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Storm Dissertation final.pdf (5.88 MB)

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Structural studies of cpTat component Tha4 in both native and synthetic membrane systems

Storm, Amanda R
http://rave.ohiolink.edu/etdc/view?acc_num=miami1385466330

Abstract Details

2013, Doctor of Philosophy, Miami University, Chemistry and Biochemistry.
The chloroplast Twin Arginine Translocation (cpTat) system facilitates transport of about half of all proteins that cross the thylakoid membrane in plant chloroplasts. Known mechanistic features of the cpTat system are drastically different from other known translocation systems, notably in its formation of a transient complex to transport fully folded proteins utilizing only the proton-motive force for energy. Tha4, one of the transmembrane cpTat components, is proposed to function as the primary pore component by potentially creating a weak 'patch' in the membrane or undergoing a conformational change to facilitate transport. Yet, there is limited knowledge about the basic topology or structure of Tha4 and little work has been done to detect any transport-stimulated conformational changes. This dissertation describes the development and use of biochemical and biophysical techniques to explore aspects of Tha4 structure in both native and synthetic membrane systems. Tha4 topology in isolated thylakoid membranes was determined under both resting state and transport conditions. A modified substituted cysteine accessibility method (SCAM) indicated that Tha4 adopts an N-in, C-out topology with a single transmembrane domain (TMD) and a tilted amphipathic helix (APH). Cysteine and protease accessibility assays performed under transport-stimulating conditions detected significant changes in the accessibility of the C-terminal APH region of Tha4 but no global changes in the over-all topology. In preparation for more detailed biophysical structural studies, procedures were developed and optimized for the over-expression and purification of Tha4 and insertion of spin-labeled protein into four different lipid systems, including a novel lipodisq nanoparticle architecture. Topology of integrated spin labeled Tha4 detected by electron paramagnetic resonance (EPR) spectroscopy was in agreement with topology observed in thylakoids. Tha4 lipodisq nanoparticles containing native thylakoid lipids were also generated and purified by nickel affinity chromatography. An additional project describes the development of methods for incorporation of the rigid TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) spin label into full length proteins using amber codon suppression and in vitro translation. Continuous-exchange cell-free translation techniques were used to improve yield of TOAC-labeled protein and co-translationally insert protein into liposomes. Issues with inefficient reagent synthesis hindered the large scale use of these methods.
Carole Dabney-Smith, PhD (Advisor)
Gary Lorigan, PhD (Committee Chair)
Christopher Makaroff, PhD (Committee Member)
Neil Danielson, PhD (Committee Member)
Rachael Morgan-Kiss, PhD (Committee Member)
118 p.
Biochemistry; Chemistry
chloroplast twin arginine translocation; cpTat; Tha4; thylakoid; native topology; electron paramagnetic resonance; EPR; lipodisq nanoparticles; TOAC; amber suppression

Recommended Citations

Citations

  • Storm, A. R. (2013). Structural studies of cpTat component Tha4 in both native and synthetic membrane systems [Doctoral dissertation, Miami University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=miami1385466330

    APA Style (7th edition)

  • Storm, Amanda. Structural studies of cpTat component Tha4 in both native and synthetic membrane systems. 2013. Miami University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=miami1385466330.

    MLA Style (8th edition)

  • Storm, Amanda. "Structural studies of cpTat component Tha4 in both native and synthetic membrane systems." Doctoral dissertation, Miami University, 2013. http://rave.ohiolink.edu/etdc/view?acc_num=miami1385466330

    Chicago Manual of Style (17th edition)

miami1385466330
595
© 2013, all rights reserved.
This open access ETD is published by Miami University and OhioLINK.