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You searched for subject:(Tha4). Showing records 1 – 3 of 3 total matches.

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Miami University

1. New, Christopher Paul. Analysis of Tha4 Function and Organization in Chloroplast Twin Arginine Transport.

Degree: PhD, Cell, Molecular and Structural Biology (CMSB), 2020, Miami University

The chloroplast Twin Arginine Translocase (cpTAT) system transports fully folded proteins across the thylakoid membrane in plant cells using only energy derived from the proton motive force (PMF). Three membrane bound component proteins: cpTatC, Hcf106, and Tha4 function together in a transient manner to accomplish transport. However, clear mechanistic details of this process remain elusive such as how cpTAT utilizes energy stored in the PMF or how the individual component proteins interact during each step of transport. In addition, prior structural characterization of (cp)TAT proteins used truncated versions of the components. This dissertation describes work to develop methods to purify full-length Hcf106 for biophysical characterization. Additionally, this dissertation details the work to determine the function of a membrane embedded glutamate in the Tha4 transmembrane helix (TMH).A series of purification trials were carried out to isolate Hcf106 fused to maltose binding protein (MBP) by the recognition sequence of tobacco etch virus protease (TEVp). Fusion protein and protease were expressed in and purified from E. coli using affinity chromatography. Multiple parameters and additives were tested during optimization of TEVp proteolysis reactions with MBP-Hcf106. TEVp and free MBP were separated from un-cleaved MBP-Hcf106 and free Hcf106 by affinity and size exclusion chromatography. Although TEVp and free MBP were removed after an optimized proteolysis reaction, free Hcf106 showed its recalcitrant nature through resistance of separation from un-cleaved MBP-Hcf106 by size exclusion chromatography in several detergent and buffer conditions.To better understand the role of the membrane embedded Tha4 glutamate 10 (E10), Tha4 variants with glutamate to alanine (E10A) or glutamate to aspartate (E10D) substitutions were used to complement loss of cpTAT function in thylakoid membranes. Sequential glutamate substitutions in the TMH of Tha4 variant E10A were unable to restore transport while aspartate substitutions were mildly able to complement loss of function. Furthermore, organization between three structural regions in Tha4 E10/A/D variants was determined by disulfide crosslinking during various transport conditions. Tha4 E10/A/D variant oligomer formation was enhanced in the presence of functional precursor with and without PMF present. An increase in TMH hydrophobicity by alanine substitution was shown to increase Tha4 stability in isolated thylakoid membranes and to promote tighter packing interactions between adjacent Tha4 monomers. The interaction data was then used to develop a model of how Tha4 E10/A/D variant tetramers pack and reorganize in the presence of precursor. Advisors/Committee Members: Dabney-Smith, Carole (Advisor), Page, Rick (Committee Chair).

Subjects/Keywords: Biochemistry; Cellular Biology; Plant Biology; Molecular Biology; chloroplast twin arginine transport; protein transport; cpTAT; TAT; Tha4; Hcf106; protein purification; maltose binding protein affinity chromatography; oligomer formation; complementation; transmembrane domain hydrophobicity

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APA (6th Edition):

New, C. P. (2020). Analysis of Tha4 Function and Organization in Chloroplast Twin Arginine Transport. (Doctoral Dissertation). Miami University. Retrieved from http://rave.ohiolink.edu/etdc/view?acc_num=miami1586878527570538

Chicago Manual of Style (16th Edition):

New, Christopher Paul. “Analysis of Tha4 Function and Organization in Chloroplast Twin Arginine Transport.” 2020. Doctoral Dissertation, Miami University. Accessed January 18, 2021. http://rave.ohiolink.edu/etdc/view?acc_num=miami1586878527570538.

MLA Handbook (7th Edition):

New, Christopher Paul. “Analysis of Tha4 Function and Organization in Chloroplast Twin Arginine Transport.” 2020. Web. 18 Jan 2021.

Vancouver:

New CP. Analysis of Tha4 Function and Organization in Chloroplast Twin Arginine Transport. [Internet] [Doctoral dissertation]. Miami University; 2020. [cited 2021 Jan 18]. Available from: http://rave.ohiolink.edu/etdc/view?acc_num=miami1586878527570538.

Council of Science Editors:

New CP. Analysis of Tha4 Function and Organization in Chloroplast Twin Arginine Transport. [Doctoral Dissertation]. Miami University; 2020. Available from: http://rave.ohiolink.edu/etdc/view?acc_num=miami1586878527570538


University of Florida

2. Celedon,Jose M. Basic Translocation Unit Composition of the Twin Arginine Translocase (TAT) System in Pea Thylakoids.

Degree: PhD, Horticultural Sciences, 2011, University of Florida

Tat (twin arginine translocation) systems transport proteins in folded conformation across prokaryote and prokaryote-derived membranes without breaching the permeability barrier. The machinery consists of three components in the photosynthetic thylakoid membrane. Multiple copies of Hcf106 and cpTatC form a receptor complex that recognizes the twin arginine signal peptide of precursor proteins. cpTatC is the primary receptor component. After precursor proteins bind to the receptor complex, Tha4, thought to facilitate transmembrane passage, assembles as a homo-oligomer with the precursor bound receptor complex and the precursor protein is transported across the membrane. Advisors/Committee Members: Cline, Kenneth C (committee chair), Settles, Andrew M (committee member), Hannah, Larkin C (committee member), Folta, Kevin M (committee member), Cain, Brian D (committee member).

Subjects/Keywords: Binding sites; Chloroplasts; Clines; Imports; Membrane proteins; Oligomers; Protein transport; Proteins; Receptors; Thylakoids; arginine  – chloroplast  – cptatc  – hcf106  – import  – membrane  – protein  – tat  – tha4  – thylakoid  – translocation  – transport  – twin

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APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6th Edition):

M, C. (2011). Basic Translocation Unit Composition of the Twin Arginine Translocase (TAT) System in Pea Thylakoids. (Doctoral Dissertation). University of Florida. Retrieved from https://ufdc.ufl.edu/UFE0043341

Chicago Manual of Style (16th Edition):

M, Celedon,Jose. “Basic Translocation Unit Composition of the Twin Arginine Translocase (TAT) System in Pea Thylakoids.” 2011. Doctoral Dissertation, University of Florida. Accessed January 18, 2021. https://ufdc.ufl.edu/UFE0043341.

MLA Handbook (7th Edition):

M, Celedon,Jose. “Basic Translocation Unit Composition of the Twin Arginine Translocase (TAT) System in Pea Thylakoids.” 2011. Web. 18 Jan 2021.

Vancouver:

M C. Basic Translocation Unit Composition of the Twin Arginine Translocase (TAT) System in Pea Thylakoids. [Internet] [Doctoral dissertation]. University of Florida; 2011. [cited 2021 Jan 18]. Available from: https://ufdc.ufl.edu/UFE0043341.

Council of Science Editors:

M C. Basic Translocation Unit Composition of the Twin Arginine Translocase (TAT) System in Pea Thylakoids. [Doctoral Dissertation]. University of Florida; 2011. Available from: https://ufdc.ufl.edu/UFE0043341

3. Storm, Amanda R. Structural studies of cpTat component Tha4 in both native and synthetic membrane systems.

Degree: PhD, Chemistry, 2013, Miami University

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. Advisors/Committee Members: Dabney-Smith, Carole (Advisor), Lorigan, Gary (Committee Chair).

Subjects/Keywords: Biochemistry; Chemistry; chloroplast twin arginine translocation; cpTat; Tha4; thylakoid; native topology; electron paramagnetic resonance; EPR; lipodisq nanoparticles; TOAC; amber suppression

…34 Figure 2.2: Complementation efficiency of cysteine-substituted Tha4… …35 Figure 2.3: Tha4 topology labeling shows a N OUT-CIN conformation… …36 Figure 2.4: Biocytin labeling of Tha4 amino terminus and TMD… …37 Figure 2.5: Tha4 emerges from the hydrophobic membrane core at the N-proximal APH… …44 Figure 3.1: Illustration of Tha4 with cysteine-substitution positions. Blue colored… 

Record DetailsSimilar RecordsGoogle PlusoneFacebookTwitterCiteULikeMendeleyreddit

APA · Chicago · MLA · Vancouver · CSE | Export to Zotero / EndNote / Reference Manager

APA (6th Edition):

Storm, A. R. (2013). Structural studies of cpTat component Tha4 in both native and synthetic membrane systems. (Doctoral Dissertation). Miami University. Retrieved from http://rave.ohiolink.edu/etdc/view?acc_num=miami1385466330

Chicago Manual of Style (16th Edition):

Storm, Amanda R. “Structural studies of cpTat component Tha4 in both native and synthetic membrane systems.” 2013. Doctoral Dissertation, Miami University. Accessed January 18, 2021. http://rave.ohiolink.edu/etdc/view?acc_num=miami1385466330.

MLA Handbook (7th Edition):

Storm, Amanda R. “Structural studies of cpTat component Tha4 in both native and synthetic membrane systems.” 2013. Web. 18 Jan 2021.

Vancouver:

Storm AR. Structural studies of cpTat component Tha4 in both native and synthetic membrane systems. [Internet] [Doctoral dissertation]. Miami University; 2013. [cited 2021 Jan 18]. Available from: http://rave.ohiolink.edu/etdc/view?acc_num=miami1385466330.

Council of Science Editors:

Storm AR. Structural studies of cpTat component Tha4 in both native and synthetic membrane systems. [Doctoral Dissertation]. Miami University; 2013. Available from: http://rave.ohiolink.edu/etdc/view?acc_num=miami1385466330

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