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2023.7.31 SrinivasanK_PhD_Dissertation.pdf (11.97 MB)

Characterizing  synthetic antigen-binding fragments for isolation of the TOC complex

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posted on 2023-07-31, 15:45 authored by Karthik SrinivasanKarthik Srinivasan

  

Protein translocation across the chloroplast outer membrane is essential for photosynthesis in all plants and certain algae. This is because most chloroplast proteins (over 90%) are encoded in the nucleus, translated in the cytoplasm, and must be imported into the chloroplasts to perform their function. The translocon at the outer chloroplast membrane (TOC) complex orchestrates this vital translocation process and consists of three components in plants: Toc75, Toc33/34 and Toc159. Our overall goal is to elucidate the architecture of the TOC complex to gain mechanistic insights into protein translocation into chloroplasts. However, the major bottleneck preventing structure determination of the TOC complex has been the inability to produce or isolate the complex to sufficient yields and purity for structural studies. We began by using phage display to screen for synthetic antigen-binding fragments (sABs) that bind to the soluble POTRA domains of Toc75 from both Arabidopsis thaliana and Pisum sativum. We then characterized the POTRA-sAB interactions using size-exclusion chromatography coupled with small angle X-ray scattering (SEC-SAXS), isothermal titration calorimetry (ITC), and X-ray crystallography. Finally, we show that we can use an affinity tagged sAB to isolate the TOC complex directly from pea biomass. This study has paved the way for high-resolution structural studies of the TOC complex from plants to understand protein translocation mechanisms. 

Funding

1R01GM127896-01 (NIH)

History

Degree Type

  • Doctor of Philosophy

Department

  • Biological Sciences

Campus location

  • West Lafayette

Advisor/Supervisor/Committee Chair

Nicholas Noinaj

Additional Committee Member 2

John Tesmer

Additional Committee Member 3

Leifu Chang

Additional Committee Member 4

Sujith Puthiyaveetil

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