On the use of fluorescence resonance energy transfer in amyloid fibril detection and protein conformational studies
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Authors
Moustouka, Caitlyn
Issue Date
2025-05
Type
Electronic thesis
Thesis
Thesis
Language
en_US
Keywords
Biology
Alternative Title
Abstract
The phenomenon known as fluorescence resonance energy transfer (FRET) has become an extremely powerful and versatile tool that is widely-used in biomolecular sciences to report on various interactions, conformational changes, and much more. This work is comprised of two independent projects that are connected through the use of FRET. First, we describe the development of a FRET-based universal amyloid detection platform harnessing the intrinsic affinity of fluorescent proteins (FPs) for the general amyloid core structure. Amyloid fibrils are a type of protein aggregate characterized by a common core structure and extreme stability. These aggregates are widely known for their pathogenic roles, as amyloid deposition is associated with numerous human diseases, but also serve many important functional roles across all domains of life. Being able to detect the presence of amyloid is central to studying their roles in both normal biological processes they are involved in, as well as the disease states they are found in. Proof of concept for our quantitative amyloid sensor platform is demonstrated using a model fibril system, PAPf39, and the subsequent application in detecting other amyloid systems is assessed. We observe differences in the sensitivity of the FRET sensor towards these different fibrils and attempt to elucidate the underlying causes. The second portion of this work describes our investigation into the effects of pressure and temperature on proteins lacking tertiary structure. The study of biological macromolecules under pressure is guided by the notion that the large majority of the Earth’s biomass exists under high hydrostatic pressure. However, high pressure is known to disrupt biological processes of organisms living at ambient pressure, and also has significant effects on biomolecular stability and interactions. These points together highlight the lack of understanding we have of the ways in which piezophilic organisms are adapted to their native environments. The study of high pressure effects on isolated proteins has widely shown that pressure denatures globular proteins primarily through the release of void spaces present in the folded structure in order to achieve a smaller volume. However, there has not been significant study of the effects of pressure on secondary structural elements at the core of these tertiary structures. Additionally, it is now well-known that intrinsically disordered proteins (IDPs) perform important biological functions, despite their lack of stable structure, and, yet, the effects of pressure on their conformational ensembles have been largely unstudied. We use FRET, among other biophysical methods, to analyze the combined pressure and temperature dependence of α-helical structure, as well as the dimensions of coiled and intrinsically disordered proteins.
Description
May2025
School of Science
School of Science
Full Citation
Publisher
Rensselaer Polytechnic Institute, Troy, NY