Date of Graduation
5-2022
Document Type
Thesis
Degree Name
Bachelor of Science
Degree Level
Undergraduate
Department
Chemistry & Biochemistry
Advisor/Mentor
Heyes, Colin
Committee Member/Reader
Allison, Neil
Committee Member/Second Reader
Billig, Noah
Committee Member/Third Reader
Potra, Adriana
Abstract
Fluorescent labeling is a technique used for visualizing functional groups contained in biomolecules by fluorescence imaging. This technique was used in this project to analyze post-translational targeting of light-harvesting chlorophyll-binding proteins (LHCP), which are the core complexes that harvest sunlight to drive photosynthetic electron transfer. This protein is synthesized in the cytosol and post-translationally targeted to the stroma of chloroplasts. CpSRP43 is a signal recognition particle (SRP) subunit unique to chloroplasts, which has been shown to interact with the stroma-soluble C-terminus of the thylakoid-bound Albino3 insertase (Alb3-Cterm). In the chloroplast stroma, targeting to thylakoids is performed via the cpSRP pathway via sequential interaction with cpSRP43/cpSRP54, cpFtsY and Alb3. Although Alb3-Cterm is mostly disordered, molecular dynamics simulations predict a transient helical secondary structure in certain regions. To analyze this interaction, various pairs of labeling sites are chosen to determine the structural changes between cpSRP43 and Alb3-Cterm. Single molecule fluorescence resonance energy transfer (smFRET) was then used to determine structural differences. This project aimed to determine what conformational changes occurs during the cpSRP43 and Alb3-Cterm interaction during post-translational targeting of LHCPs. By introducing a proline mutant to prevent helix formation in various regions, it was determined whether the secondary structure plays a role in the binding. It was also found that the smFRET mutants tested display a high-FRET population in the presence of cpSRP43 which would indicate a helical propensity in the probed region.
Keywords
FRET; smFRET; cpSRP; LHCP; Fluorescence
Citation
Tomanek, A. (2022). Investigation of the Binding Domain Interfaces of the C-terminus of the Albino3 Insertase and the 43kDa Chloroplast Signal Recognition Particle Subunit via Single Molecule Förster Resonance Energy Transfer. Chemistry & Biochemistry Undergraduate Honors Theses Retrieved from https://scholarworks.uark.edu/chbcuht/34
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