using a high-viscosity injector at APS. The resolution of the crystal structures obtained in this study was dictated by the current flux density and crystal size, but upcoming developments in beamline optics and the planned APS-U upgrade will increase the intensity by two orders of magnitude. These developments will enable structure determination from smaller and/or weakly diffracting microcrystals.
ContributorsMartin Garcia, Jose Manuel (Author) / Conrad, Chelsie (Author) / Nelson, Garrett (Author) / Stander, Natasha (Author) / Zatsepin, Nadia (Author) / Zook, James (Author) / Zhu, Lan (Author) / Geiger, James (Author) / Chun, Eugene (Author) / Kissick, David (Author) / Hilgart, Mark C. (Author) / Ogata, Craig (Author) / Ishchenko, Andrii (Author) / Nagaratnam, Nirupa (Author) / Roy Chowdhury, Shatabdi (Author) / Coe, Jesse (Author) / Subramanian, Ganesh (Author) / Schaffer, Alexander (Author) / James, Daniel (Author) / Ketwala, Gihan (Author) / Venugopalan, Nagarajan (Author) / Xu, Shenglan (Author) / Corcoran, Stephen (Author) / Ferguson, Dale (Author) / Weierstall, Uwe (Author) / Spence, John (Author) / Cherezov, Vadim (Author) / Fromme, Petra (Author) / Fischetti, Robert F. (Author) / Liu, Wei (Author) / College of Liberal Arts and Sciences (Contributor) / School of Molecular Sciences (Contributor) / Biodesign Institute (Contributor) / Applied Structural Discovery (Contributor) / Department of Physics (Contributor)
Created2017-05-24
Description
Serial femtosecond crystallography (SFX) takes advantage of extremely bright and ultrashort pulses produced by x-ray free-electron lasers (XFELs), allowing for the collection of high-resolution diffraction intensities from micrometer-sized crystals at room temperature with minimal radiation damage, using the principle of “diffraction-before-destruction.” However, de novo structure factor phase determination using XFELs has been difficult so far. We demonstrate the ability to solve the crystallographic phase problem for SFX data collected with an XFEL using the anomalous signal from native sulfur atoms, leading to a bias-free room temperature structure of the human A[subscript 2A] adenosine receptor at 1.9 Å resolution. The advancement was made possible by recent improvements in SFX data analysis and the design of injectors and delivery media for streaming hydrated microcrystals. This general method should accelerate structural studies of novel difficult-to-crystallize macromolecules and their complexes.
ContributorsBatyuk, Alexander (Author) / Galli, Lorenzo (Author) / Ishchenko, Andrii (Author) / Han, Gye Won (Author) / Gati, Cornelius (Author) / Popov, Petr A. (Author) / Lee, Ming-Yue (Author) / Stauch, Benjamin (Author) / White, Thomas A. (Author) / Barty, Anton (Author) / Aquila, Andrew (Author) / Hunter, Mark S. (Author) / Liang, Mengning (Author) / Boutet, Sebastien (Author) / Pu, Mengchen (Author) / Liu, Zhi-jie (Author) / Nelson, Garrett (Author) / James, Daniel (Author) / Li, Chufeng (Author) / Zhao, Yun (Author) / Spence, John (Author) / Liu, Wei (Author) / Fromme, Petra (Author) / Katritch, Vsevolod (Author) / Weierstall, Uwe (Author) / Stevens, Raymond C. (Author) / Cherezov, Vadim (Author) / College of Liberal Arts and Sciences (Contributor) / Department of Physics (Contributor) / Biodesign Institute (Contributor) / Applied Structural Discovery (Contributor) / School of Molecular Sciences (Contributor)
Created2016-09-23
Description
Serial femtosecond crystallography (SFX) using X-ray free-electron lasers has produced high-resolution, room temperature, time-resolved protein structures. We report preliminary SFX of Sindbis virus, an enveloped icosahedral RNA virus with ∼700 Å diameter. Microcrystals delivered in viscous agarose medium diffracted to ∼40 Å resolution. Small-angle diffuse X-ray scattering overlaid Bragg peaks and analysis suggests this results from molecular transforms of individual particles. Viral proteins undergo structural changes during entry and infection, which could, in principle, be studied with SFX. This is an important step toward determining room temperature structures from virus microcrystals that may enable time-resolved studies of enveloped viruses.
ContributorsLawrence, Robert (Author) / Conrad, Chelsie (Author) / Zatsepin, Nadia (Author) / Grant, Thomas D. (Author) / Liu, Haiguang (Author) / James, Daniel (Author) / Nelson, Garrett (Author) / Subramanian, Ganesh (Author) / Aquila, Andrew (Author) / Hunter, Mark S. (Author) / Liang, Mengning (Author) / Boutet, Sebastien (Author) / Coe, Jesse (Author) / Spence, John (Author) / Weierstall, Uwe (Author) / Liu, Wei (Author) / Fromme, Petra (Author) / Cherezov, Vadim (Author) / Hogue, Brenda (Author) / Biodesign Institute (Contributor) / Infectious Diseases and Vaccinology (Contributor) / Applied Structural Discovery (Contributor) / Department of Chemistry and Biochemistry (Contributor) / College of Liberal Arts and Sciences (Contributor) / Department of Physics (Contributor) / School of Life Sciences (Contributor)
Created2015-08-20
Description
Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) enables high-resolution protein structure determination using micrometre-sized crystals at room temperature with minimal effects from radiation damage. SFX requires a steady supply of microcrystals intersecting the XFEL beam at random orientations. An LCP–SFX method has recently been introduced in which microcrystals of membrane proteins are grown and delivered for SFX data collection inside a gel-like membrane-mimetic matrix, known as lipidic cubic phase (LCP), using a special LCP microextrusion injector. Here, it is demonstrated that LCP can also be used as a suitable carrier medium for microcrystals of soluble proteins, enabling a dramatic reduction in the amount of crystallized protein required for data collection compared with crystals delivered by liquid injectors. High-quality LCP–SFX data sets were collected for two soluble proteins, lysozyme and phycocyanin, using less than 0.1 mg of each protein.
ContributorsFromme, Raimund (Author) / Ishchenko, Andrii (Author) / Metz, Markus (Author) / Roy Chowdhury, Shatabdi (Author) / Basu, Shibom (Author) / Boutet, Sebastien (Author) / Fromme, Petra (Author) / White, Thomas A. (Author) / Barty, Anton (Author) / Spence, John (Author) / Weierstall, Uwe (Author) / Liu, Wei (Author) / Cherezov, Vadim (Author) / Biodesign Institute (Contributor) / Applied Structural Discovery (Contributor) / College of Liberal Arts and Sciences (Contributor) / Department of Physics (Contributor)
Created2015-08-04
Description
Electricity plays a special role in our lives and life. The dynamics of electrons allow light to flow through a vacuum. The equations of electron dynamics are nearly exact and apply from nuclear particles to stars. These Maxwell equations include a special term, the displacement current (of a vacuum). The displacement current allows electrical signals to propagate through space. Displacement current guarantees that current is exactly conserved from inside atoms to between stars, as long as current is defined as the entire source of the curl of the magnetic field, as Maxwell did.We show that the Bohm formulation of quantum mechanics allows the easy definition of the total current, and its conservation, without the dificulties implicit in the orthodox quantum theory. The orthodox theory neglects the reality of magnitudes, like the currents, during times that they are not being explicitly measured.We show how conservation of current can be derived without mention of the polarization or dielectric properties of matter. We point out that displacement current is handled correctly in electrical engineering by ‘stray capacitances’, although it is rarely discussed explicitly. Matter does not behave as physicists of the 1800’s thought it did. They could only measure on a time scale of seconds and tried to explain dielectric properties and polarization with a single dielectric constant, a real positive number independent of everything. Matter and thus charge moves in enormously complicated ways that cannot be described by a single dielectric constant,when studied on time scales important today for electronic technology and molecular biology. When classical theories could not explain complex charge movements, constants in equations were allowed to vary in solutions of those equations, in a way not justified by mathematics, with predictable consequences. Life occurs in ionic solutions where charge is moved by forces not mentioned or described in the Maxwell equations, like convection and diffusion. These movements and forces produce crucial currents that cannot be described as classical conduction or classical polarization. Derivations of conservation of current involve oversimplified treatments of dielectrics and polarization in nearly every textbook. Because real dielectrics do not behave in that simple way-not even approximately-classical derivations of conservation of current are often distrusted or even ignored. We show that current is conserved inside atoms. We show that current is conserved exactly in any material no matter how complex are the properties of dielectric, polarization, or conduction currents. Electricity has a special role because conservation of current is a universal law.Most models of chemical reactions do not conserve current and need to be changed to do so. On the macroscopic scale of life, conservation of current necessarily links far spread boundaries to each other, correlating inputs and outputs, and thereby creating devices.We suspect that correlations created by displacement current link all scales and allow atoms to control the machines and organisms of life. Conservation of current has a special role in our lives and life, as well as in physics. We believe models, simulations, and computations should conserve current on all scales, as accurately as possible, because physics conserves current that way. We believe models will be much more successful if they conserve current at every level of resolution, the way physics does.We surely need successful models as we try to control macroscopic functions by atomic interventions, in technology, life, and medicine. Maxwell’s displacement current lets us see stars. We hope it will help us see how atoms control life.
ContributorsEisenberg, Bob (Author) / Oriols, Xavier (Author) / Ferry, David (Author) / Ira A. Fulton Schools of Engineering (Contributor) / School of Electrical, Computer and Energy Engineering (Contributor)
Created2017-10-28
Description
We describe procedures for the preparation and delivery of membrane protein microcrystals in lipidic cubic phase for serial crystallography at X-ray free-electron lasers and synchrotron sources. These protocols can also be applied for incorporation and delivery of soluble protein microcrystals, leading to substantially reduced sample consumption compared to liquid injection.
ContributorsIshchenko, Andrii (Author) / Cherezov, Vadim (Author) / Liu, Wei (Author) / College of Liberal Arts and Sciences (Contributor)
Created2016-09-20