Barrett

Barrett, The Honors College at Arizona State University proudly showcases the work of undergraduate honors students by sharing this collection exclusively with the ASU community.

Barrett accepts high performing, academically engaged undergraduate students and works with them in collaboration with all of the other academic units at Arizona State University. All Barrett students complete a thesis or creative project which is an opportunity to explore an intellectual interest and produce an original piece of scholarly research. The thesis or creative project is supervised and defended in front of a faculty committee. Students are able to engage with professors who are nationally recognized in their fields and committed to working with honors students. Completing a Barrett thesis or creative project is an opportunity for undergraduate honors students to contribute to the ASU academic community in a meaningful way.

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Structural Analysis of the Spinach Rubisco Activase AAA+ Domain by Negative Stain Electron Microscopy

Description
Higher plant Rubisco activase (Rca) is a stromal ATPase responsible for reactivating Rubisco. It is a member of the AAA+ protein superfamily and is thought to assemble into closed-ring hexamers like other AAA+ proteins belonging to the classic clade. Progress towards modeling the interaction between Rca and Rubisco has been…

Higher plant Rubisco activase (Rca) is a stromal ATPase responsible for reactivating Rubisco. It is a member of the AAA+ protein superfamily and is thought to assemble into closed-ring hexamers like other AAA+ proteins belonging to the classic clade. Progress towards modeling the interaction between Rca and Rubisco has been slow due to limited structural information on Rca. Previous efforts in the lab were directed towards solving the structure of spinach short-form Rca using X-ray crystallography, given that it had notably high thermostability in the presence of ATP-γS, an ATP analog. However, due to disorder within the crystal lattice, an atomic resolution structure could not be obtained, prompting us to move to negative stain electron microscopy (EM), with our long-term goal being the use of cryo-electron microscopy (cryo-EM) for atomic resolution structure determination. Thus far, we have screened different Rca constructs in the presence of ATP-γS, both the full-length β-isoform and truncations containing only the AAA+ domain. Images collected on preparations of the full-length protein were amorphous, whereas images of the AAA+ domain showed well-defined ring-like assemblies under some conditions. Procedural adjustments, such as the use of previously frozen protein samples, rapid dilution, and minimizing thawing time were shown to improve complex assembly. The presence of Mn2+ was also found to improve hexamer formation over Mg2+. Calculated class averages of the AAA+ Rca construct in the presence of ATP-γS indicated a lack of homogeneity in the assemblies, showing both symmetric and asymmetric hexameric rings. To improve structural homogeneity, we tested buffer conditions containing either ADP alone or different ratios of ATP-γS to ADP, though results did not show a significant improvement in homogeneity. Multiple AAA+ domain preparations were evaluated. Because uniform protein assembly is a major requirement for structure solution by cryo-EM, more work needs to be done on screening biochemical conditions to optimize homogeneity.
ContributorsHernandez, Victoria Joan (Author) / Wachter, Rebekka (Thesis director) / Chiu, Po-Lin (Committee member) / Redding, Kevin (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2018-05
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Predicting the Binding Interactions of TNFR2 and PD-L1 on LT-ɑ, TRAF2, and CD80

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…

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
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Ionic Liquids in Simplifying Cryogenic Electron Microscopy

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…

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