Matching Items (17)

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Optimization of a Human Gastrin Receptor for LCP Crystallization

Description

The human gastrin receptor (CCKBR or CCK2R) is a class A G protein-coupled receptor (GPCR) found throughout the central nervous system, stomach, and a variety of cancer cells. CCK2R is

The human gastrin receptor (CCKBR or CCK2R) is a class A G protein-coupled receptor (GPCR) found throughout the central nervous system, stomach, and a variety of cancer cells. CCK2R is implicated in the regulation of biological processes, including anxiety, satiety, arousal, analgesia, psychosis, and cancer cell growth and proliferation. While CCK2R is an attractive drug target, few drugs have managed to effectively target the receptor, and none have been brought to market. Contributory to this is the lack of high-resolution crystal structure capable of elucidating the binding regions of CCK2R to streamlining drug screening. While GPCRs are not amenable to traditional structural analysis methodologies, the advent of lipidic cubic phase (LCP) crystallography and serial femtosecond crystallography (SFX) at X-ray free electron lasers (XFELs), has extended the applicability of X-ray crystallography to these integral membrane proteins. LCP-SFX depends on optimizing the protein of interest for extraction, purification, and crystallization. Here we report our findings regarding the optimization of CCK2R suggesting the synergistic relationship between N-terminal truncations and the insertion of a fusion protein along ICL3, in addition to a 30-residue truncation of the C-terminus. Samples were expressed in Sf9 insect cells using a Bac-to-Bac baculovirus expression system, extracted using n-Dodecyl-β-D-Maltoside detergent, and purified via TALON immobilized metal-ion affinity chromatography. The constructs were characterized via SDS-PAGE, Western blot, and size exclusion chromatography. These findings demonstrate the improvements to CCK2R’s crystallographic amenability upon these modifications, however significant improvements must be made prior to crystallization trials. Future work will involve screening C-terminal truncations, thermostabilizing point mutations, and co-crystallizing ligands. Ideally this investigation will serve as a model for future CCK2R structural analysis and contribute to a heightened interest in CCK2R as a therapeutic target.

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  • 2019-05

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Synthesis of Hybrid DNA-Protein Nanostructures

Description

While DNA and protein nanotechnologies are promising avenues for nanotechnology on their own, merging the two could create more diverse and functional structures. In order to create hybrid structures, the

While DNA and protein nanotechnologies are promising avenues for nanotechnology on their own, merging the two could create more diverse and functional structures. In order to create hybrid structures, the protein will have to undergo site-specific modification, such as the incorporation of an unnatural amino, p-azidophenylalanine (AzF), via Shultz amber codon suppression method, which can then participate in click chemistry with modified DNA. These newly synthesized structures will then be able to self-assemble into higher order structures. Thus far, a surface exposed residue on the aldolase protein has been mutated into an amber stop codon. The next steps are to express the protein with the unnatural amino acid, allow it to participate in click chemistry, and visualize the hybrid structure. If the structure is correct, it will be able to self-assemble.

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  • 2017-05

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Design, Purification, and Analysis of Histamine Family Receptors for Crystallization

Description

G protein-coupled receptors, or GPCRs, are receptors located within the membrane of cells that elicit a wide array of cellular responses through their interactions with G proteins. Recent advances in

G protein-coupled receptors, or GPCRs, are receptors located within the membrane of cells that elicit a wide array of cellular responses through their interactions with G proteins. Recent advances in the use of lipid cubic phase (LCP) for the crystallization of GPCRs, as well as increased knowledge of techniques to improve receptor stability, have led to a large increase in the number of available GPCR structures, despite historic difficulties. This project is focused on the histamine family of receptors, which are Class A GPCRs that are involved in the body’s allergic and inflammatory responses. In particular, the goal of this project was to design, express, and purify histamine receptors with the ultimate goal of crystallization. Successive rounds of optimization included the use of recombinant DNA techniques in E.coli to truncate sections of the proteins and the insertion of several fusion partner proteins to improve receptor expression and stability. All constructs were expressed in a Bac-to-Bac baculovirus expression system using Sf9 insect cells, solubilized using n-Dodecyl-β-D-Maltoside (DDM), and purified using immobilized metal affinity chromatography. Constructs were then analyzed by SDS-Page, Western blot, and size-exclusion chromatography to determine their presence, purity, and homogeneity. Along with their expression data from insect cells, the most stable and homogeneous construct from each round was used to design successive optimizations. After 3 rounds of construct design for each receptor, much work remains to produce a stable sample that has the potential to crystallize. Future work includes further optimization of the insertion site of the fusion proteins, ligand screening for co-crystallization, optimization of purification conditions, and screening of potential thermostabilizing point mutations. Success in solving a structure will allow for a more detailed understanding of the receptor function in addition to its vital use in rational drug discovery.

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  • 2016-12

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Investigating the Stability of DNA Origami Structures in Buffer Solutions

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

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.

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  • 2020-05

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The Formic Acid Cluster Distribution Observed with Femtosecond Laser Ionization

Description

Microsolvation studies have begun to shed the light on the impact that single water molecules have on the structure of a molecule. The difference in behavior that molecules show when

Microsolvation studies have begun to shed the light on the impact that single water molecules have on the structure of a molecule. The difference in behavior that molecules show when exposed to an increasing number of water molecules has been considered important but remains elusive. The cluster distributions of formic acid were studied for its known importance as an intermediate in the water gas shift reaction. Implementations of the water gas shift reaction range from a wide range of applications. Studies have proposed implementations such as variety such as making water on the manned mission to mars and as an industrial energy source. The reaction pathway of formic acid favors decarboxylation in solvated conditions but control over the pathway is an important field of study. Formic acid was introduced into a high vacuum system in the form of a cluster beam via supersonic expansion and was ionized with the second harmonic (400nm) of a pump-probe laser. Mass spectra showed a ‘magic’ 5,1 (formic acid, water) peak which showed higher intensity than was usually observed in clusters with 1 water molecule. Peak integration showed a higher relative abundance for the 5,1 cluster as well and showed the increased binding favorability of this conformation. As a result, there is an enhanced probability of molecules sticking together in this arrangement and this is due to the stable, cage-like structure that the formic acid forms when surrounding the water molecule.

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  • 2020-05

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Modulating Cyanovirin-N Lectins to Improve Glycoprotein Recognition

Description

Antiviral lectins are potential candidates for future therapies against enveloped viruses like HIV due to their ability to recognize and bind glycans displayed on their surface. Cyanovirin-N (CVN), a lectin

Antiviral lectins are potential candidates for future therapies against enveloped viruses like HIV due to their ability to recognize and bind glycans displayed on their surface. Cyanovirin-N (CVN), a lectin that specifically recognizes mannose-rich moieties, serves as a useful model for studying these glycan-recognition mechanisms. This study seeks to improve CVN's glycan-binding affinity by conjugating a boronic acid functional group to the N-terminus via N-terminal specific reductive alkylation by way of a benzaldehyde handle. However, large discrepancies were observed when attempting to confirm a successful conjugation, and further work is necessary to identify the causes and solutions for these issues.

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  • 2018-12

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The Effects of the PsaL Calcium Binding on the Oligomeric and Spectroscopic Properties in Synechocystis sp. PCC 6803

Description

In oxygenic photosynthesis, conversion of solar energy to chemical energy is catalyzed by the<br/>pigment-protein complexes Photosystem II (PSII) and Photosystem I (PSI) embedded within the<br/>thylakoid membrane of photoautotrophs. The function

In oxygenic photosynthesis, conversion of solar energy to chemical energy is catalyzed by the<br/>pigment-protein complexes Photosystem II (PSII) and Photosystem I (PSI) embedded within the<br/>thylakoid membrane of photoautotrophs. The function of these pigment-protein complexes are<br/>conserved between all photoautotrophs, however, the oligomeric structure, as well as the<br/>spectroscopic properties of the PSI complex, differ. In early evolving photoautotrophs, PSI<br/>exists in a trimeric organization, but in later evolving species this was lost and PSI exists solely<br/>as a monomer. While the reasons for a change in oligomerization are not fully understood, one<br/>of the 11 subunits within cyanobacterial PSI, PsaL, is thought to be involved in trimerization<br/>through the coordination of a calcium ion in an adjacent monomer. Recently published<br/>structures have demonstrated that PSI complexes are capable of trimerization without<br/>coordinating the calcium ion within PsaL.<br/>5 Here we explore the role the calcium ion plays in both<br/>the oligomeric and spectroscopic properties in PSI isolated from Synechocystis sp. PCC 6803.

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  • 2021-05

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A Smart Shipping Incubator for Biodosimetry Logistics in Radiation Countermeasure Operations

Description

Mass nuclear catastrophe is a serious concern for society at large when considering the rising threat of terrorism and the risks associated with harnessing nuclear energy. In the case of

Mass nuclear catastrophe is a serious concern for society at large when considering the rising threat of terrorism and the risks associated with harnessing nuclear energy. In the case of a mass nuclear/radiological event that requires hundreds of thousands of individuals to be assessed for radiation exposure, a rapid biodosimetry triage tool is crucial [1]. The Cytokinesis Block Micronucleus Assay (CBMN) is a promising cytogenetic biodosimetry assay for triage [2]; however, it requires shipping samples to a central laboratory (1-3 days) followed by a lengthy cell culture process (~3 days) before the first dose estimate can be available. The total ~ 1 week response time is too long for effective medical care intervention. A shipping incubator could cut the response time in half (~3 days) by culturing samples in transit; however, possible shipping delays beyond 2 days without the addition of a necessary reagent (Cyto-B) would ruin the integrity of the samples—for accurate CBMN assay endpoint observation, Cyto-B must be added within a 24-44 hour window after sample culture is initiated. Here, we propose a “Smart” Shipping Incubator (SSI) that can add Cyto-B while samples are in transit through a centrifugal system equipped with microfluidic capillary valve caps. The custom centrifugal system was constructed with CNC machined and 3D printed plastic parts, controlled by a custom printed circuit board (PBC) microcontroller, and housed inside a commercial shipping incubator (iQ5 from MicroQ Technologies). Teflon-coated, pre-pulled glass micropipettes (FivePhoton BioChemicals) were used as microfluidic capillary valve caps. Release of Cyto-B was characterized by a desktop centrifugal system at different tip sizes and relative centrifugal forces (RCFs). A theoretical model of Cyto-B release was also deduced to aid the optimization of the process. The CBMN assay was conducted both in the SSI with centrifugal Cyto-B release and in a standard CO2 incubator with manual addition of Cyto-B as the control. The expected mechanical shock during shipment was measured to be less than 25g. Optimal Cyto-B release was found to be at 35g RCF with a Teflon-coated 40 µm tip. Similar CBMN dose curves of micronuclei per binucleated cells (MN/BN) vs. exposed radiation (Gy) were produced for samples assessed conventionally and with the SSI. The similarities between the two methods suggest that centrifugation does not significantly affect the CBMN assay.

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  • 2019-12

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Qualification of α-keto-analogs of amino acids in orange and potato juices: A pilot study into the feasibility of traditional protein-source replacement in healthy subjects

Description

Abstract: It has been established that α-keto-analogs of amino acids can be converted into the amino acids through transamination in vivo. This discovery led to breakthroughs in treating patients who

Abstract: It has been established that α-keto-analogs of amino acids can be converted into the amino acids through transamination in vivo. This discovery led to breakthroughs in treating patients who had difficulty digesting traditional proteins, such as in chronic kidney disease (CKD) sufferers where patients have poor kidney function, which poisons the blood with ammonia products.
This pilot study aimed to ascertain the potential for keto acid supplementation in the attempt to supply adequate protein building blocks to healthy populations, with the caveats that said supplementation 1) would utilize non-synthetic methods, 2) offer an alternative to high-phosphate protein supplies such as ruminant animals, and 3) reverse the ill effects of ammonia load by reducing nitrogen intake and consuming ammonia as a fuel for the process of protein synthesis. This proposed solution turns to orange juice and certain varietals of potato juice for their familiarity to consumers, innate nutritional values, and potential for mass-production by many existing companies. The work contained here represents the first phase of experimentation: qualifying the presence of α-keto-analogues of amino acids in these types of produce which, with transamination, could yield the amino acids necessary for adequate protein intake.
Results suggest that these juices do not contain adequate α-keto-analogs of amino acids to supplement proteins in either healthy or ill individuals.

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  • 2019-05

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Purification and Characterization of TRI 05 I13S M6I

Description

2,2’ bipyridine (Bpy) can form metal complexes with divalent metals in the form of [M(Bpy-ala)¬3]+2 where M is any divalent metal. These [M(Bpy-ala)¬3]+2 complexes can have very interesting photochemical and

2,2’ bipyridine (Bpy) can form metal complexes with divalent metals in the form of [M(Bpy-ala)¬3]+2 where M is any divalent metal. These [M(Bpy-ala)¬3]+2 complexes can have very interesting photochemical and redox potentials that can be useful in more complex systems. The use of (2,2′-bipyridin-5yl)alanine (Bpy-ala) as a Noncanonical Amino Acid (NCAA) has allowed Bpy to be incorporated into an amino acid sequence which can now function in a protein scaffold. Previous studies have utilized that power of Bpy-ala to design a protein that can assemble a homotrimeric protein complex in the presence of a divalent metal. However, the issue with this design was that when the homotrimer was formed and the divalent was removed, the protein complex would not dissemble indicating that it was not metal dependent. Point mutations were made to disrupt the protein-protein interactions to favor disassembly in the absence of a divalent metal. Successfully, a mutation was made that allowed the designed protein to be metal dependent for self-assembly. Nevertheless, an issue with this design is that it poorly incorporated ruthenium(II) into the tris Bpy complex forming [Ru(Bpy-ala)¬3]+2, which was one of the main goals of the original design. This thesis sets out to form TRI 05 I13S M6I which should uphold the same metal-dependence as its predecessor and should combine ruthenium (II) into the protein complex forming [Ru(Bpy-ala)¬3]+2. The thesis shows the success of formation and expression of TRI 05 I13S M6I in Escherichia coli cells. This thesis also reports several purification steps and procedures to not only purify TRI 05 I13S M6I but also removing both the His-tag sequence and Fe(II) from the protein. The thesis also shows that TRI 05 I13S M6I does not behave like its predecessor in that it is not metal dependent for self-assembly. While this may be true, this paper also reports the incorporation of ruthenium (II) in the protein structure. Though this may be the first time that ruthenium (II) has been recorded to be in the TRI 05 protein complex with a significant signal, it is still nowhere near the optimal fluorescence that small molecule Bpy can achieve by itself. The thesis reports potential conditions and a plan of attack that should drive this project forward into achieving an optimal signal of the [Ru(Bpy-ala)¬3]+2 complex in a TRI 05 protein scaffold.

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  • 2019-05