Matching Items (17)

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Nucleoporin Gle1: Crystallographic Analysis and the Future of Neuron Disease Pathology

Description

Gle1 is an mRNP export mediator with major activity localized to the nuclear pore complex in eukaryotic cells. The protein's high preservation across vast phylogenetic distances allows us to approximate

Gle1 is an mRNP export mediator with major activity localized to the nuclear pore complex in eukaryotic cells. The protein's high preservation across vast phylogenetic distances allows us to approximate research on the properties of yeast Gle1 (yGle1) with those of human Gle1 (hGle1). Research at Vanderbilt University in 2016, which provides the research basis of this thesis, suggests that the coiled-coil domain of yGle1 is best crystallized in dicationic aqueous conditions of pH ~8.0 and 10\u201420% PEG 8000. Further exploration of crystallizable microconditions revealed a favorability toward lower pH and lower PEG concentration. Following the discovery of the protein's native crystallography conditions, a comprehensive meta-analysis of scientific literature on Gle1 was conducted on the association of Gle1 mutations with neuron disease.

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Date Created
  • 2016-12

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Construct Design, Protein Expression, and Purification of a Human Dopamine Receptor

Description

The dopamine 2 receptor (D2R) is a Class A GPCR which is essential for signaling in the nervous system, and has been implicated in numerous illnesses. While there are over

The dopamine 2 receptor (D2R) is a Class A GPCR which is essential for signaling in the nervous system, and has been implicated in numerous illnesses. While there are over 50 currently approved drugs which act on D2R, the structure has never been determined in detail. Although crystallography has historically been difficult with GPCRs, in recent years many structures have been solved using lipidic cubic phase (LCP) crystallization techniques. Sample preparation for LCP crystallization typically requires optimization of genetic constructs, recombinant expression, and purification techniques in order to produce a sample with sufficient stability and homogeneity. This study compares several genetic constructs utilizing different promoters, fusion proteins, fusion positions, and truncations in order to determine a high quality construct for LCP crystallization of
D2R. All constructs were expressed using the Bac-to-bac baculovirus expression system, then extracted with n-Dodecyl-β-D-Maltoside (DDM) and purified using metal affinity chromatography. Samples were then tested for quantity, purity, and homogeneity using SDS-PAGE, western blot, and size-exclusion chromatography. High quality samples were chosen based on insect cell expression levels, purification yield, and stability estimated by the levels of homomeric protein relative to aggregated protein. A final construct was chosen with which to continue future studies in optimization of thermal stability and crystallization conditions. Future work on this project is required to produce a sample amenable to crystallization. Screening of ligands for co-crystallization,
thermostabilizing point mutations, and potentially optimization of extraction and purification techniques prior to crystallization trials. Solving the D2R structure will lead to an increased understanding of its signaling mechanism and the mechanisms of currently approved drugs, while also providing a basis for more effective structure-based drug design.

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  • 2016-12

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Structural Discovery for Medically Relevant Proteins:Purification and Characterization of the CapBCA Membrane Protein Complex from Francisella tularensis and New Structural Insights into the Function of Catalytically Active Taspase1: A Novel Anti-Cancer

Description

Developments in structural biology has led to advancements in drug design and vaccine development. By better understanding the macromolecular structure, rational choices can be made to improve factors in such

Developments in structural biology has led to advancements in drug design and vaccine development. By better understanding the macromolecular structure, rational choices can be made to improve factors in such as binding affinity, while reducing promiscuity and off-target interactions, improving the medicines of tomorrow. The majority of diseases have a macromolecular basis where rational drug development can make a large impact. Two challenging protein targets of different medical relevance have been investigated at different stages of determining their structures with the ultimate goal of advancing in drug development. The first protein target is the CapBCA membrane protein complex, a virulence factor from the bacterium Francisella tularensis and the causative agent of tularemia and classified as a potential bioterrorism weapon by the United States. Purification of the individual protein targets from the CapBCA complex is a key and challenging step that has been, so far, a limiting factor towards the structure determination of the whole complex. Here, the purification protocols for the CapB and CapC subunits have been establish, which will allow us to progress towards biophysical and structural studies. The second protein target investigated in this thesis is the catalytically active Taspase1. Taspase1 functions as a non-oncogene addiction protease that coordinates cancer cell proliferation and apoptosis and has been found to be overexpressed in many primary human cancers. Here the structure is presented to 3.04A with the goal of rational drug design of Taspase1 inhibitors. Development of Taspase1 inhibitors has no completion in the drug discovery arena and would function as a new anti-cancer therapeutic. Solving the structures of medically relevant proteins such as these is critical towards rapidly developing treatments and prevention of old and new diseases.

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  • 2020-05

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Protein Crystallization in Unit Gravity and Microgravity Environments: Challenges and Opportunities

Description

Protein crystallization is a technique for the formation of three-dimensional protein crystals, which is widely utilized by scientists, engineers, and researchers. Protein crystallography allows for protein structures and functions to

Protein crystallization is a technique for the formation of three-dimensional protein crystals, which is widely utilized by scientists, engineers, and researchers. Protein crystallography allows for protein structures and functions to be studied. As proteins play a central role in biological systems and life itself, a deeper understanding of their structure-function properties is crucial to elucidating fundamental behaviors, such as protein folding in addition to the role that they play in emerging fields, such as, tissue engineering with application to the emerging field of regenerative medicine. However, a significant limitation toward achieving further advancements in this field is that in order to determine detailed structure of proteins from protein crystals, high-quality and larger size protein crystals are needed. Because it is difficult to produce adequate size, high-quality crystals, it remains difficult to determine the structure of many proteins. However, a new method using a microgravity environment to crystallize proteins has proven effective through various studies conducted on the International Space Station (ISS). In the presence of microgravity, free convection is essentially absent in the bulk solution where crystallization occurs, thus allowing for purely random Brownian motion to exist which favors the nucleation and growth of high-quality protein crystals. Many studies from the ISS to date have demonstrated that growing protein crystals in a microgravity environment produces larger and higher-quality crystals. This method provides new opportunities for better structure identification and analysis of proteins. Although there remains many more limitations and challenges in the field, microgravity protein crystallization holds many opportunities for the future of biotechnology and scientific development. The objective of this thesis was to study the crystallization of hen egg white lysozyme (HEWL) and determine the effects of both unit and microgravity on growth/size and quality of HEWL. Through preliminary trials using a universal ground-based reduced-gravity system, the crystallization of HEWL in a simulated microgravity environment was successfully conducted and the results reported are promising. The utility of continuous, scalable ground-based, microgravity platforms for studies on a wide range of material systems and behavior, such as, protein crystallization, has significant implications regarding its impact on many industries, including drug development as well as regenerative medicine.

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Date Created
  • 2020-12

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Allosteric Modulation and Structural Determination of G-Protein Coupled Receptors

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

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.

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Date Created
  • 2020

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Injection methods and instrumentation for serial X-ray free electron laser experiments

Description

Scientists have used X-rays to study biological molecules for nearly a century. Now with the X-ray free electron laser (XFEL), new methods have been developed to advance structural biology. These

Scientists have used X-rays to study biological molecules for nearly a century. Now with the X-ray free electron laser (XFEL), new methods have been developed to advance structural biology. These new methods include serial femtosecond crystallography, single particle imaging, solution scattering, and time resolved techniques.

The XFEL is characterized by high intensity pulses, which are only about 50 femtoseconds in duration. The intensity allows for scattering from microscopic particles, while the short pulses offer a way to outrun radiation damage. XFELs are powerful enough to obliterate most samples in a single pulse. While this allows for a “diffract and destroy” methodology, it also requires instrumentation that can position microscopic particles into the X-ray beam (which may also be microscopic), continuously renew the sample after each pulse, and maintain sample viability during data collection.

Typically these experiments have used liquid microjets to continuously renew sample. The high flow rate associated with liquid microjets requires large amounts of sample, most of which runs to waste between pulses. An injector designed to stream a viscous gel-like material called lipidic cubic phase (LCP) was developed to address this problem. LCP, commonly used as a growth medium for membrane protein crystals, lends itself to low flow rate jetting and so reduces the amount of sample wasted significantly.

This work discusses sample delivery and injection for XFEL experiments. It reviews the liquid microjet method extensively, and presents the LCP injector as a novel device for serial crystallography, including detailed protocols for the LCP injector and anti-settler operation.

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Date Created
  • 2015

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Synthesis, structures and properties of thermoelectric materials in the Zn-Sb-In system

Description

The challenging search for clean, reliable and environmentally friendly energy sources has fueled increased research in thermoelectric materials, which are capable of recovering waste heat. Among the state-of-the-art thermoelectric materials

The challenging search for clean, reliable and environmentally friendly energy sources has fueled increased research in thermoelectric materials, which are capable of recovering waste heat. Among the state-of-the-art thermoelectric materials β-Zn4Sb3 is outstanding because of its ultra-low glass-like thermal conductivity. Attempts to explore ternary phases in the Zn-Sb-In system resulted in the discovery of the new intermetallic compounds, stable Zn5Sb4In2-δ (δ=0.15) and metastable Zn9Sb6In2. Millimeter-sized crystals were grown from molten metal fluxes, where indium metal was employed as a reactive flux medium.Zn5Sb4In2-δ and Zn9Sb6In2 crystallize in new structure types featuring complex framework and the presence of structural disorder (defects and split atomic positions). The structure and phase relations between ternary Zn5Sb4In2-δ, Zn9Sb6In2 and binary Zn4Sb3 are discussed. To establish and understand structure-property relationships, thermoelectric properties measurements were carried out. The measurements suggested that Zn5Sb4In2-δ and Zn9Sb6In2 are narrow band gap semiconductors, similar to β-Zn4Sb3. Also, the peculiar low thermal conductivity of Zn4Sb3 (1 W/mK) is preserved. In the investigated temperature range 10 to 350 K Zn5Sb4In2-δ displays higher thermoelectric figure of merits than Zn4Sb3, indicating a potential significance in thermoelectric applications. Finally, the glass-like thermal conductivities of binary and ternary antimonides with complex structures are compared and the mechanism behind their low thermal conductivities is briefly discussed.

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Date Created
  • 2011

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Sample injector fabrication and delivery method development for serial crystallography using synchrotrons and X-ray free electron lasers

Description

Sample delivery is an essential component in biological imaging using serial diffraction from X-ray Free Electron Lasers (XFEL) and synchrotrons. Recent developments have made possible the near-atomic resolution structure

Sample delivery is an essential component in biological imaging using serial diffraction from X-ray Free Electron Lasers (XFEL) and synchrotrons. Recent developments have made possible the near-atomic resolution structure determination of several important proteins, including one G protein-coupled receptor (GPCR) drug target, whose structure could not easily have been determined otherwise (Appendix A). In this thesis I describe new sample delivery developments that are paramount to advancing this field beyond what has been accomplished to date. Soft Lithography was used to implement sample conservation in the Gas Dynamic Virtual Nozzle (GDVN). A PDMS/glass composite microfluidic injector was created and given the capability of millisecond fluidic switching of a GDVN liquid jet within the divergent section of a 2D Laval-like GDVN nozzle, providing a means of collecting sample between the pulses of current XFELs. An oil/water droplet immersion jet was prototyped that suspends small sample droplets within an oil jet such that the sample droplet frequency may match the XFEL pulse repetition rate. A similar device was designed to use gas bubbles for synchronized “on/off” jet behavior and for active micromixing. 3D printing based on 2-Photon Polymerization (2PP) was used to directly fabricate reproducible GDVN injectors at high resolution, introducing the possibility of systematic nozzle research and highly complex GDVN injectors. Viscous sample delivery using the “LCP injector” was improved with a method for dealing with poorly extruding sample mediums when using full beam transmission from the Linac Coherent Light Source (LCLS), and a new viscous crystal-carrying medium was characterized for use in both vacuum and atmospheric environments: high molecular weight Polyethylene Glycol.

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Date Created
  • 2015

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Time-resolved crystallography using X-ray free-electron laser

Description

Photosystem II (PSII) is a large protein-cofactor complex. The first step in

photosynthesis involves the harvesting of light energy from the sun by the antenna (made

of pigments) of the PSII trans-membrane

Photosystem II (PSII) is a large protein-cofactor complex. The first step in

photosynthesis involves the harvesting of light energy from the sun by the antenna (made

of pigments) of the PSII trans-membrane complex. The harvested excitation energy is

transferred from the antenna complex to the reaction center of the PSII, which leads to a

light-driven charge separation event, from water to plastoquinone. This phenomenal

process has been producing the oxygen that maintains the oxygenic environment of our

planet for the past 2.5 billion years.

The oxygen molecule formation involves the light-driven extraction of 4 electrons

and protons from two water molecules through a multistep reaction, in which the Oxygen

Evolving Center (OEC) of PSII cycles through 5 different oxidation states, S0 to S4.

Unraveling the water-splitting mechanism remains as a grant challenge in the field of

photosynthesis research. This requires the development of an entirely new capability, the

ability to produce molecular movies. This dissertation advances a novel technique, Serial

Femtosecond X-ray crystallography (SFX), into a new realm whereby such time-resolved

molecular movies may be attained. The ultimate goal is to make a “molecular movie” that

reveals the dynamics of the water splitting mechanism using time-resolved SFX (TRSFX)

experiments and the uniquely enabling features of X-ray Free-Electron Laser

(XFEL) for the study of biological processes.

This thesis presents the development of SFX techniques, including development of

new methods to analyze millions of diffraction patterns (~100 terabytes of data per XFEL

experiment) with the goal of solving the X-ray structures in different transition states.

ii

The research comprises significant advancements to XFEL software packages (e.g.,

Cheetah and CrystFEL). Initially these programs could evaluate only 8-10% of all the

data acquired successfully. This research demonstrates that with manual optimizations,

the evaluation success rate was enhanced to 40-50%. These improvements have enabled

TR-SFX, for the first time, to examine the double excited state (S3) of PSII at 5.5-Å. This

breakthrough demonstrated the first indication of conformational changes between the

ground (S1) and the double-excited (S3) states, a result fully consistent with theoretical

predictions.

The power of the TR-SFX technique was further demonstrated with proof-of principle

experiments on Photoactive Yellow Protein (PYP) micro-crystals that high

temporal (10-ns) and spatial (1.5-Å) resolution structures could be achieved.

In summary, this dissertation research heralds the development of the TR-SFX

technique, protocols, and associated data analysis methods that will usher into practice a

new era in structural biology for the recording of ‘molecular movies’ of any biomolecular

process.

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Date Created
  • 2015

Sample delivery enabled by 3D printing for reduced sample consumption and mix-and-inject serial crystallography at x-ray free electron lasers

Description

Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) has enabled the determination of damage-free protein structures at ambient temperatures and of reaction intermediate species with time resolution on

Serial femtosecond crystallography (SFX) with X-ray free electron lasers (XFELs) has enabled the determination of damage-free protein structures at ambient temperatures and of reaction intermediate species with time resolution on the order of hundreds of femtoseconds. However, currently available XFEL facility X-ray pulse structures waste the majority of continuously injected crystal sample, requiring a large quantity (up to grams) of crystal sample to solve a protein structure. Furthermore, mix-and-inject serial crystallography (MISC) at XFEL facilities requires fast mixing for short (millisecond) reaction time points (𝑡"), and current sample delivery methods have complex fabrication and assembly requirements.

To reduce sample consumption during SFX, a 3D printed T-junction for generating segmented aqueous-in-oil droplets was developed. The device surface properties were characterized both with and without a surface coating for improved droplet generation stability. Additionally, the droplet generation frequency was characterized. The 3D printed device interfaced with gas dynamic virtual nozzles (GDVNs) at the Linac Coherent Light Source (LCLS), and a relationship between the aqueous phase volume and the resulting crystal hit rate was developed. Furthermore, at the European XFEL (EuXFEL) a similar quantity and quality of diffraction data was collected for segmented sample delivery using ~60% less sample volume than continuous injection, and a structure of 3-deoxy-D-manno- octulosonate 8-phosphate synthase (KDO8PS) delivered by segmented injection was solved that revealed new structural details to a resolution of 2.8 Å.

For MISC, a 3D printed hydrodynamic focusing mixer for fast mixing by diffusion was developed to automate device fabrication and simplify device assembly. The mixer was characterized with numerical models and fluorescence microscopy. A variety of devices were developed to reach reaction intermediate time points, 𝑡", on the order of 100 – 103 ms. These devices include 3D printed mixers coupled to glass or 3D printed GDVNs and two designs of mixers with GDVNs integrated into the one device. A 3D printed mixer coupled to a glass GDVN was utilized at LCLS to study the oxidation of cytochrome c oxidase (CcO), and a structure of the CcO Pr intermediate was determined at 𝑡" = 8 s.

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Agent

Created

Date Created
  • 2019