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dc.rights.licenseRestricted to current Rensselaer faculty, staff and students. Access inquiries may be directed to the Rensselaer Libraries.
dc.contributorMakhatadze, George I.
dc.contributorBarquera, Blanca L.
dc.contributorColón, Wilfredo
dc.contributorRoan, Nadia R.
dc.contributorRoyer, Catherine Ann
dc.contributor.authorLoRicco, Josephine Grace
dc.date.accessioned2021-11-03T09:10:31Z
dc.date.available2021-11-03T09:10:31Z
dc.date.created2019-10-01T15:33:30Z
dc.date.issued2019-05
dc.identifier.urihttps://hdl.handle.net/20.500.13015/2411
dc.descriptionMay 2019
dc.descriptionSchool of Science
dc.description.abstractWe use PAPf39, SEM1(86-107), and insulin fibrils as our model system for exploring the interaction between green fluorescent protein (GFP) and amyloid fibrils. In order to understand what properties of GFP allows it to bind to amyloid fibrils, we characterized several GFP mutants in order to gain insights into this binding interaction. Superanionic GFPs (saGFPs) show improved binding to cationic fibrils, and stronger inhibition of PAPf39 and SEM1(86-107) aggregation.
dc.description.abstractSecond, we propose that green fluorescent protein (GFP) is able to recognize the core of amyloid fibrils, regardless of amino acid sequence. This interaction is specific to amyloid fibrils, as GFP does not bind to the monomeric forms of these peptides/proteins or to other protein aggregates. GFP is unable to bind to amyloid fibrils formed from large proteins which have a fuzzy coat surrounding the amyloid core, however if the fuzzy coat is removed by protease digestion GFP is able to recognize the amyloid core of these fibrils.
dc.description.abstractWe also hypothesize that surface aromatic residues found only on one face of the GFP beta-barrel are important for binding. Removal of surface aromatic residues (nsaGFP) weakens binding to fibrils, and also weakens inhibition of PAPf39 and SEM1(86-107) aggregation. Moving the aromatic residues to another face of the beta-barrel (Q5 GFP) improves binding to both cationic and anionic fibrils, however it reduces the ability of Q5 GFP to inhibit fibrillation. Moving the locations of the aromatic residues also was found to completely change the orientation of GFP binding to fibrils. There are still many unanswered questions about the binding of amyloid fibrils and GFP, but hopefully this work will act as a foundation for future research.
dc.description.abstractFirst, we explore a small molecule, gallic acid, that has potential to combat the fibril-enhanced transmission of HIV. We employ biophysical techniques to understand how gallic acid is able to reduce the infectivity of HIV in the presence of seminal plasma. Contrary to our initial hypothesis that gallic acid was able to disassemble mature seminal fibrils, we find that gallic acid binds to mature fibrils and modifies their surface properties rendering the fibrils less cationic. The cationic nature of the fibrils is necessary for enhancing infection. We also find that gallic acid is capable of inhibiting the aggregation of PAPf39 and SEM1(86-107) fibrils from monomeric peptide which could also contribute to reduction in infectivity in the presence of seminal amyloid fibrils.
dc.description.abstractGFP can be used to detect mature amyloid fibrils so next we tested to see if GFP could be used to monitor aggregation kinetics. SEM1(86-107), and PAP∆13 fibrillation kinetics were monitored using GFP and tracked well with kinetics assessed by traditional methods. The appearance of large aggregates bound to GFP using confocal microscopy corresponded with aggregation kinetics assessed by Thioflavin T (ThT). GFP was also capable of detecting small aggregates during the lag phase seen with ThT suggesting GFP may be able to recognize small, oligomeric aggregates. The ability of GFP to inhibit aggregation of PAPf39 and SEM1(86-107) fibrils supports the idea that GFP can bind to oligomers.
dc.description.abstractAmyloid fibrils are insoluble protein aggregates which are associated with a number of human diseases including Alzheimer’s disease, Parkinson’s disease, and type II diabetes. They are found naturally in human semen, and have been shown to enhance the infectivity of the human immunodeficiency virus (HIV) by several orders of magnitude. Although the amino acid sequences of different fibril systems vary dramatically, all fibrils consist of a highly ordered beta-sheet structure. This work focuses on two amyloid fibril systems, PAPf39 and SEM1(86-107), which are both fragments of seminal proteins that have been shown to form amyloid fibrils capable of enhancing HIV infection.
dc.language.isoENG
dc.publisherRensselaer Polytechnic Institute, Troy, NY
dc.relation.ispartofRensselaer Theses and Dissertations Online Collection
dc.subjectBiochemistry and biophysics
dc.titleBiophysical characterization of seminal amyloid fibrils : interactions with gallic acid and green fluorescent protein
dc.typeElectronic thesis
dc.typeThesis
dc.digitool.pid179699
dc.digitool.pid179700
dc.digitool.pid179701
dc.rights.holderThis electronic version is a licensed copy owned by Rensselaer Polytechnic Institute, Troy, NY. Copyright of original work retained by author.
dc.description.degreePhD
dc.relation.departmentBiochemistry and Biophysics Program


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