Matching Items (14)
Filtering by
- All Subjects: Biochemistry
- All Subjects: Protein
- Creators: Department of Physics
- Creators: Wang, Xu
- Creators: College of Liberal Arts and Sciences
- Member of: Barrett, The Honors College Thesis/Creative Project Collection
- Resource Type: Text
Description
Lyme disease is a common tick-borne illness caused by the Gram-negative bacterium Borrelia burgdorferi. An outer membrane protein of Borrelia burgdorferi, P66, has been suggested as a possible target for Lyme disease treatments. However, a lack of structural information available for P66 has hindered attempts to design medications to target the protein. Therefore, this study attempted to find methods for expressing and purifying P66 in quantities that can be used for structural studies. It was found that by using the PelB signal sequence, His-tagged P66 could be directed to the outer membrane of Escherichia coli, as confirmed by an anti-His Western blot. Further attempts to optimize P66 expression in the outer membrane were made, pending verification via Western blotting. The ability to direct P66 to the outer membrane using the PelB signal sequence is a promising first step in determining the overall structure of P66, but further work is needed before P66 is ready for large-scale purification for structural studies.
ContributorsRamirez, Christopher Nicholas (Author) / Fromme, Petra (Thesis director) / Hansen, Debra (Committee member) / Department of Physics (Contributor) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
Exploring Structure and Function of Human Cold Sensing Protein TRPM8 with ROSETTA Comparative Models
Description
Transient Receptor Potential (TRP) channels are a diverse class of ion channels notable as polymodal sensors. TRPM8 is a TRP channel implicated in cold sensation, nociception, and a variety of human diseases, including obesity and cancer. Despite sustained interest in TRPM8 since its discovery in 2001, many of the molecular mechanisms that underlie function are not yet clear. Knowledge of these properties could have implications for medicine and physiological understanding of sensation and signaling. Structures of TRP channels have proven challenging to solve, but recent Cryoelectron microscopy (Cryo-EM) structures of TRPV1 provide a basis for homology-based modeling of TRP channel structures and interactions. I present an ensemble of 11,000 Rosetta computational homology models of TRPM8 based on the recent Cryo-EM apo structure of TRPV1 (PDB code:3J5P). Site-directed mutagenesis has provided clues about which residues are most essential for modulatory ligands to bind, so the models presented provide a platform to investigate the structural basis of TRPM8 ligand modulation complementary to existing functional and structural information. Menthol and icilin appear to interact with interfacial residues in the sensor domain (S1-S4). One consensus feature of these sites is the presence of local contacts to the S4 helix, suggesting this helix may be mechanistically involved with the opening of the pore. Phosphatidylinositol 4,5-bisphosphate (PIP2)has long been known to interact with the C-terminus of TRPM8, and some of the homology models contain plausible binding pockets where PIP2 can come into contact with charged residues known to be essential for PIP2 modulation. Future in silico binding experiments could provide testable hypothesis for in vitro structural studies, and experimental data (e.g. distance constraints from electron paramagnetic resonance spectroscopy [EPR]) could further refine the models.
ContributorsHelsell, Cole Vincent Maher (Author) / Van Horn, Wade (Thesis director) / Wang, Xu (Committee member) / Barrett, The Honors College (Contributor) / School of Mathematical and Statistical Sciences (Contributor) / Department of Chemistry and Biochemistry (Contributor)
Created2015-05
Description
Identifying disease biomarkers may aid in the early detection of breast cancer and improve patient outcomes. Recent evidence suggests that tumors are immunogenic and therefore patients may launch an autoantibody response to tumor associated antigens. Single-chain variable fragments of autoantibodies derived from regional lymph node B cells of breast cancer patients were used to discover these tumor associated biomarkers on protein microarrays. Six candidate biomarkers were discovered from 22 heavy chain-only variable region antibody fragments screened. Validation tests are necessary to confirm the tumorgenicity of these antigens. However, the use of single-chain variable autoantibody fragments presents a novel platform for diagnostics and cancer therapeutics.
ContributorsSharman, M. Camila (Author) / Magee, Dewey (Mitch) (Thesis director) / Wallstrom, Garrick (Committee member) / Petritis, Brianne (Committee member) / Barrett, The Honors College (Contributor) / College of Liberal Arts and Sciences (Contributor) / Virginia G. Piper Center for Personalized Diagnostics (Contributor) / Biodesign Institute (Contributor)
Created2012-12
Description
Time spent alone is a topic that has been studied in great detail, particularly the manner in which it is spent and the effect it has during the adolescent stage of life. Similarly, stress levels in adolescents have always been a topic of interest because of the effects they could have on the individual later in adulthood. Oddly enough however, the two areas of study have never been looked at in relation to one another. This study will look at different types of alone time as possible stressors in a community sample (N=82) of adolescents transitioning to college. The data on time alone and stress levels was collected through diary reports over a period of 3 days. The analysis only yielded significant effects for females and only for specific categories. It was found that females experience the lowest amount of perceived stress when they are alone and want to be alone, they have more negative affect when their desired environment differs from their current situation, and more positive affect in both the alone incongruence and not alone congruence situations. These results indicate that only women experience stress and affect changes when they encounter different congruent and incongruent environments.
ContributorsVanderwerf, Jennifer (Author) / Doane, Leah (Thesis director) / Knight, George (Committee member) / Arbona, P. Anita (Committee member) / Barrett, The Honors College (Contributor) / College of Liberal Arts and Sciences (Contributor)
Created2012-12
Description
Understanding glycosaminoglycans’ (GAG) interaction with proteins is of growing interest for therapeutic applications. For instance, heparin is a GAG exploited for its ability to inhibit proteases, therefore inducing anticoagulation. For this reason, heparin is extracted in mass quantities from porcine intestine in the pharmaceutical field. Following a contamination in 2008, alternative sources for heparin are desired. In response, much research has been invested in the extraction of the naturally occurring polysaccharide, heparosan, from Escherichia coli K5 strain. As heparosan contains the same structural backbone as heparin, modifications can be made to produce heparin or heparin-like molecules from this source. Furthermore, isotopically labeled batches of heparosan can be produced to aid in protein-GAG interaction studies. In this study, a comparative look between extraction and purification methods of heparosan was taken. Fed-batch fermentation of this E. coli strain followed by subsequent purification yielded a final 13C/15N labeled batch of 90mg/L of heparosan which was then N-sulfated. Furthermore, a labeled sulfated disaccharide from this batch was utilized in a protein interaction study with CCL5. With NMR analysis, it was found that this heparin-like molecule interacted with CCL5 when its glucosamine residue was in a β-conformation. This represents an interaction reliant on a specific anomericity of this GAG molecule.
ContributorsHoffman, Kristin Michelle (Author) / Wang, Xu (Thesis director) / Cabirac, Gary (Committee member) / Morgan, Ashli (Committee member) / Barrett, The Honors College (Contributor) / School of International Letters and Cultures (Contributor) / School of Life Sciences (Contributor)
Created2015-05
Description
Optometry is an important field in medicine as it allows people a chance to have their vision corrected and it serves as a health screening opportunity for those who receive a dilated eye examination. One of the largest barriers to receiving a dilated eye exam is insurance coverage. Most health insurance policies have limited optometric coverage. By expanding health insurance plans to be more inclusive of optometric care, people who use these health insurance plans will have a better access of care.
ContributorsFurey, Colleen (Author) / Ruth, Alissa (Thesis director) / Mullen, Tyler (Committee member) / School of Life Sciences (Contributor) / Department of Physics (Contributor) / Barrett, The Honors College (Contributor)
Created2021-05
Description
News outlets frequently portray people with disabilities as either helpless victims or objects of motivation. Portrayal of people with disabilities has improved over the years, but there is still room to grow. News outlets tend to make disability the center of the story. A story about a disabled person is primarily about their disability, with their other accomplishments framed by it.
As one example of the victimhood narrative, ABC News used to run a special called My Extreme Affliction as part of 20/20 until 2012. As the name implies, the specials covered people with disabilities, specifically extreme versions. One 2008 episode on Tourette’s syndrome described Tourette’s like it was some sort of demonic possession. The narrator talked about children who were “prisoners in their own bodies” and a family that was at risk of being “torn apart by Tourette’s.” I have Tourette’s syndrome myself, which made ABC’s special especially uncomfortable to watch. When not wringing their metaphorical hands over the “victims” of disability, many news outlets fall into the “supercrip” narrative. They refer to people as “heroes” who “overcome” their disabilities to achieve something that ranges from impressive to utterly mundane. The main emphasis is on the disability rather than the person who has it. These articles then exploit that disability to make readers feel good. As a person with a disability, I am aware that it impacts my life, but it is not the center of my life. The tics from my Tourette’s syndrome made it difficult to speak to people when I was younger, but even then they did not rule me.
Disability coverage, however, is still incredibly important for promoting acceptance and giving people with disabilities a voice. A little over a fifth of adults in the United States have a disability (CDC: 53 million adults in the US live with a disability), so poor coverage means marginalizing or even excluding a large amount of people. Journalists should try to reach their entire audience. The news helps shape public opinion with the stories it features. Therefore, it should provide visibility for people with disabilities in order to increase acceptance. This is a matter of civil rights. People with disabilities deserve fair and accurate representation.
My personal experience with ABC’s Tourette’s special leads me to believe that the media, especially the news, needs to be more responsible in their reporting. Even the name “My Extreme Affliction” paints a poor picture of what to expect. A show that focuses on sensationalist portrayals in pursuit of views further ostracizes people with disabilities. The emphasis should be on a person and not their condition. The National Center for Disability Journalism tells reporters to “Focus on the person you are interviewing, not the disability” (Tips for interviewing people with disabilities). This people-first approach is the way to improve disability coverage: Treat people with disabilities with the same respect as any other minority group.
As one example of the victimhood narrative, ABC News used to run a special called My Extreme Affliction as part of 20/20 until 2012. As the name implies, the specials covered people with disabilities, specifically extreme versions. One 2008 episode on Tourette’s syndrome described Tourette’s like it was some sort of demonic possession. The narrator talked about children who were “prisoners in their own bodies” and a family that was at risk of being “torn apart by Tourette’s.” I have Tourette’s syndrome myself, which made ABC’s special especially uncomfortable to watch. When not wringing their metaphorical hands over the “victims” of disability, many news outlets fall into the “supercrip” narrative. They refer to people as “heroes” who “overcome” their disabilities to achieve something that ranges from impressive to utterly mundane. The main emphasis is on the disability rather than the person who has it. These articles then exploit that disability to make readers feel good. As a person with a disability, I am aware that it impacts my life, but it is not the center of my life. The tics from my Tourette’s syndrome made it difficult to speak to people when I was younger, but even then they did not rule me.
Disability coverage, however, is still incredibly important for promoting acceptance and giving people with disabilities a voice. A little over a fifth of adults in the United States have a disability (CDC: 53 million adults in the US live with a disability), so poor coverage means marginalizing or even excluding a large amount of people. Journalists should try to reach their entire audience. The news helps shape public opinion with the stories it features. Therefore, it should provide visibility for people with disabilities in order to increase acceptance. This is a matter of civil rights. People with disabilities deserve fair and accurate representation.
My personal experience with ABC’s Tourette’s special leads me to believe that the media, especially the news, needs to be more responsible in their reporting. Even the name “My Extreme Affliction” paints a poor picture of what to expect. A show that focuses on sensationalist portrayals in pursuit of views further ostracizes people with disabilities. The emphasis should be on a person and not their condition. The National Center for Disability Journalism tells reporters to “Focus on the person you are interviewing, not the disability” (Tips for interviewing people with disabilities). This people-first approach is the way to improve disability coverage: Treat people with disabilities with the same respect as any other minority group.
ContributorsMackrell, Marguerite (Author) / Gilger, Kristin (Thesis director) / Doig, Steve (Committee member) / Walter Cronkite School of Journalism & Mass Comm (Contributor) / School of Politics and Global Studies (Contributor) / Barrett, The Honors College (Contributor)
Created2019-05
Description
Proteins continually and naturally incur evolutionary selection through mutagenesis that optimizes their fitness, which is primarily determined by their function. It is known that allosteric regulation alters a protein's conformational dynamics leading to functional changes. We have computationally introduced a mutation at a predicted regulatory site of a short, 46 residue-long, protein interaction module composed of a WW domain and corresponding polyproline ligand (PDB id: 1k9r). The dynamic flexibility index (DFI) was computed for the binding site of the wild type and mutant WW domains to quantify the mutations effect on the rigidity of the binding pocket. DFI is used as a metric to quantify the resilience of a given position to perturbation along the chain. Using steered molecular dynamics (SMD), we also measure the effect of the point mutation on allosteric regulation by approximating the binding free energy of the system calculated using Jarzynski's Equality. Calculation of the DFI shows that the overall flexibility of the protein complex increases as a result of the distal point mutation. Total change in DFI percentile of the binding site showed a 0.067 increase suggesting an allosteric, loss of function mutation. Furthermore, we see that the change in the binding free energy is greater for that of the mutated complex supporting the idea that an increase in flexibility is correlated to a decrease in proteinlig and binding affinity. We show that sequence mutation of an allosteric site affects the mechanical stability and functionality of the binding pocket.
ContributorsMarianchuk, Tegan (Author) / Ozkan, Sefika (Thesis director) / Ros, Robert (Committee member) / Barrett, The Honors College (Contributor) / Department of Physics (Contributor)
Created2018-05
Description
Integrin is a protein in cells that manage cell adhesion. They are crucial to the biochemical functions of cells. L 2 is one type of integrin. Its I domain is responsible for ligand binding. Scientists understand how Alpha L I domain binds Mg2+ at a pH of 7 but not in acidic environments. Knowing the specificity of integrin at a lower pH is important because when tissues become inflamed, they release acidic compounds. We have cloned, expressed, and purified L I-domain and using NMR analysis, we determined that wild type Alpha L I domain does not bind to Mg2+ at a pH of 5.
ContributorsALAM, RAHAT (Author) / Wang, Xu (Thesis director) / Podolnikova, Nataly (Committee member) / School of Molecular Sciences (Contributor) / Barrett, The Honors College (Contributor)
Created2017-05
Description
This qualitative study sought to investigate the potential reaction between the 3,3',5,5'-tetramethylbenzidine (TMB) radical and LAF-1 RGG, the N-terminus domain of an RNA helicase which functions as a coacervating intrinsically disordered protein. The study was performed by adding horseradish peroxidase to a solution containing TMB and either LAF-1 or tyrosine in various concentrations, and monitoring the output through UV-Vis spectroscopy. The reacted species was also analyzed via MALDI-TOF mass spectrometry. UV-Vis spectroscopic monitoring showed that in the presence of LAF-1 or tyrosine, the reaction between HRP and TMB occurred more quickly than the control, as well as in the highest concentration of LAF-1, the evolution of a peak at 482 nm. The analysis through MALDI-TOF spectrometry showed the development of a second peak likely due to the reaction between LAF-1 and TMB, as the Δ between the peaks is 229 Da and the size of the TMB species is 240 Da.
ContributorsDavis, Morgan (Author) / Ghirlanda, Giovanna (Thesis director) / Heyden, Matthias (Committee member) / Mazor, Yuval (Committee member) / Barrett, The Honors College (Contributor) / Department of Physics (Contributor) / School of Molecular Sciences (Contributor)
Created2022-12